Objectives

Human airway epithelial cells are the principal target of human rhinovirus (HRV), a common cold pathogen that triggers the majority of asthma exacerbations. The objectives of this study were 1) to evaluate an in vitro air liquid interface cultured human airway epithelial cell model for HRV infection, and 2) to identify gene expression patterns associated with asthma intrinsically and/or after HRV infection using this model.

Methods

Air-liquid interface (ALI) human airway epithelial cell cultures were prepared from 6 asthmatic and 6 non-asthmatic donors. The effects of rhinovirus RV-A16 on ALI cultures were compared. Genome-wide gene expression changes in ALI cultures following HRV infection at 24 hours post exposure were further analyzed using RNA-seq technology. Cellular gene expression and cytokine/chemokine secretion were further evaluated by qPCR and a Luminex-based protein assay, respectively.

Main results

ALI cultures were readily infected by HRV. RNA-seq analysis of HRV infected ALI cultures identified sets of genes associated with asthma specific viral responses. These genes are related to inflammatory pathways, epithelial structure and remodeling and cilium assembly and function, including those described previously (e.g. CCL5, CXCL10 and CX3CL1, MUC5AC, CDHR3), and novel ones that were identified for the first time in this study (e.g. CCRL1).

Conclusions

ALI-cultured human airway epithelial cells challenged with HRV are a useful translational model for the study of HRV-induced responses in airway epithelial cells, given that gene expression profile using this model largely recapitulates some important patterns of gene responses in patients during clinical HRV infection. Furthermore, our data emphasize that both abnormal airway epithelial structure and inflammatory signaling are two important asthma signatures, which can be further exacerbated by HRV infection.

Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.

Recurrent mutations in the spliceosome are observed in several human cancers, but their functional and therapeutic significance remains elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3' splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3' ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3' ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1^K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1^K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1^K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

Given the skyrocketing costs to develop new drugs, repositioning of approved drugs, such as histone deacetylase (HDAC) inhibitors, may be a promising strategy to develop novel therapies. However, a gap exists in the understanding and advancement of these agents to meaningful translation for which new indications may emerge. To address this, we performed systems-level analyses of 33 independent HDAC inhibitor microarray studies. Based on network analysis, we identified enrichment for pathways implicated in metabolic syndrome and diabetes (insulin receptor signaling, lipid metabolism, immunity and trafficking). Integration with ENCODE ChIP-seq datasets identified suppression of EP300 target genes implicated in diabetes. Experimental validation indicates reversal of diabetes-associated EP300 target genes in primary vascular endothelial cells derived from a diabetic individual following inhibition of HDACs (by SAHA), EP300, or EP300 knockdown. Our computational systems biology approach provides an adaptable framework for the prediction of novel therapeutics for existing disease.

To understand better the factors contributing to keratoconus (KTCN), we performed comprehensive transcriptome profiling of human KTCN corneas for the first time using an RNA-Seq approach. Twenty-five KTCN and 25 non-KTCN corneas were enrolled in this study. After RNA extraction, total RNA libraries were prepared and sequenced. The discovery RNA-Seq analysis (in eight KTCN and eight non-KTCN corneas) was conducted first, after which the replication RNA-Seq experiment was performed on a second set of samples (17 KTCN and 17 non-KTCN corneas). Over 82% of the genes and almost 75% of the transcripts detected as differentially expressed in KTCN and non-KTCN corneas were confirmed in the replication study using another set of samples. We used these differentially expressed genes to generate a network of KTCN-deregulated genes. We found an extensive disruption of collagen synthesis and maturation pathways, as well as downregulation of the core elements of the TGF-β, Hippo, and Wnt signaling pathways influencing corneal organization. This first comprehensive transcriptome profiling of human KTCN corneas points further to a complex etiology of KTCN.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by interstitial remodelling, leading to compromised lung function. Cellular senescence markers are detectable within IPF lung tissue and senescent cell deletion rejuvenates pulmonary health in aged mice. Whether and how senescent cells regulate IPF or if their removal may be an efficacious intervention strategy is unknown. Here we demonstrate elevated abundance of senescence biomarkers in IPF lung, with p16 expression increasing with disease severity. We show that the secretome of senescent fibroblasts, which are selectively killed by a senolytic cocktail, dasatinib plus quercetin (DQ), is fibrogenic. Leveraging the bleomycin-injury IPF model, we demonstrate that early-intervention suicide-gene-mediated senescent cell ablation improves pulmonary function and physical health, although lung fibrosis is visibly unaltered. DQ treatment replicates benefits of transgenic clearance. Thus, our findings establish that fibrotic lung disease is mediated, in part, by senescent cells, which can be targeted to improve health and function.

Glucocorticoids, commonly used asthma controller medications, decrease symptoms in most patients, but some remain symptomatic despite high-dose treatment. The physiological basis underlying the glucocorticoid response, especially in asthma patients with severe, refractory disease, is not fully understood. We sought to identify differences between the transcriptomic response of airway smooth muscle (ASM) cells derived from donors with fatal asthma and donors without asthma to glucocorticoid exposure and to compare ASM-specific changes with those observed in other cell types. In cells derived from nine donors with fatal asthma and eight donors without asthma, RNA sequencing was used to measure ASM transcriptome changes after exposure to budesonide (100 nM 24 h) or control vehicle (DMSO). Differential expression results were obtained for this dataset, as well as 13 publicly available glucocorticoid-response transcriptomic datasets corresponding to seven cell types. Specific genes were differentially expressed in response to glucocorticoid exposure (7,835 and 6,957 in ASM cells derived from donors with fatal asthma and donors without asthma, respectively; adjusted P value < 0.05). Transcriptomic changes in response to glucocorticoid exposure were similar in ASM derived from donors with fatal asthma and donors without asthma, with enriched ontological pathways that included cytokine- and chemokine-related categories. A comparison of glucocorticoid-induced changes in the nonasthma ASM transcriptome with those observed in six other cell types showed that ASM has a distinct glucocorticoid-response signature that is also present in ASM cells from donors with fatal asthma.

Chronic hepatitis B virus (HBV) infection leads to liver fibrosis, which is a major risk factor in hepatocellular carcinoma (HCC) and an independent risk factor of recurrence after HCC tumor resection. The HBV genome can be inserted into the human genome, and chronic inflammation may trigger somatic mutations. However, how HBV integration and other genomic changes contribute to the risk of tumor recurrence with regards to the different degree of liver fibrosis is not clearly understood.We sequenced mRNAs of 21 pairs of tumor and distant non-neoplastic liver tissues of HBV-HCC patients and performed comprehensive genomic analyses of our RNAseq data and public available HBV-HCC sequencing data.We developed a robust pipeline for sensitively identifying HBV integration sites based on sequencing data. Simulations showed that our method outperformed existing methods. Applying it to our data, 374 and 106 HBV host genes were identified in non-neoplastic liver and tumor tissues, respectively. When applying it to other RNA sequencing datasets, consistently more HBV integrations were identified in non-neoplastic liver than in tumor tissues. HBV host genes identified in non-neoplastic liver samples significantly overlapped with known tumor suppressor genes. More significant enrichment of tumor suppressor genes was observed among HBV host genes identified from patients with tumor recurrence, indicating the potential risk of tumor recurrence driven by HBV integration in non-neoplastic liver tissues. We also compared SNPs of each sample with SNPs in a cancer census database and inferred samples' pathogenic SNP loads. Pathogenic SNP loads in non-neoplastic liver tissues were consistently higher than those in normal liver tissues. Additionally, HBV host genes identified in non-neoplastic liver tissues significantly overlapped with pathogenic somatic mutations, suggesting that HBV integration and somatic mutations targeting the same set of genes are important to tumorigenesis. HBV integrations and pathogenic mutations showed distinct patterns between low and high liver fibrosis patients with regards to tumor recurrence.The results suggest that HBV integrations and pathogenic SNPs in non-neoplastic tissues are important for tumorigenesis and different recurrence risk models are needed for patients with low and high degrees of liver fibrosis.

Chronic obstructive pulmonary disease (COPD) is a serious global health problem characterized by chronic airway inflammation, progressive airflow limitation and destruction of lung parenchyma. Remodeling of the bronchial airways in COPD includes changes in both the bronchial epithelium and the subepithelial extracellular matrix (ECM). To explore the impact of an aberrant ECM on epithelial cell phenotype in COPD we developed a new ex vivo model, in which normal human bronchial epithelial (NHBE) cells repopulate and differentiate on decellularized human bronchial scaffolds derived from COPD patients and healthy individuals. By using transcriptomics, we show that bronchial ECM from COPD patients induces differential gene expression in primary NHBE cells when compared to normal bronchial ECM. The gene expression profile indicated altered activity of upstream mediators associated with COPD pathophysiology, including hepatocyte growth factor, transforming growth factor beta 1 and platelet-derived growth factor B, which suggests that COPD-related changes in the bronchial ECM contribute to the defective regenerative ability in the airways of COPD patients.

Purpose: The blockade of immune checkpoints such as PD-L1 and PD-1 is being exploited therapeutically in several types of malignancies. Here, we aimed to understand the contribution of the genetics of lung cancer to the ability of tumor cells to escape immunosurveillance checkpoints.Experimental Design: More than 150 primary non-small cell lung cancers, including pulmonary sarcomatoid carcinomas, were tested for levels of the HLA-I complex, PD-L1, tumor-infiltrating CD8+ lymphocytes, and alterations in main lung cancer genes. Correlations were validated in cancer cell lines using appropriate treatments to activate or inhibit selected pathways. We also performed RNA sequencing to assess changes in gene expression after these treatments.Results:MET-oncogenic activation tended to associate with positive PD-L1 immunostaining, whereas STK11 mutations were correlated with negative immunostaining. In MET-altered cancer cells, MET triggered a transcriptional increase of PD-L1 that was independent of the IFNγ-mediated JAK/STAT pathway. The activation of MET also upregulated other immunosuppressive genes (PDCD1LG2 and SOCS1) and transcripts involved in angiogenesis (VEGFA and NRP1) and in cell proliferation. We also report recurrent inactivating mutations in JAK2 that co-occur with alterations in MET and STK11, which prevented the induction of immunoresponse-related genes following treatment with IFNγ.Conclusions: We show that MET activation promotes the expression of several negative checkpoint regulators of the immunoresponse, including PD-L1. In addition, we report inactivation of JAK2 in lung cancer cells that prevented the response to IFNγ. These alterations are likely to facilitate tumor growth by enabling immune tolerance and may affect the response to immune checkpoint inhibitors. Clin Cancer Res; 24(18); 4579-87. ©2018 AACR.

Cutaneous T-cell lymphoma (CTCL) develops from clonally expanded CD4+ T cells in a background of chronic inflammation. Although dendritic cells (DCs) stimulate T cells and are present in skin, cutaneous T cells in CTCL do not respond with effective antitumor immunity. We evaluated primary T-cell and DC émigrés from epidermal and dermal explant cultures of skin biopsies from CTCL patients (n = 37) and healthy donors (n = 5). Compared with healthy skin, CD4+ CTCL populations contained more T cells expressing PD-1, CTLA-4, and LAG-3. CD8+ CTCL populations contained more T cells expressing CTLA-4 and LAG-3. CTCL populations also contained more T cells expressing the inducible T-cell costimulator (ICOS), a marker of T-cell activation. DC émigrés from healthy or CTCL skin biopsies expressed PD-L1, indicating that maturation during migration resulted in PD-L1 expression irrespective of disease. Most T cells did not express PD-L1. Using skin samples from 49 additional CTCL patients for an unsupervised analysis of genome-wide mRNA expression profiles corroborated that advanced T3/T4-stage samples expressed more checkpoint inhibition mRNA compared with T1/T2 stage patients or healthy controls. Exhaustion of activated T cells is therefore a hallmark of both CD4+ and CD8+ T cells isolated from the lesional skin of patients with CTCL, with increasing expression as the disease progresses. These results justify identification of antigens driving T-cell exhaustion and the evaluation of immune checkpoint inhibition to reverse T-cell exhaustion earlier in the treatment of CTCL. Cancer Immunol Res; 6(8); 900-9. ©2018 AACR.

Tuberculous meningitis (TBM) is the most severe form of TB with high rates of mortality and morbidity. Here we conduct RNA-sequencing on whole blood as well as on ventricular and lumbar cerebrospinal fluid (CSF) of pediatric patients treated for TBM. Differential transcript expression of TBM cases are compared with healthy controls in whole blood and with non-TB cerebral infection controls in CSF. Whole blood RNA-Seq analysis demonstrates a distinct immune response pattern in TBM, with significant increase in both canonical and non-canonical inflammasome activation and decrease in T-cell activation. In ventricular CSF, a significant enrichment associated with neuronal excitotoxicity and cerebral damage is detected in TBM. Finally, compartmental comparison in TBM indicates that the ventricular profile represents brain injury whereas the lumbar profile represents protein translation and cytokine signaling. Together, transcriptomic analysis shows that disease processes differ between the periphery and the central nervous system, and within brain compartments.

TLR4 activation initiates a signaling cascade leading to the production of type I IFNs and of the downstream IFN-stimulated genes (ISGs). Recently, a number of IFN-induced long non-coding RNAs (lncRNAs) that feed-back regulate the IFN response have been identified. Dysregulation of this process, collectively known as the "Interferon (IFN) Response," represents a common molecular basis in the development of autoimmune and autoinflammatory disorders. Concurrently, alteration of lncRNA profile has been described in several type I IFN-driven autoimmune diseases. In particular, both TLR activation and the upregulation of ISGs in peripheral blood mononuclear cells have been identified as possible contributors to the pathogenesis of systemic sclerosis (SSc), a connective tissue disease characterized by vascular abnormalities, immune activation, and fibrosis. However, hitherto, a potential link between specific lncRNA and the presence of a type I IFN signature remains unclear in SSc. In this study, we identified, by RNA sequencing, a group of lncRNAs related to the IFN and anti-viral response consistently modulated in a type I IFN-dependent manner in human monocytes in response to TLR4 activation by LPS. Remarkably, these lncRNAs were concurrently upregulated in a total of 46 SSc patients in different stages of their disease as compared to 18 healthy controls enrolled in this study. Among these lncRNAs, Negative Regulator of the IFN Response (NRIR) was found significantly upregulated in vivo in SSc monocytes, strongly correlating with the IFN score of SSc patients. Weighted Gene Co-expression Network Analysis showed that NRIR-specific modules, identified in the two datasets, were enriched in "type I IFN" and "viral response" biological processes. Protein coding genes common to the two distinct NRIR modules were selected as putative NRIR target genes. Fifteen in silico-predicted NRIR target genes were experimentally validated in NRIR-silenced monocytes. Remarkably, induction of CXCL10 and CXCL11, two IFN-related chemokines associated with SSc pathogenesis, was reduced in NRIR-knockdown monocytes, while their plasmatic level was increased in SSc patients. Collectively, our data show that NRIR affects the expression of ISGs and that dysregulation of NRIR in SSc monocytes may account, at least in part, for the type I IFN signature present in SSc patients.

RNA-sequencing (RNA-seq) is a powerful technique to investigate the complexity of gene expression in the human brain. We used RNA-seq to survey the brain transcriptome in high-quality postmortem dorsolateral prefrontal cortex from 11 individuals diagnosed with bipolar disorder (BD) and from 11 age- and gender-matched controls. Deep sequencing was performed, with over 350 million reads per specimen. At a false discovery rate of <5%, we detected five differentially expressed (DE) genes and 12 DE transcripts, most of which have not been previously implicated in BD. Among these, Prominin 1/CD133 and ATP-binding cassette-sub-family G-member2 (ABCG2) have important roles in neuroplasticity. We also show for the first time differential expression of long noncoding RNAs (lncRNAs) in BD. DE transcripts include those of serine/arginine-rich splicing factor 5 (SRSF5) and regulatory factor X4 (RFX4), which along with lncRNAs have a role in mammalian circadian rhythms. The DE genes were significantly enriched for several Gene Ontology categories. Of these, genes involved with GTPase binding were also enriched for BD-associated SNPs from previous genome-wide association studies, suggesting that differential expression of these genes is not simply a consequence of BD or its treatment. Many of these findings were replicated by microarray in an independent sample of 60 cases and controls. These results highlight common pathways for inherited and non-inherited influences on disease risk that may constitute good targets for novel therapies.

Unlabelled

Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the "window period" of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets.

Importance

Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies.

Previous preclinical studies and a phase I clinical trial suggested that myo-inositol may be a safe and effective lung cancer chemopreventive agent. We conducted a randomized, double blind, placebo-controlled phase IIb study to determine the chemopreventive effects of myo-inositol in smokers with bronchial dysplasia. Smokers with ≥1 site of dysplasia identified by autofluorescence bronchoscopy-directed biopsy were randomly assigned to receive oral placebo or myo-inositol, 9 g once a day for 2 weeks, and then twice a day for 6 months. The primary endpoint was change in dysplasia rate after 6 months of intervention on a per-participant basis. Other trial endpoints reported herein include Ki-67 labeling index, blood and bronchoalveolar lavage fluid (BAL) levels of proinflammatory, oxidant/antioxidant biomarkers, and an airway epithelial gene expression signature for PI3K activity. Seventy-four (n = 38 myo-inositol and n = 36 placebo) participants with a baseline and 6-month bronchoscopy were included in all efficacy analyses. The complete response and the progressive disease rates were 26.3% versus 13.9% and 47.4% versus 33.3%, respectively, in the myo-inositol and placebo arms (P = 0.76). Compared with placebo, myo-inositol intervention significantly reduced IL6 levels in BAL over 6 months (P = 0.03). Among those with a complete response in the myo-inositol arm, there was a significant decrease in a gene expression signature reflective of PI3K activation within the cytologically normal bronchial airway epithelium (P = 0.002). The heterogeneous response to myo-inositol suggests a targeted therapy approach based on molecular alterations is needed in future clinical trials to determine the efficacy of myo-inositol as a chemopreventive agent. Cancer Prev Res; 9(12); 906-14. ©2016 AACR.

Medical image biomarkers of cancer promise improvements in patient care through advances in precision medicine. Compared to genomic biomarkers, image biomarkers provide the advantages of being non-invasive, and characterizing a heterogeneous tumor in its entirety, as opposed to limited tissue available via biopsy. We developed a unique radiogenomic dataset from a Non-Small Cell Lung Cancer (NSCLC) cohort of 211 subjects. The dataset comprises Computed Tomography (CT), Positron Emission Tomography (PET)/CT images, semantic annotations of the tumors as observed on the medical images using a controlled vocabulary, and segmentation maps of tumors in the CT scans. Imaging data are also paired with results of gene mutation analyses, gene expression microarrays and RNA sequencing data from samples of surgically excised tumor tissue, and clinical data, including survival outcomes. This dataset was created to facilitate the discovery of the underlying relationship between tumor molecular and medical image features, as well as the development and evaluation of prognostic medical image biomarkers.

Esthesioneuroblastoma (ENB) is a rare cancer of the olfactory mucosa, with no established molecular stratification to date. We report similarities of ENB with tumors arising in the neural crest and perform integrative analysis of these tumors. We propose a molecular-based subtype classification of ENB as basal or neural, both of which have distinct pathological, transcriptomic, proteomic, and immune features. Among the basal subtype, we uncovered an IDH2 R172 mutant-enriched subgroup (∼35%) harboring a CpG island methylator phenotype reminiscent of IDH2 mutant gliomas. Compared with the basal ENB methylome, the neural ENB methylome shows genome-wide reprogramming with loss of DNA methylation at the enhancers of axonal guidance genes. Our study reveals insights into the molecular pathogenesis of ENB and provides classification information of potential therapeutic relevance.

Aim

The aim of our study was to identify pharmacodynamic biomarkers and assess differential effects of tumor necrosis factor (TNF)- and non-TNF-targeting agents on rheumatoid arthritis (RA) patients with an inadequate response to anti-TNF agents (anti-TNF-IR) in comparison with biologic-naïve patients.

Methods

EARTH EXPLORER 2, a phase IIb trial, evaluated golimumab, an anti-TNF antibody, and mavrilimumab, an granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor antibody, in disease-modifying antirheumatic drug (DMARD)-IR and anti-TNF-IR patients. Our current study assessed peripheral protein markers and gene expression levels in association with clinical response post-treatment in two disease strata.

Results

Serum proteomics results indicated the existence of specific pharmacodynamic markers for golimumab and mavrilimumab, regardless of prior anti-TNF treatment. In contrast, both antibodies induced early and sustained suppression of RA disease markers, including interleukin (IL)-6, C-reactive protein, IL2RA, and matrix metalloproteinase 1, in DMARD-IR patients. Golimumab-induced early changes rapidly returned toward baseline concentrations in anti-TNF-IR patients, whereas mavrilimumab-induced changes were maintained through to day 169. RNA sequencing demonstrated gene expression changes at day 169 after administration of mavrilimumab but not golimumab in anti-TNF-IR patients. Additionally, receiver operating characteristic curve and regression analysis showed the association of early IL-6 change and subsequent clinical responses to golimumab in anti-TNF-IR patients.

Conclusion

Our results revealed golimumab- and mavrilimumab-specific pharmacodynamic biomarkers, and demonstrated differential biomarker-treatment relationships in anti-TNF-IR and DMARD-IR patients, respectively. Early IL-6 change after anti-TNF antibody treatment may be a potential predictive biomarker for selection of different treatment regimens in anti-TNF-IR patients.

Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a syndrome of unknown etiology characterized by profound fatigue exacerbated by physical activity, also known as post-exertional malaise (PEM). Previously, we did not detect evidence of immune dysregulation or virus reactivation outside of PEM periods. Here we sought to determine whether cardiopulmonary exercise stress testing of ME/CFS patients could trigger such changes. ME/CFS patients (n = 14) and matched sedentary controls (n = 11) were subjected to cardiopulmonary exercise on 2 consecutive days and followed up to 7 days post-exercise, and longitudinal whole blood samples analyzed by RNA-seq. Although ME/CFS patients showed significant worsening of symptoms following exercise versus controls, with 8 of 14 ME/CFS patients showing reduced oxygen consumption ([Formula: see text]) on day 2, transcriptome analysis yielded only 6 differentially expressed gene (DEG) candidates when comparing ME/CFS patients to controls across all time points. None of the DEGs were related to immune signaling, and no DEGs were found in ME/CFS patients before and after exercise. Virome composition (P = 0.746 by chi-square test) and number of viral reads (P = 0.098 by paired t-test) were not significantly associated with PEM. These observations do not support transcriptionally-mediated immune cell dysregulation or viral reactivation in ME/CFS patients during symptomatic PEM episodes.

Objective

Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose at resectable stage. Recent studies have suggested that extracellular vesicles (EVs) contain long RNAs. The aim of this study was to develop a diagnostic (d-)signature for the detection of PDAC based on EV long RNA (exLR) profiling.

Design

We conducted a case-control study with 501 participants, including 284 patients with PDAC, 100 patients with chronic pancreatitis (CP) and 117 healthy subjects. The exLR profile of plasma samples was analysed by exLR sequencing. The d-signature was identified using a support vector machine algorithm and a training cohort (n=188) and was validated using an internal validation cohort (n=135) and an external validation cohort (n=178).

Results

We developed a d-signature that comprised eight exLRs, including FGA, KRT19, HIST1H2BK, ITIH2, MARCH2, CLDN1, MAL2 and TIMP1, for PDAC detection. The d-signature showed high accuracy, with an area under the receiver operating characteristic curve (AUC) of 0.960, 0.950 and 0.936 in the training, internal validation and external validation cohort, respectively. The d-signature was able to identify resectable stage I/II cancer with an AUC of 0.949 in the combined three cohorts. In addition, the d-signature showed superior performance to carbohydrate antigen 19-9 in distinguishing PDAC from CP (AUC 0.931 vs 0.873, p=0.028).

Conclusion

This study is the first to characterise the plasma exLR profile in PDAC and to report an exLR signature for the detection of pancreatic cancer. This signature may improve the prognosis of patients who would have otherwise missed the curative treatment window.

Viral pandemics, such as the one caused by SARS-CoV-2, pose an imminent threat to humanity. Because of its recent emergence, there is a paucity of information regarding viral behavior and host response following SARS-CoV-2 infection. Here we offer an in-depth analysis of the transcriptional response to SARS-CoV-2 compared with other respiratory viruses. Cell and animal models of SARS-CoV-2 infection, in addition to transcriptional and serum profiling of COVID-19 patients, consistently revealed a unique and inappropriate inflammatory response. This response is defined by low levels of type I and III interferons juxtaposed to elevated chemokines and high expression of IL-6. We propose that reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production are the defining and driving features of COVID-19.

To increase our understanding of psoriasis, we used high-throughput complementary DNA sequencing (RNA-seq) to assay the transcriptomes of lesional psoriatic and normal skin. We sequenced polyadenylated RNA-derived complementary DNAs from 92 psoriatic and 82 normal punch biopsies, generating an average of ∼38 million single-end 80-bp reads per sample. Comparison of 42 samples examined by both RNA-seq and microarray revealed marked differences in sensitivity, with transcripts identified only by RNA-seq having much lower expression than those also identified by microarray. RNA-seq identified many more differentially expressed transcripts enriched in immune system processes. Weighted gene coexpression network analysis (WGCNA) revealed multiple modules of coordinately expressed epidermal differentiation genes, overlapping significantly with genes regulated by the long noncoding RNA TINCR, its target gene, staufen-1 (STAU1), the p63 target gene ZNF750, and its target KLF4. Other coordinately expressed modules were enriched for lymphoid and/or myeloid signature transcripts and genes induced by IL-17 in keratinocytes. Dermally expressed genes were significantly downregulated in psoriatic biopsies, most likely because of expansion of the epidermal compartment. These results show the power of WGCNA to elucidate gene regulatory circuits in psoriasis, and emphasize the influence of tissue architecture in both differential expression and coexpression analysis.

Background

Although analysis pipelines have been developed to use RNA-seq to identify long non-coding RNAs (lncRNAs), inference of their biological and pathological relevance remains a challenge. As a result, most transcriptome studies of autoimmune disease have only assessed protein-coding transcripts.

Results

We used RNA-seq data from 99 lesional psoriatic, 27 uninvolved psoriatic, and 90 normal skin biopsies, and applied computational approaches to identify and characterize expressed lncRNAs. We detect 2,942 previously annotated and 1,080 novel lncRNAs which are expected to be skin specific. Notably, over 40% of the novel lncRNAs are differentially expressed and the proportions of differentially expressed transcripts among protein-coding mRNAs and previously-annotated lncRNAs are lower in psoriasis lesions versus uninvolved or normal skin. We find that many lncRNAs, in particular those that are differentially expressed, are co-expressed with genes involved in immune related functions, and that novel lncRNAs are enriched for localization in the epidermal differentiation complex. We also identify distinct tissue-specific expression patterns and epigenetic profiles for novel lncRNAs, some of which are shown to be regulated by cytokine treatment in cultured human keratinocytes.

Conclusions

Together, our results implicate many lncRNAs in the immunopathogenesis of psoriasis, and our results provide a resource for lncRNA studies in other autoimmune diseases.

Autoimmune diseases affect 7.5% of the US population, and they are among the leading causes of death and disability. A notable feature of many autoimmune diseases is their greater prevalence in females than in males, but the underlying mechanisms of this have remained unclear. Through the use of high-resolution global transcriptome analyses, we demonstrated a female-biased molecular signature associated with susceptibility to autoimmune disease and linked this to extensive sex-dependent co-expression networks. This signature was independent of biological age and sex-hormone regulation and was regulated by the transcription factor VGLL3, which also had a strong female-biased expression. On a genome-wide level, VGLL3-regulated genes had a strong association with multiple autoimmune diseases, including lupus, scleroderma and Sjögren's syndrome, and had a prominent transcriptomic overlap with inflammatory processes in cutaneous lupus. These results identified a VGLL3-regulated network as a previously unknown inflammatory pathway that promotes female-biased autoimmunity. They demonstrate the importance of studying immunological processes in females and males separately and suggest new avenues for therapeutic development.

While immunotherapies are rapidly becoming mainstays of cancer treatment, significant gaps remain in our understanding of how to optimally target them, alone or in combination. Here we describe a novel method to monitor levels of immune cells and pathways in expression data from solid tumors using pre-defined groups or modules of co-regulated immune genes. We show that expression of an interconnected sub-network of type I interferon-stimulated genes (ISGs) in melanomas at the time of diagnosis significantly predicted patient survival, as did, to a lesser extent, sub-networks of T helper/T regulatory and NK/T Cytotoxic cell genes. As a group, poor prognosis tumors with reduced ISG and immune gene levels exhibited significant copy number loss of the interferon gene cluster located at chromosome 9p21.3. Our studies demonstrate a link between type I interferon action and immune cell levels in melanomas, and suggest that therapeutic approaches augmenting both activities may be most beneficial.

Background

Surveillance of integrity of the basic elements of the cell including DNA, RNA, and proteins is a critical element of cellular physiology. Mechanisms of surveillance of DNA and protein integrity are well understood. Surveillance of structural RNAs making up the vast majority of RNA in a cell is less well understood. Here, we sought to explore integrity of processing of structural RNAs in relapsing remitting multiple sclerosis (RRMS) and other inflammatory diseases.

Results

We employed mononuclear cells obtained from subjects with RRMS and cell lines. We used quantitative-PCR and whole genome RNA sequencing to define defects in structural RNA surveillance and siRNAs to deplete target proteins. We report profound defects in surveillance of structural RNAs in RRMS exemplified by elevated levels of poly(A) + Y1-RNA, poly(A) + 18S rRNA and 28S rRNAs, elevated levels of misprocessed 18S and 28S rRNAs and levels of the U-class of small nuclear RNAs. Multiple sclerosis is also associated with genome-wide defects in mRNA splicing. Ro60 and La proteins, which exist in ribonucleoprotein particles and play different roles in quality control of structural RNAs, are also deficient in RRMS. In cell lines, silencing of the genes encoding Ro60 and La proteins gives rise to these same defects in surveillance of structural RNAs.

Conclusions

Our results establish that profound defects in structural RNA surveillance exist in RRMS and establish a causal link between Ro60 and La proteins and integrity of structural RNAs.

Myelin-reactive T cells have been identified in patients with multiple sclerosis (MS) and healthy subjects with comparable frequencies, but the contribution of these autoreactive T cells to disease pathology remains unknown. A total of 13,324 T cell libraries generated from blood of 23 patients and 22 healthy controls were interrogated for reactivity to myelin antigens. Libraries derived from CCR6(+) myelin-reactive T cells from patients with MS exhibited significantly enhanced production of interferon-γ (IFN-γ), interleukin-17 (IL-17), and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to healthy controls. Single-cell clones isolated by major histocompatibility complex/peptide tetramers from CCR6(+) T cell libraries also secreted more proinflammatory cytokines, whereas clones isolated from controls secreted more IL-10. The transcriptomes of myelin-specific CCR6(+) T cells from patients with MS were distinct from those derived from healthy controls and, notably, were enriched in T helper cell 17 (TH17)-induced experimental autoimmune encephalitis gene signatures, and gene signatures derived from TH17 cells isolated other human autoimmune diseases. These data, although not causal, imply that functional differences between antigen-specific T cells from MS and healthy controls are fundamental to disease development and support the notion that IL-10 production from myelin-reactive T cells may act to limit disease progression or even pathogenesis.

Background & aims

Etrolizumab is a humanized monoclonal antibody against the β7 integrin subunit that has shown efficacy vs placebo in patients with moderate to severely active ulcerative colitis (UC). Patients with colon tissues that expressed high levels of the integrin αE gene (ITGAE) appeared to have the best response. We compared differences in colonic expression of ITGAE and other genes between patients who achieved clinical remission with etrolizumab vs those who did.

Methods

We performed a retrospective analysis of data collected from 110 patients with UC who participated in a phase 2 placebo-controlled trial of etrolizumab, as well as from 21 patients with UC or without inflammatory bowel disease (controls) enrolled in an observational study at a separate site. Colon biopsies were collected from patients in both studies and analyzed by immunohistochemistry and gene expression profiling. Mononuclear cells were isolated and analyzed by flow cytometry. We identified biomarkers associated with response to etrolizumab. In the placebo-controlled trial, clinical remission was defined as total Mayo Clinic Score ≤2, with no individual subscore >1, and mucosal healing was defined as endoscopic score ≤1.

Results

Colon tissues collected at baseline from patients who had a clinical response to etrolizumab expressed higher levels of T-cell-associated genes than patients who did not respond (P < .05). Colonic CD4(+) integrin αE(+) cells from patients with UC expressed higher levels of granzyme A messenger RNA (GZMA mRNA) than CD4(+) αE(-) cells (P < .0001); granzyme A and integrin αE protein were detected in the same cells. Of patients receiving 100 mg etrolizumab, a higher proportion of those with high levels of GZMA mRNA (41%) or ITGAE mRNA (38%) than those with low levels of GZMA (6%) or ITGAE mRNA (13%) achieved clinical remission (P < .05) and mucosal healing (41% GZMA(high) vs 19% GZMA(low) and 44% ITGAE(high) vs 19% ITGAE(low)). Compared with ITGAE(low) and GZMA(low) patients, patients with ITGAE(high) and GZMA(high) had higher baseline numbers of epithelial crypt-associated integrin αE(+) cells (P < .01 for both), but a smaller number of crypt-associated integrin αE(+) cells after etrolizumab treatment (P < .05 for both). After 10 weeks of etrolizumab treatment, expression of genes associated with T-cell activation and genes encoding inflammatory cytokines decreased by 40%-80% from baseline (P < .05) in patients with colon tissues expressing high levels of GZMA at baseline.

Conclusions

Levels of GZMA and ITGAE mRNAs in colon tissues can identify patients with UC who are most likely to benefit from etrolizumab; expression levels decrease with etrolizumab administration in biomarker(high) patients. Larger, prospective studies of markers are needed to assess their clinical value.

We used RNA sequencing to study and characterize the long noncoding RNA (lncRNA) transcriptome in lesional skin from psoriasis patients before (PP) and after treatment (PT) with adalimumab and in normal skin from healthy individuals (NN). To this end, we sequenced total RNA from 18 psoriasis patients and 16 healthy controls. We merged three lncRNA reference datasets to create a single combined reference of 67,157 lncRNA transcripts with no overlaps. We identified differential expression of 971 lncRNAs between PP and NN, 157 between PP and PT, and 377 between PT and NN. Using differentially expressed lncRNAs between PP and NN, we identified a molecular lncRNA signature that distinguishes psoriatic skin from healthy skin. Furthermore, we performed an unsupervised hierarchical analysis that revealed distinct clustering of PP samples from NN. A coding noncoding network analysis revealed a large network of highly correlated lncRNA and protein coding transcripts that provided insight into the potential functions of unannotated lncRNAs. To the best of our knowledge, this description of both polyadenylated as well as nonpolyadenylated lncRNA transcripts in psoriasis has not been previously reported. Our findings highlight the potential importance of lncRNAs in the biology of psoriasis and response to treatment.

Background

Psoriasis is an immune-mediated, inflammatory disorder of the skin characterized by chronic inflammation and hyperproliferation of the epidermis. Differential expression analysis of microarray or RNA-seq data have shown that thousands of coding and non-coding genes are differentially expressed between psoriatic and healthy control skin. However, differential expression analysis may fail to detect perturbations in gene coexpression networks. Sensitive detection of such networks may provide additional insight into important disease-associated pathways. In this study, we applied weighted gene coexpression network analysis (WGCNA) on RNA-seq data from psoriasis patients and healthy controls.

Results

RNA-seq was performed on skin samples from 18 psoriasis patients (pre-treatment and post-treatment with the TNF-α inhibitor adalimumab) and 16 healthy controls, generating an average of 52.3 million 100-bp paired-end reads per sample. Using WGCNA, we identified 3 network modules that were significantly correlated with psoriasis and 6 network modules significantly correlated with biologic treatment, with only 16 % of the psoriasis-associated and 5 % of the treatment-associated coexpressed genes being identified by differential expression analysis. In a majority of these correlated modules, more than 50 % of coexpressed genes were long non-coding RNAs (lncRNA). Enrichment analysis of these correlated modules revealed that short-chain fatty acid metabolism and olfactory signaling are amongst the top pathways enriched for in modules associated with psoriasis, while regulation of leukocyte mediated cytotoxicity and regulation of cell killing are amongst the top pathways enriched for in modules associated with biologic treatment. A putative autoantigen, LL37, was coexpressed in the module most correlated with psoriasis.

Conclusions

This study has identified several networks of coding and non-coding genes associated with psoriasis and biologic drug treatment, including networks enriched for short-chain fatty acid metabolism and olfactory receptor activity, pathways that were not previously identified through differential expression analysis and may be dysregulated in psoriatic skin. As these networks are comprised mostly of non-coding genes, it is likely that non-coding genes play critical roles in the regulation of pathways involved in the pathogenesis of psoriasis.

Stratifying patients on the basis of molecular signatures could facilitate development of therapeutics that target pathways specific to a particular disease or tissue location. Previous studies suggest that pathogenesis of rheumatoid arthritis (RA) is similar in all affected joints. Here we show that distinct DNA methylation and transcriptome signatures not only discriminate RA fibroblast-like synoviocytes (FLS) from osteoarthritis FLS, but also distinguish RA FLS isolated from knees and hips. Using genome-wide methods, we show differences between RA knee and hip FLS in the methylation of genes encoding biological pathways, such as IL-6 signalling via JAK-STAT pathway. Furthermore, differentially expressed genes are identified between knee and hip FLS using RNA-sequencing. Double-evidenced genes that are both differentially methylated and expressed include multiple HOX genes. Joint-specific DNA signatures suggest that RA disease mechanisms might vary from joint to joint, thus potentially explaining some of the diversity of drug responses in RA patients.

Systemic lupus erythematosus (SLE) patients exhibit immense heterogeneity which is challenging from the diagnostic perspective. Emerging high throughput sequencing technologies have been proved to be a useful platform to understand the complex and dynamic disease processes. SLE patients categorised based on autoantibody specificities are reported to have differential immuno-regulatory mechanisms. Therefore, we performed RNA-seq analysis to identify transcriptomics of SLE patients with distinguished autoantibody specificities. The SLE patients were segregated into three subsets based on the type of autoantibodies present in their sera (anti-dsDNA+ group with anti-dsDNA autoantibody alone; anti-ENA+ group having autoantibodies against extractable nuclear antigens (ENA) only, and anti-dsDNA+ENA+ group having autoantibodies to both dsDNA and ENA). Global transcriptome profiling for each SLE patients subsets was performed using Illumina® Hiseq-2000 platform. The biological relevance of dysregulated transcripts in each SLE subsets was assessed by ingenuity pathway analysis (IPA) software. We observed that dysregulation in the transcriptome expression pattern was clearly distinct in each SLE patients subsets. IPA analysis of transcripts uniquely expressed in different SLE groups revealed specific biological pathways to be affected in each SLE subsets. Multiple cytokine signaling pathways were specifically dysregulated in anti-dsDNA+ patients whereas Interferon signaling was predominantly dysregulated in anti-ENA+ patients. In anti-dsDNA+ENA+ patients regulation of actin based motility by Rho pathway was significantly affected. The granulocyte gene signature was a common feature to all SLE subsets; however, anti-dsDNA+ group showed relatively predominant expression of these genes. Dysregulation of Plasma cell related transcripts were higher in anti-dsDNA+ and anti-ENA+ patients as compared to anti-dsDNA+ ENA+. Association of specific canonical pathways with the uniquely expressed transcripts in each SLE subgroup indicates that specific immunological disease mechanisms are operative in distinct SLE patients' subsets. This 'sub-grouping' approach could further be useful for clinical evaluation of SLE patients and devising targeted therapeutics.

Background

Ulcerative colitis (UC) only involves the colonic mucosa. Yet, nearly 50% of patients with UC who undergo total proctocolectomy with ileal pouch anal anastomosis develop UC-like inflammation of the ileal pouch (pouchitis). By contrast, patients with familial adenomatous polyposis (FAP) with ileal pouch anal anastomosis develop pouchitis far less frequently. We hypothesized that pathogenic events associated with the development of UC are recapitulated by colonic-metaplastic transcriptomic reprogramming of the UC pouch.

Methods

We prospectively sampled pouch and prepouch ileum mucosal biopsies in patients with UC with ileal pouch anal anastomosis 4, 8, and 12 months after their pouch was in continuity. Mucosal samples were also obtained from patients with FAP. Transcriptional profiles of the UC and FAP pouch and prepouch ileum were investigated via RNA sequencing and compared with data from a previously published microarray study.

Results

Unlike patients with FAP, subjects with UC exhibited a large set of differentially expressed genes between the pouch and prepouch ileum as early as 4 months after pouch functionalization. Functional pathway analysis of differentially expressed genes in the UC pouch revealed an enhanced state of immune/inflammatory response and extracellular matrix remodeling. Moreover, >70% of differentially expressed genes mapped to published inflammatory bowel diseases microarray data sets displayed directional changes consistent with active UC but not with Crohn's disease.

Conclusions

The UC pouch, well before histologic inflammation, already displays a systems-level gain of colon-associated genes and loss of ileum-associated genes. Patients with UC exhibit a unique transcriptomic response to ileal pouch creation that can be observed well before disease and may in part explain their susceptibility to the development of pouchitis.

The inflammatory CD40-CD40L pathway is implicated in various autoimmune diseases, but the activity status of this pathway in various stages of rheumatoid arthritis (RA) progression is unknown. In this study, we used gene signatures of CD40L stimulation derived from human immature dendritic cells and naive B cells to assess the expression of CD40-downstream genes in synovial tissues from anti-citrullinated protein Ab-positive arthralgia, undifferentiated arthritis (UA), early RA, and established RA cohorts in comparison with healthy donors. Interestingly, the expression of CD40LG and active full-length CD40 was increased in the disease tissues, whereas that of a dominant-negative CD40 isoform was decreased. Gene set variation analysis revealed that CD40L-responsive genes in immature dendritic cells and naive B cells were significantly enriched in synovial tissues from UA, early RA, and established RA patients. Additionally, CD40L-induced naive B cell genes were also significantly enriched in synovial tissues from arthralgia patients. In our efforts to characterize downstream mediators of CD40L signaling, we have identified GPR120 and KDM6B as novel components of the pathway. In conclusion, our data suggest that therapeutic CD40-CD40L blocking agents may prove efficacious not only in early and established RA, but also in inhibiting the progression of the disease from arthralgia or UA to RA.

Objectives

This study sought to investigate the genome-wide transcriptional effects of a combination of disease modifying anti-rheumatic drugs (tDMARD; methotrexate, sulfasalazine and hydroxychloroquine) in synovial tissues obtained from early rheumatoid arthritis (RA) patients. While combination DMARD strategies have been investigated for clinical efficacy, very little data exists on the potential molecular mechanism of action. We hypothesized that tDMARD would impact multiple biological pathways, but the specific pathways were unknown.

Methods

Paired synovial biopsy samples from early RA patients before and after 6 months of tDMARD therapy were collected by arthroscopy (n = 19). These biopsies as well as those from subjects with normal synovium (n = 28) were profiled by total RNA sequencing.

Results

Large differences in gene expression between RA and control biopsies (over 5000 genes) were identified. Despite clinical efficacy, the expression of a restricted set of less than 300 genes was reversed after 6 months of treatment. Many genes remained elevated, even in patients who achieved low disease activity. Interestingly, tDMARD downregulated genes included those involved in T cell activation and signaling and plasmablast/plasma cell differentiation and function.

Conclusions

We have identified transcriptomic signatures that characterize synovial tissue from RA patients with early disease. Analysis after 6 months of tDMARD treatment highlight consistent alterations in expression of genes related to T cell activation and plasmablast/plasma cell differentiation. These results provide novel insight into the biology of early RA and the mechanism of tDMARD action and may help identify novel drug targets to improve rates of treatment-induced disease remission.

Background

Here we integrate verified signals from previous genetic association studies with gene expression and pathway analysis for discovery of new candidate genes and signaling networks, relevant for rheumatoid arthritis (RA).

Method

RNA-sequencing-(RNA-seq)-based expression analysis of 377 genes from previously verified RA-associated loci was performed in blood cells from 5 newly diagnosed, non-treated patients with RA, 7 patients with treated RA and 12 healthy controls. Differentially expressed genes sharing a similar expression pattern in treated and untreated RA sub-groups were selected for pathway analysis. A set of "connector" genes derived from pathway analysis was tested for differential expression in the initial discovery cohort and validated in blood cells from 73 patients with RA and in 35 healthy controls.

Results

There were 11 qualifying genes selected for pathway analysis and these were grouped into two evidence-based functional networks, containing 29 and 27 additional connector molecules. The expression of genes, corresponding to connector molecules was then tested in the initial RNA-seq data. Differences in the expression of ERBB2, TP53 and THOP1 were similar in both treated and non-treated patients with RA and an additional nine genes were differentially expressed in at least one group of patients compared to healthy controls. The ERBB2, TP53. THOP1 expression profile was successfully replicated in RNA-seq data from peripheral blood mononuclear cells from healthy controls and non-treated patients with RA, in an independent collection of samples.

Conclusion

Integration of RNA-seq data with findings from association studies, and consequent pathway analysis implicate new candidate genes, ERBB2, TP53 and THOP1 in the pathogenesis of RA.

Proliferative diabetic retinopathy (PDR) is a common cause of blindness in the developed world's working adult population and affects those with type 1 and type 2 diabetes. We identified Runt-related transcription factor 1 (RUNX1) as a gene upregulated in CD31⁺ vascular endothelial cells obtained from human PDR fibrovascular membranes (FVMs) via transcriptomic analysis. In vitro studies using human retinal microvascular endothelial cells (HRMECs) showed increased RUNX1 RNA and protein expression in response to high glucose, whereas RUNX1 inhibition reduced HRMEC migration, proliferation, and tube formation. Immunohistochemical staining for RUNX1 showed reactivity in vessels of patient-derived FVMs and angiogenic tufts in the retina of mice with oxygen-induced retinopathy, suggesting that RUNX1 upregulation is a hallmark of aberrant retinal angiogenesis. Inhibition of RUNX1 activity with the Ro5-3335 small molecule resulted in a significant reduction of neovascular tufts in oxygen-induced retinopathy, supporting the feasibility of targeting RUNX1 in aberrant retinal angiogenesis.

Psoriasis is driven by focal disruptions of the immune-homeostasis in human skin. Local relapse following cessation of therapy is common and unpredictable, which complicates clinical management of psoriasis. We have previously shown that pathogenic resident T cells accumulate in active and resolved psoriasis, but whether these cells drive psoriasiform tissue reactions is less clear. Here, we activated T cells within skin explants using the pan-T cell activating antibody OKT-3. To explore if T cells induced different tissue response patterns in healthy and psoriasis afflicted skin, transcriptomic analyses were performed with RNA-sequencing and Nanostring. Core tissue responses dominated by IFN-induced pathways were triggered regardless of the inflammatory status of the skin. In contrast, pathways induced by IL-17A, including Defensin beta 2 and keratinocyte differentiation markers, were activated in psoriasis samples. An integrated analysis of IL-17A and IFN-related responses revealed that IL-17 dominated tissue response correlated with early relapse following UVB treatment. Stratification of tissue responses to T cell activation in resolved lesions could potentially offer individualized prediction of disease relapse during long-term immunomodulatory treatment.

Objectives

Several common and rare risk variants have been reported for systemic sclerosis (SSc), but the effector cell(s) mediating the function of these genetic variants remains to be elucidated. While innate immune cells have been proposed as the critical targets to interfere with the disease process underlying SSc, no studies have comprehensively established their effector role. Here we investigated the contribution of monocyte-derived macrophages (MDMs) in mediating genetic susceptibility to SSc.

Methods

We carried out RNA sequencing and genome-wide genotyping in MDMs from 57 patients with SSc and 15 controls. Our differential expression and expression quantitative trait locus (eQTL) analysis in SSc was further integrated with epigenetic, expression and eQTL data from skin, monocytes, neutrophils and lymphocytes.

Results

We identified 602 genes upregulated and downregulated in SSc macrophages that were significantly enriched for genes previously implicated in SSc susceptibility (P=5×10^-4), and 270 cis-regulated genes in MDMs. Among these, GSDMA was reported to carry an SSc risk variant (rs3894194) regulating expression of neighbouring genes in blood. We show that GSDMA is upregulated in SSc MDMs (P=8.4×10^-4) but not in the skin, and is a significant eQTL in SSc macrophages and lipopolysaccharide/interferon gamma (IFNγ)-stimulated monocytes. Furthermore, we identify an SSc macrophage transcriptome signature characterised by upregulation of glycolysis, hypoxia and mTOR signalling and a downregulation of IFNγ response pathways.

Conclusions

Our data further establish the link between macrophages and SSc, and suggest that the contribution of the rs3894194 risk variant to SSc susceptibility can be mediated by GSDMA expression in macrophages.

Although the aetiology of systemic lupus erythematosus (SLE) is unclear, dysregulated B cell responses have been implicated. Here we show that an unusual CD11chiT-bet+ B cell subset, with a unique expression profile including chemokine receptors consistent with migration to target tissues, is expanded in SLE patients, present in nephrotic kidney, enriched for autoreactive specificities and correlates with defined clinical manifestations. IL-21 can potently induce CD11chiT-bet+ B cells and promote the differentiation of these cells into Ig-secreting autoreactive plasma cells. While murine studies have identified a role for T-bet-expressing B cells in autoimmunity, this study describes and exemplifies the importance of CD11chiT-bet+ B cells in human SLE.

Background

The genetic and immunological factors that contribute to differences in susceptibility and progression between sub-types of inflammatory and autoimmune diseases continue to be elucidated. Inflammatory bowel disease and juvenile idiopathic arthritis are both clinically heterogeneous and known to be due in part to abnormal regulation of gene activity in diverse immune cell types. Comparative genomic analysis of these conditions is expected to reveal differences in underlying genetic mechanisms of disease.

Methods

We performed RNA-Seq on whole blood samples from 202 patients with oligoarticular, polyarticular, or systemic juvenile idiopathic arthritis, or with Crohn's disease or ulcerative colitis, as well as healthy controls, to characterize differences in gene expression. Gene ontology analysis combined with Blood Transcript Module and Blood Informative Transcript analysis was used to infer immunological differences. Comparative expression quantitative trait locus (eQTL) analysis was used to quantify disease-specific regulation of transcript abundance.

Results

A pattern of differentially expressed genes and pathways reveals a gradient of disease spanning from healthy controls to oligoarticular, polyarticular, and systemic juvenile idiopathic arthritis (JIA); Crohn's disease; and ulcerative colitis. Transcriptional risk scores also provide good discrimination of controls, JIA, and IBD. Most eQTL are found to have similar effects across disease sub-types, but we also identify disease-specific eQTL at loci associated with disease by GWAS.

Conclusion

JIA and IBD are characterized by divergent peripheral blood transcriptomes, the genetic regulation of which displays limited disease specificity, implying that disease-specific genetic influences are largely independent of, or downstream of, eQTL effects.

It has long been recognized that anatomic location is an important feature for defining distinct subtypes of plaque psoriasis. However, little is known about the molecular differences between scalp, palmoplantar, and conventional plaque psoriasis. To investigate the molecular heterogeneity of these psoriasis subtypes, we performed RNA-seq and flow cytometry on skin samples from individuals with scalp, palmoplantar, and conventional plaque psoriasis, along with samples from healthy control patients. We performed differential expression analysis and network analysis using weighted gene coexpression network analysis (WGCNA). Our analysis revealed a core set of 763 differentially expressed genes common to all sub-types of psoriasis. In contrast, we identified 605, 632, and 262 genes uniquely differentially expressed in conventional, scalp, and palmoplantar psoriasis, respectively. WGCNA and pathway analysis revealed biological processes for the core genes as well as subtype-specific genes. Flow cytometry analysis revealed a shared increase in the percentage of CD4+ T regulatory cells in all psoriasis subtypes relative to controls, whereas distinct psoriasis subtypes displayed differences in IL-17A, IFN-gamma, and IL-22 production. This work reveals the molecular heterogeneity of plaque psoriasis and identifies subtype-specific signaling pathways that will aid in the development of therapy that is appropriate for each subtype of plaque psoriasis.

Atopic dermatitis (AD) affects up to 20% of children and adults worldwide. To gain a deeper understanding of the pathophysiology of AD, we conducted a large-scale transcriptomic study of AD with deeply sequenced RNA-sequencing samples using long (126-bp) paired-end reads. In addition to the comparisons against previous transcriptomic studies, we conducted in-depth analysis to obtain a high-resolution view of the global architecture of the AD transcriptome and contrasted it with that of psoriasis from the same cohort. By using 147 RNA samples in total, we found striking correlation between dysregulated genes in lesional psoriasis and lesional AD skin with 81% of AD dysregulated genes being shared with psoriasis. However, we described disease-specific molecular and cellular features, with AD skin showing dominance of IL-13 pathways, but with near undetectable IL-4 expression. We also demonstrated greater disease heterogeneity and larger proportion of dysregulated long noncoding RNAs in AD, and illustrated the translational impact, including skin-type classification and drug-target prediction. This study is by far the largest study comparing the AD and psoriasis transcriptomes using RNA sequencing and demonstrating the shared inflammatory components, as well as specific discordant cytokine signatures of these two skin diseases.

Applying tissue-specific deconvolution of transcriptional networks to identify their master regulators (MRs) in neuropsychiatric disorders has been largely unexplored. Here, using two schizophrenia (SCZ) case-control RNA-seq datasets, one on postmortem dorsolateral prefrontal cortex (DLPFC) and another on cultured olfactory neuroepithelium, we deconvolved the transcriptional networks and identified TCF4 as a top candidate MR that may be dysregulated in SCZ. We validated TCF4 as a MR through enrichment analysis of TCF4-binding sites in induced pluripotent stem cell (hiPSC)-derived neurons and in neuroblastoma cells. We further validated the predicted TCF4 targets by knocking down TCF4 in hiPSC-derived neural progenitor cells (NPCs) and glutamatergic neurons (Glut_Ns). The perturbed TCF4 gene network in NPCs was more enriched for pathways involved in neuronal activity and SCZ-associated risk genes, compared to Glut_Ns. Our results suggest that TCF4 may serve as a MR of a gene network dysregulated in SCZ at early stages of neurodevelopment.

Genome-wide association studies (GWASs) have revealed 59 genomic loci associated with type 1 diabetes (T1D). Functional interpretation of the SNPs located in the noncoding region of these loci remains challenging. We perform epigenomic profiling of two enhancer marks, H3K4me1 and H3K27ac, using primary TH1 and TREG cells isolated from healthy and T1D subjects. We uncover a large number of deregulated enhancers and altered transcriptional circuitries in both cell types of T1D patients. We identify four SNPs (rs10772119, rs10772120, rs3176792, rs883868) in linkage disequilibrium (LD) with T1D-associated GWAS lead SNPs that alter enhancer activity and expression of immune genes. Among them, rs10772119 and rs883868 disrupt the binding of retinoic acid receptor α (RARA) and Yin and Yang 1 (YY1), respectively. Loss of binding by YY1 also results in the loss of long-range enhancer-promoter interaction. These findings provide insights into how noncoding variants affect the transcriptomes of two T-cell subtypes that play critical roles in T1D pathogenesis.

Background

The chronic remitting and relapsing intestinal inflammation characteristic of Crohn's disease frequently leads to fibrosis and subsequent stenosis of the inflamed region. Approximately a third of all Crohn's disease patients require resection at some stage in their disease course. As the pathogenesis of Crohn's disease associated fibrosis is largely unknown, a strong necessity exists to better understand the pathophysiology thereof.

Methods

In this study, we investigated changes of the DNA methylome and transcriptome of ileum-derived fibroblasts associated to the occurrence of Crohn's disease associated fibrosis. Eighteen samples were included in a DNA methylation array and twenty-one samples were used for RNA sequencing.

Results

Most differentially methylated regions and differentially expressed genes were observed when comparing stenotic with non-inflamed samples. By contrast, few differences were observed when comparing Crohn's disease with non-Crohn's disease, or inflamed with non-inflamed tissue. Integrative methylation and gene expression analyses revealed dysregulation of genes associated to the PRKACA and E2F1 network, which is involved in cell cycle progression, angiogenesis, epithelial to mesenchymal transition, and bile metabolism.

Conclusion

Our research provides evidence that the methylome and the transcriptome are systematically dysregulated in stenosis-associated fibroblasts.

Background

The cellular effects of androgen are transduced through the androgen receptor, which controls the expression of genes that regulate biosynthetic processes, cell growth, and metabolism. Androgen signaling also impacts DNA damage signaling through mechanisms involving gene expression and transcription-associated DNA damaging events. Defining the contributions of androgen signaling to DNA repair is important for understanding androgen receptor function, and it also has translational implications.

Methods

We generated RNA-seq data from multiple prostate cancer lines and used bioinformatic analyses to characterize androgen-regulated gene expression. We compared the results from cell lines with gene expression data from prostate cancer xenografts, and patient samples, to query how androgen signaling and prostate cancer progression influences the expression of DNA repair genes. We performed whole genome sequencing to help characterize the status of the DNA repair machinery in widely used prostate cancer lines. Finally, we tested a DNA repair enzyme inhibitor for effects on androgen-dependent transcription.

Results

Our data indicates that androgen signaling regulates a subset of DNA repair genes that are largely specific to the respective model system and disease state. We identified deleterious mutations in the DNA repair genes RAD50 and CHEK2. We found that inhibition of the DNA repair enzyme MRE11 with the small molecule mirin inhibits androgen-dependent transcription and growth of prostate cancer cells.

Conclusions

Our data supports the view that crosstalk between androgen signaling and DNA repair occurs at multiple levels, and that DNA repair enzymes in addition to PARPs, could be actionable targets in prostate cancer.

Objective

To investigate miR-155 in the SOD1 mouse model and human sporadic and familial amyotrophic lateral sclerosis (ALS).

Methods

NanoString microRNA, microglia and immune gene profiles, protein mass spectrometry, and RNA-seq analyses were measured in spinal cord microglia, splenic monocytes, and spinal cord tissue from SOD1 mice and in spinal cord tissue of familial and sporadic ALS. miR-155 was targeted by genetic ablation or by peripheral or centrally administered anti-miR-155 inhibitor in SOD1 mice.

Results

In SOD1 mice, we found loss of the molecular signature that characterizes homeostatic microglia and increased expression of miR-155. There was loss of the microglial molecules P2ry12, Tmem119, Olfml3, transcription factors Egr1, Atf3, Jun, Fos, and Mafb, and the upstream regulators Csf1r, Tgfb1, and Tgfbr1, which are essential for microglial survival. Microglia biological functions were suppressed including phagocytosis. Genetic ablation of miR-155 increased survival in SOD1 mice by 51 days in females and 27 days in males and restored the abnormal microglia and monocyte molecular signatures. Disease severity in SOD1 males was associated with early upregulation of inflammatory genes, including Apoe in microglia. Treatment of adult microglia with apolipoprotein E suppressed the M0-homeostatic unique microglia signature and induced an M1-like phenotype. miR-155 expression was increased in the spinal cord of both familial and sporadic ALS. Dysregulated proteins that we identified in human ALS spinal cord were restored in SOD1(G93A) /miR-155(-/-) mice. Intraventricular anti-miR-155 treatment derepressed microglial miR-155 targeted genes, and peripheral anti-miR-155 treatment prolonged survival.

Interpretation

We found overexpression of miR-155 in the SOD1 mouse and in both sporadic and familial human ALS. Targeting miR-155 in SOD1 mice restores dysfunctional microglia and ameliorates disease. These findings identify miR-155 as a therapeutic target for the treatment of ALS.

Joint pain causes significant morbidity in osteoarthritis (OA). The aetiology of joint pain in OA is not well understood. The synovial membrane as an innervated joint structure represents a potential source of peripheral pain in OA. Here we analyse, using a hypothesis-free next generation RNA sequencing, the differences in protein-coding and non-coding transcriptomes in knee synovial tissues from OA patients with high knee pain (n = 5) compared with OA patients with low knee pain (n = 5), as evaluated by visual analogue scale (VAS). We conduct Gene Ontology and pathway analyses on differentially expressed mRNA genes. We identify new protein-coding, long non-coding RNA and microRNA candidates that can be associated with OA joint pain. Top enriched genes in painful OA knees encode neuronal proteins that are known to promote neuronal survival under cellular stress or participate in calcium-dependent synaptic exocytosis and modulation of GABA(γ-aminobutyric acid)ergic activity. Our study uncovers transcriptome changes associated with pain in synovial microenvironment of OA knees. This sets a firm ground for future mechanistic studies and drug discovery to alleviate joint pain in OA.

The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this, we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that, although iHTNs maintain a fetal identity, they respond appropriately to metabolic hormones ghrelin and leptin. Notably, OBS iHTNs retained disease signatures and phenotypes of high BMI, exhibiting dysregulated respiratory function, ghrelin-leptin signaling, axonal guidance, glutamate receptors, and endoplasmic reticulum (ER) stress pathways. Thus, human iHTNs provide a powerful platform to study obesity and gene-environment interactions.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivoVHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1β heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression.Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305. ©2017 AACR.See related commentary by Ricketts and Linehan, p. 1221This article is highlighted in the In This Issue feature, p. 1201.

Tumor recurrence remains the main reason for breast cancer-associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in ∼10-30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor-associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9-IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.

Disruption of epigenetic mechanisms has been intimately linked to the etiology of human cancer. Understanding how these epigenetic mechanisms (including DNA methylation [5mC], hydroxymethylation [5hmC], and histone post-translational modifications) work in concert to drive cancer initiation and progression remains unknown. Hepatocellular carcinoma (HCC) is increasing in frequency in Western countries but lacks efficacious treatments. The epigenome of HCC remains understudied. To better understand the epigenetic underpinnings of HCC, we performed a genome-wide assessment of 5mC, 5hmC, four histone modifications linked to promoter/enhancer function (H3K4me1, H3K27ac, H3K4me3, and H3K27me3), and transcription across normal, cirrhotic, and HCC liver tissue. Implementation of bioinformatic strategies integrated these epigenetic marks with each other and with transcription to provide a comprehensive epigenetic profile of how and when the liver epigenome is perturbed during progression to HCC. Our data demonstrate significant deregulation of epigenetic regulators combined with disruptions in the epigenome hallmarked by profound loss of 5hmC, locus-specific gains in 5mC and 5hmC, and markedly altered histone modification profiles, particularly remodeling of enhancers. Data integration demonstrates that these marks collaborate to influence transcription (e.g., hyper-5hmC in HCC-gained active enhancers is linked to elevated expression) of genes regulating HCC proliferation. Two such putative epigenetic driver loci identified through our integrative approach, COMT and FMO3, increase apoptosis and decrease cell viability in liver-derived cancer cell lines when ectopically re-expressed. Conclusion: Altogether, integration of multiple epigenetic parameters is a powerful tool for identifying epigenetically regulated drivers of HCC and elucidating how epigenome deregulation contributes to liver disease and HCC.

Dysregulated IL-23/IL-17 responses have been linked to psoriatic arthritis and other forms of spondyloarthritides (SpA). RORγt, the key Thelper17 (Th17) cell transcriptional regulator, is also expressed by subsets of innate-like T cells, including invariant natural killer T (iNKT) and γδ-T cells, but their contribution to SpA is still unclear. Here we describe the presence of particular RORγt⁺T-bet^loPLZF^- iNKT and γδ-hi T cell subsets in healthy peripheral blood. RORγt⁺ iNKT and γδ-hi T cells show IL-23 mediated Th17-like immune responses and were clearly enriched within inflamed joints of SpA patients where they act as major IL-17 secretors. SpA derived iNKT and γδ-T cells showed unique and Th17-skewed phenotype and gene expression profiles. Strikingly, RORγt inhibition blocked γδ17 and iNKT17 cell function while selectively sparing IL-22⁺ subsets. Overall, our findings highlight a unique diversity of human RORγt⁺ T cells and underscore the potential of RORγt antagonism to modulate aberrant type 17 responses.

Circulating in China and 158 other countries and areas, the ongoing COVID-19 outbreak has caused devastating mortality and posed a great threat to public health. However, efforts to identify effectively supportive therapeutic drugs and treatments has been hampered by our limited understanding of host immune response for this fatal disease. To characterize the transcriptional signatures of host inflammatory response to SARS-CoV-2 (HCoV-19) infection, we carried out transcriptome sequencing of the RNAs isolated from the bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients. Our results reveal distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release such as CCL2/MCP-1, CXCL10/IP-10, CCL3/MIP-1A, and CCL4/MIP1B. Furthermore, SARS-CoV-2 induced activation of apoptosis and P53 signalling pathway in lymphocytes may be the cause of patients' lymphopenia. The transcriptome dataset of COVID-19 patients would be a valuable resource for clinical guidance on anti-inflammatory medication and understanding the molecular mechansims of host response.

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development. Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology (ART) practices. Our data reveal that the global methylation levels of high-quality blastocysts are similar (0.30 ± 0.02, mean ± SD), while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts. The proportion of blastocysts with a methylation level falling within the range of 0.30 ± 0.02 in different grades correlates with the live birth rate for that grade. Moreover, abnormal methylated regions are associated with the failure of embryonic development. Furthermore, we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts. Our data indicate that global abnormal methylome reprogramming often occurs in human embryos, and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.

The dynamics of the chromatin regulatory landscape during human early embryogenesis remains unknown. Using DNase I hypersensitive site (DHS) sequencing, we report that the chromatin accessibility landscape is gradually established during human early embryogenesis. Interestingly, the DHSs with OCT4 binding motifs are enriched at the timing of zygotic genome activation (ZGA) in humans, but not in mice. Consistently, OCT4 contributes to ZGA in humans, but not in mice. We further find that lower CpG promoters usually establish DHSs at later stages. Similarly, younger genes tend to establish promoter DHSs and are expressed at later embryonic stages, while older genes exhibit these features at earlier stages. Moreover, our data show that human active transposons SVA and HERV-K harbor DHSs and are highly expressed in early embryos, but not in differentiated tissues. In summary, our data provide an evolutionary developmental view for understanding the regulation of gene and transposon expression.

Zika virus (ZIKV) has elicited global concern due to its unique biological features, unusual transmission routes, and unexpected clinical outcomes. Although ZIKV transmission through anal intercourse has been reported in humans, it remains unclear if ZIKV is detectable in the stool, if it can infect the host through the anal canal mucosa, and what the pathogenesis of such a route of infection might be in the mouse model. Herein, we demonstrate that ZIKV RNA can be recovered from stools in multiple mouse models, as well as from the stool of a ZIKV patient. Remarkably, intra-anal (i.a.) inoculation with ZIKV leads to efficient infection in both Ifnar1^-/- and immunocompetent mice, characterized by extensive viral replication in the blood and multiple organs, including the brain, small intestine, testes, and rectum, as well as robust humoral and innate immune responses. Moreover, i.a. inoculation of ZIKV in pregnant mice resulted in transplacental infection and delayed fetal development. Overall, our results identify the anorectal mucosa as a potential site of ZIKV infection in mice, reveal the associated pathogenesis of i.a. infection, and highlight the complexity of ZIKV transmission through anal intercourse.

Azithromycin (AZM) is a widely used antibiotic, with additional antiviral and anti-inflammatory properties that remain poorly understood. Although Zika virus (ZIKV) poses a significant threat to global health, there are currently no vaccines or effective therapeutics against it. Herein, we report that AZM effectively suppresses ZIKV infection in vitro by targeting a late stage in the viral life cycle. Besides that, AZM upregulates the expression of host type I and III interferons and several of their downstream interferon-stimulated genes (ISGs) in response to ZIKV infection. In particular, we found that AZM upregulates the expression of MDA5 and RIG-I, pathogen recognition receptors (PRRs) induced by ZIKV infection, and increases the levels of phosphorylated TBK1 and IRF3. Interestingly, AZM treatment upregulates phosphorylation of TBK1, without inducing phosphorylation of IRF3 by itself. These findings highlight the potential use of AZM as a broad antiviral agent to combat viral infection and prevent ZIKV associated devastating clinical outcomes, such as congenital microcephaly.

Ischemic stroke (IS) is one of the main causes of morbidity and disability worldwide due to its complex mechanism. Anemia was characterized as a risk factor of IS because the direct connection between central nervous system, blood supply, and tissue oxygen delivery. As the key oxygen-carrying molecule in the blood, hemoglobin (Hb) may be decisive in the destiny of penumbral area or influence the brain recovery and neurologic function, which could finally affect the outcome of IS. However, more detailed information on the expression levels of Hb related genes were still lacking possibly because the concentration of Hb was determined by the genes' expression several hours ago, which may make the research more difficult to perform. This time gap between gene expressions and protein concentration could make these genes predictive bio-markers for IS outcome. In this study, we choose 28 IS patients, of which 12 were suffering from anemia. Statistical analysis results showed that the outcomes of the patients were different when dividing them into two groups characterized by Hb concentration. 2 sex and age matched patients were first chosen to perform RNA-seq analysis on, on two occasions at two different time points, after which the Hb counts were tested at least 24 h after sequencing. Results showed that the outcome of anemia patients was poor compared with non-anemia patients. Two other patients were then chosen for analysis which excluded the coincidence of other factors. The results showed that the low value of Hb under 13 g/dL in men were closely related to the poor outcome of IS patients. Differently expressed Hb related genes were tested and six genes were shown to be positively correlated with the recovery degree of IS patients: ELANE, FGF23, HBB, PIEZO1, RASA4, and PRTN3. Gene CPM was shown to be negatively correlated with clinical outcomes. All of the seven genes were validated to be related to strokes using real-time PCR or literature searches. Taken together, these genes could be considered as new predictors for the recovery of IS patients.

Whether cell types exposed to a high level of environmental insults possess cell type-specific prosurvival mechanisms or enhanced DNA damage repair capacity is not well understood. BRN2 is a tissue-restricted POU domain transcription factor implicated in neural development and several cancers. In melanoma, BRN2 plays a key role in promoting invasion and regulating proliferation. Here we found, surprisingly, that rather than interacting with transcription cofactors, BRN2 is instead associated with DNA damage response proteins and directly binds PARP1 and Ku70/Ku80. Rapid PARP1-dependent BRN2 association with sites of DNA damage facilitates recruitment of Ku80 and reprograms DNA damage repair by promoting Ku-dependent nonhomologous end-joining (NHEJ) at the expense of homologous recombination. BRN2 also suppresses an apoptosis-associated gene expression program to protect against UVB-, chemotherapy- and vemurafenib-induced apoptosis. Remarkably, BRN2 expression also correlates with a high single-nucleotide variation prevalence in human melanomas. By promoting error-prone DNA damage repair via NHEJ and suppressing apoptosis of damaged cells, our results suggest that BRN2 contributes to the generation of melanomas with a high mutation burden. Our findings highlight a novel role for a key transcription factor in reprogramming DNA damage repair and suggest that BRN2 may impact the response to DNA-damaging agents in BRN2-expressing cancers.

Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing (RNA-Seq) analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

Defining the transcriptional dynamics of a temporal process such as cell differentiation is challenging owing to the high variability in gene expression between individual cells. Time-series gene expression analyses of bulk cells have difficulty distinguishing early and late phases of a transcriptional cascade or identifying rare subpopulations of cells, and single-cell proteomic methods rely on a priori knowledge of key distinguishing markers. Here we describe Monocle, an unsupervised algorithm that increases the temporal resolution of transcriptome dynamics using single-cell RNA-Seq data collected at multiple time points. Applied to the differentiation of primary human myoblasts, Monocle revealed switch-like changes in expression of key regulatory factors, sequential waves of gene regulation, and expression of regulators that were not known to act in differentiation. We validated some of these predicted regulators in a loss-of function screen. Monocle can in principle be used to recover single-cell gene expression kinetics from a wide array of cellular processes, including differentiation, proliferation and oncogenic transformation.

The causes of embryonic arrest during pre-implantation development are poorly understood. Attempts to correlate patterns of oocyte gene expression with successful embryo development have been hampered by the lack of reliable and nondestructive predictors of viability at such an early stage. Here we report that zygote viscoelastic properties can predict blastocyst formation in humans and mice within hours after fertilization, with >90% precision, 95% specificity and 75% sensitivity. We demonstrate that there are significant differences between the transcriptomes of viable and non-viable zygotes, especially in expression of genes important for oocyte maturation. In addition, we show that low-quality oocytes may undergo insufficient cortical granule release and zona-hardening, causing altered mechanics after fertilization. Our results suggest that embryo potential is largely determined by the quality and maturation of the oocyte before fertilization, and can be predicted through a minimally invasive mechanical measurement at the zygote stage.

Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

Background

Intra-tumoral genetic and functional heterogeneity correlates with cancer clinical prognoses. However, the mechanisms by which intra-tumoral heterogeneity impacts therapeutic outcome remain poorly understood. RNA sequencing (RNA-seq) of single tumor cells can provide comprehensive information about gene expression and single-nucleotide variations in individual tumor cells, which may allow for the translation of heterogeneous tumor cell functional responses into customized anti-cancer treatments.

Results

We isolated 34 patient-derived xenograft (PDX) tumor cells from a lung adenocarcinoma patient tumor xenograft. Individual tumor cells were subjected to single cell RNA-seq for gene expression profiling and expressed mutation profiling. Fifty tumor-specific single-nucleotide variations, including KRAS(G12D), were observed to be heterogeneous in individual PDX cells. Semi-supervised clustering, based on KRAS(G12D) mutant expression and a risk score representing expression of 69 lung adenocarcinoma-prognostic genes, classified PDX cells into four groups. PDX cells that survived in vitro anti-cancer drug treatment displayed transcriptome signatures consistent with the group characterized by KRAS(G12D) and low risk score.

Conclusions

Single-cell RNA-seq on viable PDX cells identified a candidate tumor cell subgroup associated with anti-cancer drug resistance. Thus, single-cell RNA-seq is a powerful approach for identifying unique tumor cell-specific gene expression profiles which could facilitate the development of optimized clinical anti-cancer strategies.

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.

The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.

Background

Intratumoral heterogeneity hampers the success of marker-based anticancer treatment because the targeted therapy may eliminate a specific subpopulation of tumor cells while leaving others unharmed. Accordingly, a rational strategy minimizing survival of the drug-resistant subpopulation is essential to achieve long-term therapeutic efficacy.

Results

Using single-cell RNA sequencing (RNA-seq), we examine the intratumoral heterogeneity of a pair of primary renal cell carcinoma and its lung metastasis. Activation of drug target pathways demonstrates considerable variability between the primary and metastatic sites, as well as among individual cancer cells within each site. Based on the prediction of multiple drug target pathway activation, we derive a combinatorial regimen co-targeting two mutually exclusive pathways for the metastatic cancer cells. This combinatorial strategy shows significant increase in the treatment efficacy over monotherapy in the experimental validation using patient-derived xenograft platforms in vitro and in vivo.

Conclusions

Our findings demonstrate the investigational application of single-cell RNA-seq in the design of an anticancer regimen. The approach may overcome intratumoral heterogeneity which hampers the success of precision medicine.

Long non-coding RNAs (lncRNAs) are a diverse and poorly conserved category of transcripts that have expanded greatly in primates, particularly in the brain. We identified an lncRNA, which has acquired 16 microRNA response elements for miR-143-3p in the Catarrhini branch of primates. This lncRNA, termed LncND (neurodevelopment), is expressed in neural progenitor cells and then declines in neurons. Binding and release of miR-143-3p by LncND control the expression of Notch receptors. LncND expression is enriched in radial glia cells (RGCs) in the ventricular and subventricular zones of developing human brain. Downregulation in neuroblastoma cells reduced cell proliferation and induced neuronal differentiation, an effect phenocopied by miR-143-3p overexpression. Gain of function of LncND in developing mouse cortex led to an expansion of PAX6+ RGCs. These findings support a role for LncND in miRNA-mediated regulation of Notch signaling within the neural progenitor pool in primates that may have contributed to the expansion of cerebral cortex.

Single-cell transcriptome profiling of tumour tissue isolates allows the characterization of heterogeneous tumour cells along with neighbouring stromal and immune cells. Here we adopt this powerful approach to breast cancer and analyse 515 cells from 11 patients. Inferred copy number variations from the single-cell RNA-seq data separate carcinoma cells from non-cancer cells. At a single-cell resolution, carcinoma cells display common signatures within the tumour as well as intratumoral heterogeneity regarding breast cancer subtype and crucial cancer-related pathways. Most of the non-cancer cells are immune cells, with three distinct clusters of T lymphocytes, B lymphocytes and macrophages. T lymphocytes and macrophages both display immunosuppressive characteristics: T cells with a regulatory or an exhausted phenotype and macrophages with an M2 phenotype. These results illustrate that the breast cancer transcriptome has a wide range of intratumoral heterogeneity, which is shaped by the tumour cells and immune cells in the surrounding microenvironment.

Single-cell RNA sequencing (scRNA-seq) can be used to characterize variation in gene expression levels at high resolution. However, the sources of experimental noise in scRNA-seq are not yet well understood. We investigated the technical variation associated with sample processing using the single-cell Fluidigm C1 platform. To do so, we processed three C1 replicates from three human induced pluripotent stem cell (iPSC) lines. We added unique molecular identifiers (UMIs) to all samples, to account for amplification bias. We found that the major source of variation in the gene expression data was driven by genotype, but we also observed substantial variation between the technical replicates. We observed that the conversion of reads to molecules using the UMIs was impacted by both biological and technical variation, indicating that UMI counts are not an unbiased estimator of gene expression levels. Based on our results, we suggest a framework for effective scRNA-seq studies.

Single-cell RNA-seq enables the quantitative characterization of cell types based on global transcriptome profiles. We present single-cell consensus clustering (SC3), a user-friendly tool for unsupervised clustering, which achieves high accuracy and robustness by combining multiple clustering solutions through a consensus approach (http://bioconductor.org/packages/SC3). We demonstrate that SC3 is capable of identifying subclones from the transcriptomes of neoplastic cells collected from patients.

Single-cell transcriptomics has been used for analysis of heterogeneous populations of cells during developmental processes and for analysis of tumor cell heterogeneity. More recently, analysis of pseudotime (PT) dynamics of heterogeneous cell populations has been established as a powerful concept to study developmental processes. Here we perform PT analysis of 3 melanoma short-term cultures with different genetic backgrounds to study specific and concordant properties of PT dynamics of selected cellular programs with impact on melanoma progression. Overall, in our setting of melanoma cells PT dynamics towards higher tumor malignancy appears to be largely driven by cell cycle genes. Single cells of all three short-term cultures show a bipolar expression of microphthalmia-associated transcription factor (MITF) and AXL receptor tyrosine kinase (AXL) signatures. Furthermore, opposing gene expression changes are observed for genes regulated by epigenetic mechanisms suggesting epigenetic reprogramming during melanoma progression. The three melanoma short-term cultures show common themes of PT dynamics such as a stromal signature at initiation, bipolar expression of the MITF/AXL signature and opposing regulation of poised and activated promoters. Differences are observed at the late stage of PT dynamics with high, low or intermediate MITF and anticorrelated AXL signatures. These findings may help to identify targets for interference at different stages of tumor progression.

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

Through the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of EZH2-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon EZH2 deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in EZH2-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. EZH2-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. EZH2-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, EZH2 is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.

As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue.

High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth, but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron-glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections, forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation, whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth, human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together, these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression.

After entering tissues, monocytes differentiate into cells that share functional features with either macrophages or dendritic cells (DCs). How monocyte fate is directed toward monocyte-derived macrophages (mo-Macs) or monocyte-derived DCs (mo-DCs) and which transcription factors control these differentiation pathways remains unknown. Using an in vitro culture model yielding human mo-DCs and mo-Macs closely resembling those found in vivo in ascites, we show that IRF4 and MAFB were critical regulators of monocyte differentiation into mo-DCs and mo-Macs, respectively. Activation of the aryl hydrocarbon receptor (AHR) promoted mo-DC differentiation through the induction of BLIMP-1, while impairing differentiation into mo-Macs. AhR deficiency also impaired the in vivo differentiation of mouse mo-DCs. Finally, AHR activation correlated with mo-DC infiltration in leprosy lesions. These results establish that mo-DCs and mo-Macs are controlled by distinct transcription factors and show that AHR acts as a molecular switch for monocyte fate specification in response to micro-environmental factors.

CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the TEMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4+ T cells in the central memory (TCM) and effector memory (TEM) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared with TCM and TEM cells and that most of CD4-TEMRA were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity.

Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

The current understanding of mammalian kidney development is largely based on mouse models. Recent landmark studies revealed pervasive differences in renal embryogenesis between mouse and human. The scarcity of detailed gene expression data in humans therefore hampers a thorough understanding of human kidney development and the possible developmental origin of kidney diseases. In this paper, we present a single-cell transcriptomics study of the human fetal kidney. We identified 22 cell types and a host of marker genes. Comparison of samples from different developmental ages revealed continuous gene expression changes in podocytes. To demonstrate the usefulness of our data set, we explored the heterogeneity of the nephrogenic niche, localized podocyte precursors, and confirmed disease-associated marker genes. With close to 18,000 renal cells from five different developmental ages, this study provides a rich resource for the elucidation of human kidney development, easily accessible through an interactive web application.

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4+ T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3neg CD4+ T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

Spermatogenesis has been intensely studied in rodents but remains poorly understood in humans. Here, we used single-cell RNA sequencing to analyze human testes. Clustering analysis of neonatal testes reveals several cell subsets, including cell populations with characteristics of primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). In adult testes, we identify four undifferentiated spermatogonia (SPG) clusters, each of which expresses specific marker genes. We identify protein markers for the most primitive SPG state, allowing us to purify this likely SSC-enriched cell subset. We map the timeline of male germ cell development from PGCs through fetal germ cells to differentiating adult SPG stages. We also define somatic cell subsets in both neonatal and adult testes and trace their developmental trajectories. Our data provide a blueprint of the developing human male germline and supporting somatic cells. The PGC-like and SSC markers are candidates to be used for SSC therapy to treat infertility.

γδ T lymphocytes represent ∼1% of human peripheral blood mononuclear cells and even more cells in most tissues of vertebrates. Although they have important anticancer functions, most current single-cell RNA sequencing (scRNA-seq) studies do not identify γδ T lymphocytes because their transcriptomes at the single-cell level are unknown. Here we show that high-resolution clustering of large scRNA-seq datasets and a combination of gene signatures allow the specific detection of human γδ T lymphocytes and identification of their T cell receptor (TCR)Vδ1 and TCRVδ2 subsets in large datasets from complex cell mixtures. In t-distributed stochastic neighbor embedding plots from blood and tumor samples, the few γδ T lymphocytes appear collectively embedded between cytotoxic CD8 T and NK cells. Their TCRVδ1 and TCRVδ2 subsets form close yet distinct subclusters, respectively neighboring NK and CD8 T cells because of expression of shared and distinct cytotoxic maturation genes. Similar pseudotime maturation trajectories of TCRVδ1 and TCRVδ2 γδ T lymphocytes were discovered, unveiling in both subsets an unattended pool of terminally differentiated effector memory cells with preserved proliferative capacity, a finding confirmed by in vitro proliferation assays. Overall, the single-cell transcriptomes of thousands of individual γδ T lymphocytes from different CMV+ and CMV- donors reflect cytotoxic maturation stages driven by the immunological history of donors. This landmark study establishes the rationale for identification, subtyping, and deep characterization of human γδ T lymphocytes in further scRNA-seq studies of complex tissues in physiological and disease conditions.

PURPOSE:In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites. EXPERIMENTAL DESIGN:Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed. RESULTS:Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells. CONCLUSIONS:These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion.

After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5^pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5^pos basal-like state. Activated murine Krt5^pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2^pos LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.

Single-cell RNA sequencing (scRNA-seq) has been widely applied to discover new cell types by detecting sub-populations in a heterogeneous group of cells. Since scRNA-seq experiments have lower read coverage/tag counts and introduce more technical biases compared to bulk RNA-seq experiments, the limited number of sampled cells combined with the experimental biases and other dataset specific variations presents a challenge to cross-dataset analysis and discovery of relevant biological variations across multiple cell populations. In this paper, we introduce a method of variance-driven multitask clustering of single-cell RNA-seq data (scVDMC) that utilizes multiple single-cell populations from biological replicates or different samples. scVDMC clusters single cells in multiple scRNA-seq experiments of similar cell types and markers but varying expression patterns such that the scRNA-seq data are better integrated than typical pooled analyses which only increase the sample size. By controlling the variance among the cell clusters within each dataset and across all the datasets, scVDMC detects cell sub-populations in each individual experiment with shared cell-type markers but varying cluster centers among all the experiments. Applied to two real scRNA-seq datasets with several replicates and one large-scale droplet-based dataset on three patient samples, scVDMC more accurately detected cell populations and known cell markers than pooled clustering and other recently proposed scRNA-seq clustering methods. In the case study applied to in-house Recessive Dystrophic Epidermolysis Bullosa (RDEB) scRNA-seq data, scVDMC revealed several new cell types and unknown markers validated by flow cytometry. MATLAB/Octave code available at https://github.com/kuanglab/scVDMC.

Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.

Aims

The mechanisms coordinating maturation with an environment-driven metabolic shift, a critical step in determining the developmental potential of human in vitro maturation (IVM) oocytes, remain to be elucidated. Here we explored the key genes regulating human oocyte maturation using single-cell RNA sequencing and illuminated the compensatory mechanism from a metabolic perspective by analyzing gene expression.

Results

Three key genes that encode CoA-related enzymes were screened from the RNA sequencing data. Two of them, ACAT1 and HADHA, were closely related to the regulation of substrate production in the Krebs cycle. Dysfunction of the Krebs cycle was induced by decreases in the activity of specific enzymes. Furthermore, the activator of these enzymes, the calcium concentration, was also decreased because of the failure of influx of exogenous calcium. Although release of endogenous calcium from the endoplasmic reticulum and mitochondria met the requirement for maturation, excessive release resulted in aneuploidy and developmental incompetence. High nicotinamide nucleotide transhydrogenase expression induced NADPH dehydrogenation to compensate for the NADH shortage resulting from the dysfunction of the Krebs cycle. Importantly, high NADP⁺ levels activated DPYD to enhance the repair of DNA double-strand breaks to maintain euploidy.

Innovation

The present study shows for the first time that exposure to the in vitro environment can lead to the decline of energy metabolism in human oocytes during maturation but that a compensatory action maintains their developmental competence.

Conclusion

In vitro maturation of human oocytes is mediated through a cascade of competing and compensatory actions driven by genes encoding enzymes.

Death of photoreceptors is a common cause of age-related and inherited retinal dystrophies, and thus their replenishment from renewable stem cell sources is a highly desirable therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to not only differentiate into cells of the retina but also self-organize into tissue with structure akin to the human retina as part of three-dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells through application of cell surface markers or fluorescent reporter approaches and shown to have a similar transcriptome to fetal photoreceptors. In this study, we investigated the transcriptional profile of CRX-expressing photoreceptor precursors derived from human pluripotent stem cells and their engraftment capacity in an animal model of retinitis pigmentosa (Pde6brd1), which is characterized by rapid photoreceptor degeneration. Single cell RNA-Seq analysis revealed the presence of a dominant cell cluster comprising 72% of the cells, which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the Pde6brd1 mice, the CRX⁺ cells settled next to the inner nuclear layer and made connections with the inner neurons of the host retina, and approximately one-third of them expressed the pan cone marker, Arrestin 3, indicating further maturation upon integration into the host retina. Together, our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells-derived CRX⁺ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors. Stem Cells 2019;37:609-622.

Megakaryocytic-erythroid progenitors (MEPs) give rise to the cells that produce red blood cells and platelets. Although the mechanisms underlying megakaryocytic (MK) and erythroid (E) maturation have been described, those controlling their specification from MEPs are unknown. Single-cell RNA sequencing of primary human MEPs, common myeloid progenitors (CMPs), megakaryocyte progenitors, and E progenitors revealed a distinct transitional MEP signature. Inferred regulatory transcription factors (TFs) were associated with differential expression of cell cycle regulators. Genetic manipulation of selected TFs validated their role in lineage specification and demonstrated coincident modulation of the cell cycle. Genetic and pharmacologic modulation demonstrated that cell cycle activation is sufficient to promote E versus MK specification. These findings, obtained from healthy human cells, lay a foundation to study the mechanisms underlying benign and malignant disease states of the megakaryocytic and E lineages.

Lung adenocarcinoma (LUAD)-derived Wnts increase cancer cell proliferative/stemness potential, but whether they impact the immune microenvironment is unknown. Here we show that LUAD cells use paracrine Wnt1 signaling to induce immune resistance. In TCGA, Wnt1 correlates strongly with tolerogenic genes. In another LUAD cohort, Wnt1 inversely associates with T cell abundance. Altering Wnt1 expression profoundly affects growth of murine lung adenocarcinomas and this is dependent on conventional dendritic cells (cDCs) and T cells. Mechanistically, Wnt1 leads to transcriptional silencing of CC/CXC chemokines in cDCs, T cell exclusion and cross-tolerance. Wnt-target genes are up-regulated in human intratumoral cDCs and decrease upon silencing Wnt1, accompanied by enhanced T cell cytotoxicity. siWnt1-nanoparticles given as single therapy or part of combinatorial immunotherapies act at both arms of the cancer-immune ecosystem to halt tumor growth. Collectively, our studies show that Wnt1 induces immunologically cold tumors through cDCs and highlight its immunotherapeutic targeting.

Objectives

The purpose of this study was to determine the level of heterogeneity in high grade serous ovarian cancer (HGSOC) by analyzing RNA expression in single epithelial and cancer associated stromal cells. In addition, we explored the possibility of identifying subgroups based on pathway activation and pre-defined signatures from cancer stem cells and chemo-resistant cells.

Methods

A fresh, HGSOC tumor specimen derived from ovary was enzymatically digested and depleted of immune infiltrating cells. RNA sequencing was performed on 92 single cells and 66 of these single cell datasets passed quality control checks. Sequences were analyzed using multiple bioinformatics tools, including clustering, principle components analysis, and geneset enrichment analysis to identify subgroups and activated pathways. Immunohistochemistry for ovarian cancer, stem cell and stromal markers was performed on adjacent tumor sections.

Results

Analysis of the gene expression patterns identified two major subsets of cells characterized by epithelial and stromal gene expression patterns. The epithelial group was characterized by proliferative genes including genes associated with oxidative phosphorylation and MYC activity, while the stromal group was characterized by increased expression of extracellular matrix (ECM) genes and genes associated with epithelial-to-mesenchymal transition (EMT). Neither group expressed a signature correlating with published chemo-resistant gene signatures, but many cells, predominantly in the stromal subgroup, expressed markers associated with cancer stem cells.

Conclusions

Single cell sequencing provides a means of identifying subpopulations of cancer cells within a single patient. Single cell sequence analysis may prove to be critical for understanding the etiology, progression and drug resistance in ovarian cancer.

Human cancers are complex ecosystems composed of cells with distinct phenotypes, genotypes, and epigenetic states, but current models do not adequately reflect tumor composition in patients. We used single-cell RNA sequencing (RNA-seq) to profile 430 cells from five primary glioblastomas, which we found to be inherently variable in their expression of diverse transcriptional programs related to oncogenic signaling, proliferation, complement/immune response, and hypoxia. We also observed a continuum of stemness-related expression states that enabled us to identify putative regulators of stemness in vivo. Finally, we show that established glioblastoma subtype classifiers are variably expressed across individual cells within a tumor and demonstrate the potential prognostic implications of such intratumoral heterogeneity. Thus, we reveal previously unappreciated heterogeneity in diverse regulatory programs central to glioblastoma biology, prognosis, and therapy.

A major obstacle to curing chronic myeloid leukemia (CML) is the intrinsic resistance of CML stem cells (CMLSCs) to the drug imatinib mesylate (IM). Prosurvival genes that are preferentially expressed in CMLSCs compared with normal hematopoietic stem cells (HSCs) represent potential therapeutic targets for selectively eradicating CMLSCs. However, the discovery of such preferentially expressed genes has been hampered by the inability to completely separate CMLSCs from HSCs, which display a very similar set of surface markers. To overcome this challenge, and to minimize confounding effects of individual differences in gene expression profiles, we performed single-cell RNA-seq on CMLSCs and HSCs that were isolated from the same patient and distinguished based on the presence or absence of BCR-ABL. Among genes preferentially expressed in CMLSCs is PIM2, which encodes a prosurvival serine-threonine kinase that phosphorylates and inhibits the proapoptotic protein BAD. We show that IM resistance of CMLSCs is due, at least in part, to maintenance of BAD phosphorylation by PIM2. We find that in CMLSCs, PIM2 expression is promoted by both a BCR-ABL-dependent (IM-sensitive) STAT5-mediated pathway and a BCR-ABL-independent (IM-resistant) STAT4-mediated pathway. Combined treatment with IM and a PIM inhibitor synergistically increases apoptosis of CMLSCs, suppresses colony formation, and significantly prolongs survival in a mouse CML model, with a negligible effect on HSCs. Our results reveal a therapeutically targetable mechanism of IM resistance in CMLSCs. The experimental approach that we describe can be generally applied to other malignancies that harbor oncogenic fusion proteins or other characteristic genetic markers.

Increasing evidence suggests the presence of minor cell subpopulations in prostate cancer that are androgen independent and poised for selection as dominant clones after androgen deprivation therapy. In this study, we investigated this phenomenon by stratifying cell subpopulations based on transcriptome profiling of 144 single LNCaP prostate cancer cells treated or untreated with androgen after cell-cycle synchronization. Model-based clustering of 397 differentially expressed genes identified eight potential subpopulations of LNCaP cells, revealing a previously unappreciable level of cellular heterogeneity to androgen stimulation. One subpopulation displayed stem-like features with a slower cell doubling rate, increased sphere formation capability, and resistance to G2-M arrest induced by a mitosis inhibitor. Advanced growth of this subpopulation was associated with enhanced expression of 10 cell-cycle-related genes (CCNB2, DLGAP5, CENPF, CENPE, MKI67, PTTG1, CDC20, PLK1, HMMR, and CCNB1) and decreased dependence upon androgen receptor signaling. In silico analysis of RNA-seq data from The Cancer Genome Atlas further demonstrated that concordant upregulation of these genes was linked to recurrent prostate cancers. Analysis of receiver operating characteristic curves implicates aberrant expression of these genes and could be useful for early identification of tumors that subsequently develop biochemical recurrence. Moreover, this single-cell approach provides a better understanding of how prostate cancer cells respond heterogeneously to androgen deprivation therapies and reveals characteristics of subpopulations resistant to this treatment.Significance: Illustrating the challenge in treating cancers with targeted drugs, which by selecting for drug resistance can drive metastatic progression, this study characterized the plasticity and heterogeneity of prostate cancer cells with regard to androgen dependence, defining the character or minor subpopulations of androgen-independent cells that are poised for clonal selection after androgen-deprivation therapy. Cancer Res; 78(4); 853-64. ©2017 AACR.

Tumor-stromal communication within the microenvironment contributes to initiation of metastasis and may present a therapeutic opportunity. Using serial single-cell RNA sequencing in an orthotopic mouse prostate cancer model, we find up-regulation of prolactin receptor as cancer cells that have disseminated to the lungs expand into micrometastases. Secretion of the ligand prolactin by adjacent lung stromal cells is induced by tumor cell production of the COX-2 synthetic product prostaglandin E2 (PGE2). PGE2 treatment of fibroblasts activates the orphan nuclear receptor NR4A (Nur77), with prolactin as a major transcriptional target for the NR4A-retinoid X receptor (RXR) heterodimer. Ectopic expression of prolactin receptor in mouse cancer cells enhances micrometastasis, while treatment with the COX-2 inhibitor celecoxib abrogates prolactin secretion by fibroblasts and reduces tumor initiation. Across multiple human cancers, COX-2, prolactin, and prolactin receptor show consistent differential expression in tumor and stromal compartments. Such paracrine cross-talk may thus contribute to the documented efficacy of COX-2 inhibitors in cancer suppression.

Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4⁺ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.

Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle toward HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV-infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression.

Adult stem-cells may serve as the cell-of-origin for cancer, yet their unbiased identification in single cell RNA sequencing data is challenging due to the high dropout rate. In the case of breast, the existence of a bipotent stem-like state is also controversial. Here we apply a marker-free algorithm to scRNA-Seq data from the human mammary epithelium, revealing a high-potency cell-state enriched for an independent mammary stem-cell expression module. We validate this stem-like state in independent scRNA-Seq data. Our algorithm further predicts that the stem-like state is bipotent, a prediction we are able to validate using FACS sorted bulk expression data. The bipotent stem-like state correlates with clinical outcome in basal breast cancer and is characterized by overexpression of YBX1 and ENO1, two modulators of basal breast cancer risk. This study illustrates the power of a marker-free computational framework to identify a novel bipotent stem-like state in the mammary epithelium.

The development of the digestive tract is critical for proper food digestion and nutrient absorption. Here, we analyse the main organs of the digestive tract, including the oesophagus, stomach, small intestine and large intestine, from human embryos between 6 and 25 weeks of gestation as well as the large intestine from adults using single-cell RNA-seq analyses. In total, 5,227 individual cells are analysed and 40 cell types clearly identified. Their crucial biological features, including developmental processes, signalling pathways, cell cycle, nutrient digestion and absorption metabolism, and transcription factor networks, are systematically revealed. Moreover, the differentiation and maturation processes of the large intestine are thoroughly investigated by comparing the corresponding transcriptome profiles between embryonic and adult stages. Our work offers a rich resource for investigating the gene regulation networks of the human fetal digestive tract and adult large intestine at single-cell resolution.

Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.

We used single cell RNA-Seq to examine molecular heterogeneity in multiple myeloma (MM) in 597 CD138 positive cells from bone marrow aspirates of 15 patients at different stages of disease progression. 790 genes were selected by coefficient of variation (CV) method and organized cells into four groups (L1-L4) using unsupervised clustering. Plasma cells from each patient clustered into at least two groups based on gene expression signature. The L1 group contained cells from all MGUS patients having the lowest expression of genes involved in the oxidative phosphorylation, Myc targets, and mTORC1 signaling pathways (p < 1.2 × 10^-14). In contrast, the expression level of these pathway genes increased progressively and were the highest in L4 group containing only cells from MM patients with t(4;14) translocations. A 44 genes signature of consistently overexpressed genes among the four groups was associated with poorer overall survival in MM patients (APEX trial, p < 0.0001; HR, 1.83; 95% CI, 1.33-2.52), particularly those treated with bortezomib (p < 0.0001; HR, 2.00; 95% CI, 1.39-2.89). Our study, using single cell RNA-Seq, identified the most significantly affected molecular pathways during MM progression and provided a novel signature predictive of patient prognosis and treatment stratification.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Background

Intra-tumor heterogeneity stems from genetic, epigenetic, functional, and environmental differences among tumor cells. A major source of genetic heterogeneity comes from DNA sequence differences and/or whole chromosome and focal copy number variations (CNVs). Whole chromosome CNVs are caused by chromosomal instability (CIN) that is defined by a persistently high rate of chromosome mis-segregation. Accordingly, CIN causes constantly changing karyotypes that result in extensive cell-to-cell genetic heterogeneity. How the genetic heterogeneity caused by CIN influences gene expression in individual cells remains unknown.

Methods

We performed single-cell RNA sequencing on a chromosomally unstable glioblastoma cancer stem cell (CSC) line and a control normal, diploid neural stem cell (NSC) line to investigate the impact of CNV due to CIN on gene expression. From the gene expression data, we computationally inferred large-scale CNVs in single cells. Also, we performed copy number adjusted differential gene expression analysis between NSCs and glioblastoma CSCs to identify copy number dependent and independent differentially expressed genes.

Results

Here, we demonstrate that gene expression across large genomic regions scales proportionally to whole chromosome copy number in chromosomally unstable CSCs. Also, we show that the differential expression of most genes between normal NSCs and glioblastoma CSCs is largely accounted for by copy number alterations. However, we identify 269 genes whose differential expression in glioblastoma CSCs relative to normal NSCs is independent of copy number. Moreover, a gene signature derived from the subset of genes that are differential expressed independent of copy number in glioblastoma CSCs correlates with tumor grade and is prognostic for patient survival.

Conclusions

These results demonstrate that CIN is directly responsible for gene expression changes and contributes to both genetic and transcriptional heterogeneity among glioblastoma CSCs. These results also demonstrate that the expression of some genes is buffered against changes in copy number, thus preserving some consistency in gene expression levels from cell-to-cell despite the continuous change in karyotype driven by CIN. Importantly, a gene signature derived from the subset of genes whose expression is buffered against copy number alterations correlates with tumor grade and is prognostic for patient survival that could facilitate patient diagnosis and treatment.

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.

The mammalian prefrontal cortex comprises a set of highly specialized brain areas containing billions of cells and serves as the centre of the highest-order cognitive functions, such as memory, cognitive ability, decision-making and social behaviour. Although neural circuits are formed in the late stages of human embryonic development and even after birth, diverse classes of functional cells are generated and migrate to the appropriate locations earlier in development. Dysfunction of the prefrontal cortex contributes to cognitive deficits and the majority of neurodevelopmental disorders; there is therefore a need for detailed knowledge of the development of the prefrontal cortex. However, it is still difficult to identify cell types in the developing human prefrontal cortex and to distinguish their developmental features. Here we analyse more than 2,300 single cells in the developing human prefrontal cortex from gestational weeks 8 to 26 using RNA sequencing. We identify 35 subtypes of cells in six main classes and trace the developmental trajectories of these cells. Detailed analysis of neural progenitor cells highlights new marker genes and unique developmental features of intermediate progenitor cells. We also map the timeline of neurogenesis of excitatory neurons in the prefrontal cortex and detect the presence of interneuron progenitors in early developing prefrontal cortex. Moreover, we reveal the intrinsic development-dependent signals that regulate neuron generation and circuit formation using single-cell transcriptomic data analysis. Our screening and characterization approach provides a blueprint for understanding the development of the human prefrontal cortex in the early and mid-gestational stages in order to systematically dissect the cellular basis and molecular regulation of prefrontal cortex function in humans.

Human pluripotent stem cells (hPSCs) provide powerful models for studying cellular differentiations and unlimited sources of cells for regenerative medicine. However, a comprehensive single-cell level differentiation roadmap for hPSCs has not been achieved.We use high throughput single-cell RNA-sequencing (scRNA-seq), based on optimized microfluidic circuits, to profile early differentiation lineages in the human embryoid body system. We present a cellular-state landscape for hPSC early differentiation that covers multiple cellular lineages, including neural, muscle, endothelial, stromal, liver, and epithelial cells. Through pseudotime analysis, we construct the developmental trajectories of these progenitor cells and reveal the gene expression dynamics in the process of cell differentiation. We further reprogram primed H9 cells into naïve-like H9 cells to study the cellular-state transition process. We find that genes related to hemogenic endothelium development are enriched in naïve-like H9. Functionally, naïve-like H9 show higher potency for differentiation into hematopoietic lineages than primed cells.Our single-cell analysis reveals the cellular-state landscape of hPSC early differentiation, offering new insights that can be harnessed for optimization of differentiation protocols.

We recently demonstrated that the major effector function of neonatal CD4⁺ T cells is to produce CXCL8, a prototypic cytokine of innate immune cells. In this article, we show that CXCL8 expression, prior to proliferation, is common in newly arising T cells (so-called "recent thymic emigrants") in adults, as well as in babies. This effector potential is acquired in the human thymus, prior to TCR signaling, but rather than describing end-stage differentiation, such cells, whether isolated from neonates or adults, can further differentiate into IFN-γ-producing CD4⁺ T cells. Thus, the temporal transition of host defense from innate to adaptive immunity is unexpectedly mirrored at the cellular level by the capacity of human innate-like CXCL8-producing CD4⁺ T cells to transition directly into Th1 cells.

Study question

What effect does maternal age have on the human oocyte's molecular response to in vitro oocyte maturation?

Summary answer

Although polyadenylated transcript abundance is similar between young and advanced maternal age (AMA) germinal vesicle (GV) oocytes, metaphase II (MII) oocytes exhibit a divergent transcriptome resulting from a differential response to in vitro oocyte maturation.

What is known already

Microarray studies considering maternal age or maturation stage have shown that either of these factors will affect oocyte polyadenylated transcript abundance in human oocytes. However, studies considering both human oocyte age and multiple stages simultaneously are limited to a single study that examined transcript levels for two genes by qPCR. Thus, polyadenylated RNA sequencing (RNA-Seq) could provide novel insight into age-associated aberrations in gene expression in GV and MII oocytes.

Study design, size, duration

The effect of maternal age (longitudinal analysis) on polyadenylated transcript abundance at different stages was analyzed by examining single GV and single in vitro matured MII oocytes derived from five young (YNG; < 30 years; average age 26.8; range 20-29) and five advanced maternal age (AMA; ≥40 years; average age 41.6 years; range 40-43 years) patients. Thus, a total of 10 YNG (5 GV and 5 MII) and 10 AMA (5 GV and 5 MII) oocytes were individually processed for RNA-Seq analysis.

Participants/materials, settings, methods

Patients undergoing infertility treatment at the Colorado Center for Reproductive Medicine (Lone Tree, CO, USA) underwent ovarian stimulation with FSH and received hCG for final follicular maturation prior to ultrasound guided oocyte retrieval. Unused GV oocytes obtained at retrieval were donated for transcriptome analysis. Single oocytes were stored (at -80°C in PicoPure RNA Extraction Buffer; Thermo Fisher Scientific, USA) immediately upon verification of immaturity or after undergoing in vitro oocyte maturation (24 h incubation), representing GV and MII samples, respectively. After isolating RNA and generating single oocyte RNA-Seq libraries (SMARTer Ultra Low Input RNA HV kit; Clontech, USA), Illumina sequencing (100 bp paired-end reads on HiSeq 2500) and bioinformatics analysis (CLC Genomics Workbench, DESeq2, weighted gene correlation network analysis (WGCNA), Ingenuity Pathway Analysis) were performed.

Main results and the role of chance

A total of 12 770 genes were determined to be expressed in human oocytes (reads per kilobase per million mapped reads (RPKM) > 0.4 in at least three of five replicates for a minimum of one sample type). Differential gene expression analysis between YNG and AMA oocytes (within stage) identified 1 and 255 genes that significantly differed (adjusted P < 0.1 and log2 fold change >1) in polyadenylated transcript abundance for GV and MII oocytes, respectively. These genes included CDK1, NLRP5 and PRDX1, which have been reported to affect oocyte developmental potential. Despite the similarity in transcript abundance between GV oocytes irrespective of age, divergent expression patterns emerged during oocyte maturation. These age-specific differentially expressed genes were enriched (FDR < 0.05) for functions and pathways associated with mitochondria, cell cycle and cytoskeleton. Gene modules generated by WGCNA (based on gene expression) and patient traits related to oocyte quality (e.g. age and blastocyst development) were correlated (P < 0.05) and enriched (FDR < 0.05) for functions and pathways associated with oocyte maturation.

Large scale data

Raw data from this study can be accessed through GSE95477.

Limitations, reasons for caution

The human oocytes used in the current study were obtained from patients with varying causes of infertility (e.g. decreased oocyte quality and oocyte quality-independent factors), possibly affecting oocyte gene expression. Oocytes in this study were retrieved at the GV stage following hCG administration and the MII oocytes were derived by IVM of patient oocytes. Although the approach has the benefit of identifying intrinsic differences between samples, it may not be completely representative of in vivo matured oocytes.

Wider implications of the findings

Transcriptome profiles of YNG and AMA oocytes, particularly at the MII stage, suggest that aberrant transcript abundance may contribute to the age-associated decline in fertility.

Study funding/competing interest(s)

J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.

Lung epithelial lineages have been difficult to maintain in pure form in vitro, and lineage-specific reporters have proven invaluable for monitoring their emergence from cultured pluripotent stem cells (PSCs). However, reporter constructs for tracking proximal airway lineages generated from PSCs have not been previously available, limiting the characterization of these cells. Here, we engineer mouse and human PSC lines carrying airway secretory lineage reporters that facilitate the tracking, purification, and profiling of this lung subtype. Through bulk and single-cell-based global transcriptomic profiling, we find PSC-derived airway secretory cells are susceptible to phenotypic plasticity exemplified by the tendency to co-express both a proximal airway secretory program as well as an alveolar type 2 cell program, which can be minimized by inhibiting endogenous Wnt signaling. Our results provide global profiles of engineered lung cell fates, a guide for improving their directed differentiation, and a human model of the developing airway.

Single-cell RNA-seq (scRNA-seq) of pancreatic islets have reported on α- and β-cell gene expression in mice and subjects of predominantly European ancestry. We aimed to assess these findings in East-Asian islet-cells. 448 islet-cells were captured from three East-Asian non-diabetic subjects for scRNA-seq. Hierarchical clustering using pancreatic cell lineage genes was used to assign cells into cell-types. Differentially expressed transcripts between α- and β-cells were detected using ANOVA and in silico replications of mouse and human islet cell genes were performed. We identified 118 α, 105 β, 6 δ endocrine cells and 47 exocrine cells. Besides INS and GCG, 26 genes showed differential expression between α- and β-cells. 10 genes showed concordant expression as reported in rodents, while FAM46A was significantly discordant. Comparing our East-Asian data with data from primarily European subjects, we replicated several genes implicated in nuclear receptor activations, acute phase response pathway, glutaryl-CoA/tryptophan degradations and EIF2/AMPK/mTOR signaling. Additionally, we identified protein ubiquitination to be associated among East-Asian β-cells. We report on East-Asian α- and β-cell gene signatures and substantiate several genes/pathways. We identify expression signatures in East-Asian β-cells that perhaps reflects increased susceptibility to cell-death and warrants future validations to fully appreciate their role in East-Asian diabetes pathogenesis.

The significance of islet Ag-reactive T cells found in peripheral blood of type 1 diabetes (T1D) subjects is unclear, partly because similar cells are also found in healthy control (HC) subjects. We hypothesized that key disease-associated cells would show evidence of prior Ag exposure, inferred from expanded TCR clonotypes, and essential phenotypic properties in their transcriptomes. To test this, we developed single-cell RNA sequencing procedures for identifying TCR clonotypes and transcript phenotypes in individual T cells. We applied these procedures to analysis of islet Ag-reactive CD4⁺ memory T cells from the blood of T1D and HC individuals after activation with pooled immunodominant islet peptides. We found extensive TCR clonotype sharing in Ag-activated cells, especially from individual T1D subjects, consistent with in vivo T cell expansion during disease progression. The expanded clonotype from one T1D subject was detected at repeat visits spanning >15 mo, demonstrating clonotype stability. Notably, we found no clonotype sharing between subjects, indicating a predominance of "private" TCR specificities. Expanded clones from two T1D subjects recognized distinct IGRP peptides, implicating this molecule as a trigger for CD4⁺ T cell expansion. Although overall transcript profiles of cells from HC and T1D subjects were similar, profiles from the most expanded clones were distinctive. Our findings demonstrate that islet Ag-reactive CD4⁺ memory T cells with unique Ag specificities and phenotypes are expanded during disease progression and can be detected by single-cell analysis of peripheral blood.

Esophageal squamous cell carcinoma (ESCC) is a tumor composed of heterogeneous cells that easily become radioresistant, which leads to tumor recurrence. The most commonly used treatment for ESCC is fractionated irradiation (FIR) therapy that utilizes ionizing radiation to directly induce cytotoxic cell death. However, this treatment may not be able to eliminate all cancer cells due to high adaptive evolution. To determine whether the transcriptome dynamics during ESCC recurrence formation are associated with FIR response, an in vitro cell culture model for ESCC radioresistance that mimics the common radiotherapy process in patients with ESCC was established in the present study. High‑throughput sequencing analysis of in vitro cultured ESCC cells was performed using different cumulative irradiation doses, as well as tumor samples from FIR‑treated patients with ESCC before and after the development of radioresistance. Radioresistance‑associated genes and signaling pathways that were aberrantly expressed in radioresistant ESCC cells were identified, including autophagy‑related 9B (regulation of autophagy), DNA damage‑inducible transcript 4, myoglobin and plasminogen activator tissue type, which are associated with response to hypoxia, Bcl2‑binding component 3, tumor protein P63 and interferon γ‑inducible protein 16, which are associated with DNA damage response. The heterogeneity and dynamic gene expression of ESCC cells during acquired radioresistance were further studied in primary (41 single cells), 12 Gy FIR‑treated (87 single cells) and 30 Gy FIR‑treated (89 single cells) cancer cells using a single‑cell RNA sequencing approach. The results of the present study comprehensively characterized the transcriptome dynamics during acquired radioresistance in an in vitro model of ESCC and patient tumor samples at the population and single cell level. Single‑cell RNA sequencing revealed the heterogeneity of irradiated ESCC cells and an increase in the radioresistant ESCC cell subpopulation during acquired radioresistance. Overall, these results are of potential clinical relevance as they identify a number of signaling molecules associated with radioresistance, as well as opportunities for the development of novel therapeutic options for the treatment of ESCC.

The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome-a neurodevelopmental disorder that is caused by mutations in the Ca_V1.2 calcium channel-interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM⁺ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.

The development of the mammalian retina is a complicated process involving the generation of distinct types of neurons from retinal progenitor cells (RPCs) in a spatiotemporal-specific manner. The progression of RPCs during retinogenesis includes RPC proliferation, cell-fate commitment, and specific neuronal differentiation. In this study, by performing single-cell RNA sequencing of cells isolated from human embryonic stem cell (hESC)-derived 3D retinal organoids, we successfully deconstructed the temporal progression of RPCs during early human retinogenesis. We identified two distinctive subtypes of RPCs with unique molecular profiles, namely multipotent RPCs and neurogenic RPCs. We found that genes related to the Notch and Wnt signaling pathways, as well as chromatin remodeling, were dynamically regulated during RPC commitment. Interestingly, our analysis identified that CCND1, a G₁-phase cell-cycle regulator, was coexpressed with ASCL1 in a cell-cycle-independent manner. Temporally controlled overexpression of CCND1 in retinal organoids demonstrated a role for CCND1 in promoting early retinal neurogenesis. Together, our results revealed critical pathways and novel genes in early retinogenesis of humans.

Human mononuclear phagocytes comprise phenotypically and functionally overlapping subsets of dendritic cells (DCs) and monocytes, but the extent of their heterogeneity and distinct markers for subset identification remains elusive. By integrating high-dimensional single-cell protein and RNA expression data, we identified distinct markers to delineate monocytes from conventional DC2 (cDC2s). Using CD88 and CD89 for monocytes and HLA-DQ and FcεRIα for cDC2s allowed for their specific identification in blood and tissues. We also showed that cDC2s could be subdivided into phenotypically and functionally distinct subsets based on CD5, CD163, and CD14 expression, including a distinct subset of circulating inflammatory CD5^-CD163⁺CD14⁺ cells related to previously defined DC3s. These inflammatory DC3s were expanded in systemic lupus erythematosus patients and correlated with disease activity. These findings further unravel the heterogeneity of DC subpopulations in health and disease and may pave the way for the identification of specific DC subset-targeting therapies.

Previous studies have shown that mouse dermis is composed of functionally distinct fibroblast lineages. To explore the extent of fibroblast heterogeneity in human skin, we used a combination of comparative spatial transcriptional profiling of human and mouse dermis and single-cell transcriptional profiling of human dermal fibroblasts. We show that there are at least four distinct fibroblast populations in adult human skin, not all of which are spatially segregated. We define markers permitting their isolation and show that although marker expression is lost in culture, different fibroblast subpopulations retain distinct functionality in terms of Wnt signaling, responsiveness to IFN-γ, and ability to support human epidermal reconstitution when introduced into decellularized dermis. These findings suggest that ex vivo expansion or in vivo ablation of specific fibroblast subpopulations may have therapeutic applications in wound healing and diseases characterized by excessive fibrosis.

The heart is the central organ of the circulatory system, and its proper development is vital for maintaining human life. Here, we used single-cell RNA sequencing to profile the gene expression landscapes of ∼4,000 cardiac cells from human embryos and identified four major types of cells: cardiomyocytes (CMs), cardiac fibroblasts, endothelial cells (ECs), and valvar interstitial cells (VICs). Atrial and ventricular CMs acquired distinct features early in heart development. Furthermore, both CMs and fibroblasts show stepwise changes in gene expression. As development proceeds, VICs may be involved in the remodeling phase, and ECs display location-specific characteristics. Finally, we compared gene expression profiles between humans and mice and identified a series of unique features of human heart development. Our study lays the groundwork for elucidating the mechanisms of in vivo human cardiac development and provides potential clues to understand cardiac regeneration.

In this study, we aimed to determine the mechanisms underlying the initial extramedullary translocation of myeloma cells from bone marrow into peripheral blood. We found that clonal circulating plasma cells (cPCs) are more frequently detected by flow cytometry in extramedullary plasmacytoma (EMP) patients and worsen their prognosis. It is technically much easier to collect single cPCs using FACS than it is to perform EMP biopsy. Therefore, combining EMP imaging with cPC detection may be a promising strategy for prognostic stratification. Here, using single-cell transcriptome analysis, we found that the chemokine CXCL12, a key molecule involved in CXCR4-dependent cell retention in the bone marrow, is abnormally upregulated in cPCs and might initially enable cPCs to evade bone marrow retention and translocate into the bloodstream.

Multiple sclerosis (MS) is a disabling disease of the CNS. Inflammatory features of MS include lymphocyte accumulations in the CNS and cerebrospinal fluid (CSF). The preclinical events leading to established MS are still enigmatic. Here we compared gene expression patterns of CSF cells from MS-discordant monozygotic twin pairs. Six "healthy" co-twins, who carry a maximal familial risk for developing MS, showed subclinical neuroinflammation (SCNI) with small MRI lesions. Four of these subjects had oligoclonal bands (OCBs). By single-cell RNA sequencing of 2752 CSF cells, we identified clonally expanded CD8+ T cells, plasmablasts, and, to a lesser extent, CD4+ T cells not only from MS patients but also from subjects with SCNI. In contrast to nonexpanded T cells, clonally expanded T cells showed characteristics of activated tissue-resident memory T (TRM) cells. The TRM-like phenotype was detectable already in cells from SCNI subjects but more pronounced in cells from patients with definite MS. Expanded plasmablast clones were detected only in MS and SCNI subjects with OCBs. Our data provide evidence for very early concomitant activation of 3 components of the adaptive immune system in MS, with a notable contribution of clonally expanded TRM-like CD8+ cells.

Alternative splicing (AS) generates isoform diversity for cellular identity and homeostasis in multicellular life. Although AS variation has been observed among single cells, little is known about the biological or evolutionary significance of such variation. We developed Expedition, a computational framework consisting of outrigger, a de novo splice graph transversal algorithm to detect AS; anchor, a Bayesian approach to assign modalities; and bonvoyage, a visualization tool using non-negative matrix factorization to display modality changes. Applying Expedition to single pluripotent stem cells undergoing neuronal differentiation, we discover that up to 20% of AS exons exhibit bimodality. Bimodal exons are flanked by more conserved intronic sequences harboring distinct cis-regulatory motifs, constitute much of cell-type-specific splicing, are highly dynamic during cellular transitions, preserve reading frame, and reveal intricacy of cell states invisible to conventional gene expression analysis. Systematic AS characterization in single cells redefines our understanding of AS complexity in cell biology.

Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here we performed single-cell RNA-seq analysis of over 2,000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis, and cell-cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of the bone morphogenic protein (BMP) and Notch signaling pathways. Our work provides key insights into the crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo.

Background

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages.

Results

We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation.

Conclusion

As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

Naive human embryonic stem cells (hESCs) can be derived from primed hESCs or directly from blastocysts, but their X chromosome state has remained unresolved. Here, we show that the inactive X chromosome (X_i) of primed hESCs was reactivated in naive culture conditions. Like cells of the blastocyst, the resulting naive cells contained two active X chromosomes with XIST expression and chromosome-wide transcriptional dampening and initiated XIST-mediated X inactivation upon differentiation. Both establishment of and exit from the naive state (differentiation) happened via an XIST-negative X_aX_a intermediate. Together, these findings identify a cell culture system for functionally exploring the two X chromosome dosage compensation processes in early human development: X dampening and X inactivation. However, remaining differences between naive hESCs and embryonic cells related to mono-allelic XIST expression and non-random X inactivation highlight the need for further culture improvement. As the naive state resets X_i abnormalities seen in primed hESCs, it may provide cells better suited for downstream applications.

Organismal function is, to a great extent, determined by interactions among their fundamental building blocks, the cells. In this work, we studied the cell-cell interactome of fetal placental trophoblast cells and maternal endometrial stromal cells, using single-cell transcriptomics. The placental interface mediates the interaction between two semiallogenic individuals, the mother and the fetus, and is thus the epitome of cell interactions. To study these, we inferred the cell-cell interactome by assessing the gene expression of receptor-ligand pairs across cell types. We find a highly cell-type-specific expression of G-protein-coupled receptors, implying that ligand-receptor profiles could be a reliable tool for cell type identification. Furthermore, we find that uterine decidual cells represent a cell-cell interaction hub with a large number of potential incoming and outgoing signals. Decidual cells differentiate from their precursors, the endometrial stromal fibroblasts, during uterine preparation for pregnancy. We show that decidualization (even in vitro) enhances the ability to communicate with the fetus, as most of the receptors and ligands up-regulated during decidualization have their counterpart expressed in trophoblast cells. Among the signals transmitted, growth factors and immune signals dominate, and suggest a delicate balance of enhancing and suppressive signals. Finally, this study provides a rich resource of gene expression profiles of term intravillous and extravillous trophoblasts, including the transcriptome of the multinucleated syncytiotrophoblast.

The placenta is crucial for a successful pregnancy and the health of both the fetus and the pregnant woman. However, how the human trophoblast lineage is regulated, including the categorization of the placental cell subtypes is poorly understood. Here we performed single-cell RNA sequencing (RNA-seq) on sorted placental cells from first- and second-trimester human placentas. New subtypes of cells of the known cytotrophoblast cells (CTBs), extravillous trophoblast cells (EVTs), Hofbauer cells, and mesenchymal stromal cells were identified and cell-type-specific gene signatures were defined. Functionally, this study revealed many previously unknown functions of the human placenta. Notably, 102 polypeptide hormone genes were found to be expressed by various subtypes of placental cells, which suggests a complex and significant role of these hormones in regulating fetal growth and adaptations of maternal physiology to pregnancy. These results document human placental trophoblast differentiation at single-cell resolution and thus advance our understanding of human placentation during the early stage of pregnancy.

The cellular complexity of human brain development has been intensively investigated, although a regional characterization of the entire human cerebral cortex based on single-cell transcriptome analysis has not been reported. Here, we performed RNA-seq on over 4,000 individual cells from 22 brain regions of human mid-gestation embryos. We identified 29 cell sub-clusters, which showed different proportions in each region and the pons showed especially high percentage of astrocytes. Embryonic neurons were not as diverse as adult neurons, although they possessed important features of their destinies in adults. Neuron development was unsynchronized in the cerebral cortex, as dorsal regions appeared to be more mature than ventral regions at this stage. Region-specific genes were comprehensively identified in each neuronal sub-cluster, and a large proportion of these genes were neural disease related. Our results present a systematic landscape of the regionalized gene expression and neuron maturation of the human cerebral cortex.

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from one nonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders.

The developmental pathway of the neural retina (NR) and retinal pigment epithelium (RPE) has been revealed by extensive research in mice. However, the molecular mechanisms underlying the development of the human NR and RPE, as well as the interactions between these two tissues, have not been well defined. Here, we analyzed 2,421 individual cells from human fetal NR and RPE using single-cell RNA sequencing (RNA-seq) technique and revealed the tightly regulated spatiotemporal gene expression network of human retinal cells. We identified major cell classes of human fetal retina and potential crucial transcription factors for each cell class. We dissected the dynamic expression patterns of visual cycle- and ligand-receptor interaction-related genes in the RPE and NR. Moreover, we provided a map of disease-related genes for human fetal retinal cells and highlighted the importance of retinal progenitor cells as potential targets of inherited retinal diseases. Our findings captured the key in vivo features of the development of the human NR and RPE and offered insightful clues for further functional studies.

Healthy renal function depends on normal nephrogenesis during embryonic development. However, a comprehensive gene expression profile of human fetal kidney development remains largely unexplored. Here, using a single-cell RNA-sequencing technique, we analyzed >3,000 human fetal renal cells spanning 4 months of development in utero. Unsupervised analysis identified two progenitor subtypes during cap mesenchyme development, suggesting a mechanism for sustaining their progenitor states. Furthermore, we identified critical transcriptional regulators and signaling pathways involved in the segmentation of nephron tubules. We explored the development of the highly heterogeneous collecting duct epithelia and dissected the metabolic gene repertoire and the extracellular matrix composition of the glomerular mesangium. The results provide insights on the molecular basis and regulatory events in human renal development. Moreover, the cell-type-specific expression features of causal genes in congenital renal diseases may be helpful in the treatment of these diseases.

The rapid improvements in single cell sequencing technologies and analyses afford greater scope for dissecting organoid cultures composed of multiple cell types and create an opportunity to interrogate these models to understand tissue biology, cellular behavior and interactions. To this end, retinal organoids generated from human embryonic stem cells (hESCs) were analyzed by single cell RNA-sequencing (scRNA-Seq) at three time points of differentiation. Combinatorial data from all time points revealed the presence of nine clusters, five of which corresponded to key retinal cell types: retinal pigment epithelium (RPE), retinal ganglion cells (RGCs), cone and rod photoreceptors, and Müller glia. The remaining four clusters expressed genes typical of mitotic cells, extracellular matrix components and those involved in homeostasis. The cell clustering analysis revealed the decreasing presence of mitotic cells and RGCs, formation of a distinct RPE cluster, the emergence of cone and rod photoreceptors from photoreceptor precursors, and an increasing number of Müller glia cells over time. Pseudo-time analysis resembled the order of cell birth during retinal development, with the mitotic cluster commencing the trajectory and the large majority of Müller glia completing the time line. Together, these data demonstrate the feasibility and potential of scRNA-Seq to dissect the inherent complexity of retinal organoids and the orderly birth of key retinal cell types. Stem Cells 2019;37:593-598.

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors.

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells' DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression. Binding of p300 and RNAPII are key features of these constituents. We determine two key parameters of enhancer activity in single cells: their penetrance in a population and their contribution to expression in these cells. Through combinatorial interrogation, we find that simultaneous repression of multiple weak constituents can alter super-enhancer activity in a manner greatly exceeding repression of individual constituents.

Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting.

Altered glycolysis is a characteristic of many cancers, and can also be associated with changes in stem cell-like cancer (SCLC) cell populations. We therefore set out to directly examine the effect of glycolysis on SCLC cell phenotype, using a model where glycolysis is stably reduced by adapting the cells to a sugar source other than glucose. Restricting glycolysis using this approach consistently resulted in cells with increased oncogenic potential; including an increase in SCLC cells, proliferation in 3D matrigel, invasiveness, chemoresistance, and altered global gene expression. Tumorigenicity in vivo was also markedly increased. SCLC cells exhibited increased dependence upon alternate metabolic pathways. They also became c-KIT dependent, indicating that their apparent state of maturation is regulated by glycolysis. Single-cell mRNA sequencing identified altered networks of metabolic-, stem- and signaling- gene expression within SCLC-enriched populations in response to glycolytic restriction. Therefore, reduced glycolysis, which may occur in niches within tumors where glucose availability is limiting, can promote tumor aggressiveness by increasing SCLC cell populations, but can also introduce novel, potentially exploitable, vulnerabilities in SCLC cells.

Organoids derived from human pluripotent stem cells are a potentially powerful tool for high-throughput screening (HTS), but the complexity of organoid cultures poses a significant challenge for miniaturization and automation. Here, we present a fully automated, HTS-compatible platform for enhanced differentiation and phenotyping of human kidney organoids. The entire 21-day protocol, from plating to differentiation to analysis, can be performed automatically by liquid-handling robots, or alternatively by manual pipetting. High-content imaging analysis reveals both dose-dependent and threshold effects during organoid differentiation. Immunofluorescence and single-cell RNA sequencing identify previously undetected parietal, interstitial, and partially differentiated compartments within organoids and define conditions that greatly expand the vascular endothelium. Chemical modulation of toxicity and disease phenotypes can be quantified for safety and efficacy prediction. Screening in gene-edited organoids in this system reveals an unexpected role for myosin in polycystic kidney disease. Organoids in HTS formats thus establish an attractive platform for multidimensional phenotypic screening.

Podocyte injury is central to many forms of kidney disease, but transcriptional signatures reflecting podocyte injury and compensation mechanisms are challenging to analyze in vivo. Human kidney organoids derived from pluripotent stem cells (PSCs), a potentially new model for disease and regeneration, present an opportunity to explore the transcriptional plasticity of podocytes. Here, transcriptional profiling of more than 12,000 single cells from human PSC-derived kidney organoid cultures was used to identify robust and reproducible cell lineage gene expression signatures shared with developing human kidneys based on trajectory analysis. Surprisingly, the gene expression signature characteristic of developing glomerular epithelial cells was also observed in glomerular tissue from a kidney disease cohort. This signature correlated with proteinuria and inverse eGFR, and it was confirmed in an independent podocytopathy cohort. Three genes in particular were further characterized as potentially novel components of the glomerular disease signature. We conclude that cells in human PSC-derived kidney organoids reliably recapitulate the developmental transcriptional program of podocytes and other cell lineages in the human kidney and that transcriptional profiles seen in developing podocytes are reactivated in glomerular disease. Our findings demonstrate an approach to identifying potentially novel molecular programs involved in the pathogenesis of glomerulopathies.

Viral infection is usually studied at the population level by averaging over millions of cells. However, infection at the single-cell level is highly heterogeneous, with most infected cells giving rise to no or few viral progeny while some cells produce thousands. Analysis of Herpes Simplex virus 1 (HSV-1) infection by population-averaged measurements has taught us a lot about the course of viral infection, but has also produced contradictory results, such as the concurrent activation and inhibition of type I interferon signaling during infection. Here, we combine live-cell imaging and single-cell RNA sequencing to characterize viral and host transcriptional heterogeneity during HSV-1 infection of primary human cells. We find extreme variability in the level of viral gene expression among individually infected cells and show that these cells cluster into transcriptionally distinct sub-populations. We find that anti-viral signaling is initiated in a rare group of abortively infected cells, while highly infected cells undergo cellular reprogramming to an embryonic-like transcriptional state. This reprogramming involves the recruitment of β-catenin to the host nucleus and viral replication compartments, and is required for late viral gene expression and progeny production. These findings uncover the transcriptional differences in cells with variable infection outcomes and shed new light on the manipulation of host pathways by HSV-1.

Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

In vitro differentiation of human stem cells can produce pancreatic β-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro β-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to β-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo β-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the β-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro β-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine.

The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

Maternal-effect genes are especially critical for early embryonic development after fertilization and until massive activation of the embryonic genome occurs. By applying a tandem mass tag (TMT)-labeled quantitative proteomics combined with RNA sequencing approach, the proteome of the buffalo was quantitatively analyzed during parthenogenesis of mature oocytes and the two-cell stage embryo. Of 1908 quantified proteins, 123 differed significantly. The transcriptome was analyzed eight stages (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst) of Buffalo using the RNA sequencing approach, and a total of 3567 unique genes were identified to be differently expressed between all consecutive stages of pre-implantation development. Validation of proteomics results (TUBB3, CTNNA1, CDH3, MAP2K1), which are involved in tight junction and gap junction, revealing that the maternal expression of the proteins possibly plays a role in the formation of cellular junctions firstly after parthenogenetic activation. Correlation and hierarchical analyses of transcriptional profiles and the expression of NPM2 and NLRP5 mRNA of buffalo in vitro developed oocytes and parthenogenetic embryos indicated that the "maternal-to-zygotic transition" (MZT) process might exist in the model of parthenogenesis, which is similar to a normally fertilized embryo, and may occur between the 8-cell to 16-cell stage. These data provide a rich resource for further studies on maternal proteins and genes and are conducive to improving nuclear transfer technology.

Purpose

To better characterize retinal endothelial barrier properties through analysis of individual transcriptomes of primary bovine retinal microvascular endothelial cells (RMECs).

Methods

Individual RMECs were captured on the Fluidigm C1 system, cDNA libraries were prepared using a Nextera XT kit, and sequencing was performed on a NextSeq system (Illumina). Data analysis was performed using R packages Scater, SC3, and Seurat, and the browser application Automated Single-cell Analysis Pipeline (ASAP). Alternative splicing events in single cells were quantified with Outrigger. Cytoscape was used for network analyses.

Results

Application of a single-cell RNA sequencing (scRNA-seq) analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main differences related to proliferation status. Expression of markers from along the arteriovenous tree suggested that most cells originated from capillaries. Average gene expression levels across all cells were used to develop an in silico model of the inner blood-retina barrier incorporating junctional proteins not previously reported within the retinal vasculature. Correlation of barrier gene expression among individual cells revealed a subgroup of genes highly correlated with PECAM-1 at the center of the correlation network. Numerous alternative splicing events involving exons within microvascular barrier genes were observed, and in many cases, individual cells expressed one isoform exclusively.

Conclusions

We optimized a workflow for single-cell transcriptomics in primary RMECs. The results provide fundamental insights into the genes involved in formation of the retinal-microvascular barrier.

The regulatory mechanisms by which neurons coordinate their physiology and connectivity are not well understood. The Drosophila olfactory receptor neurons (ORNs) provide an excellent system to investigate this question. Each ORN type expresses a unique olfactory receptor, or a combination thereof, and sends their axons to a stereotyped glomerulus. Using single-cell RNA sequencing, we identified 33 transcriptomic clusters for ORNs and mapped 20 to their glomerular types, demonstrating that transcriptomic clusters correspond well with anatomically and physiologically defined ORN types. Each ORN type expresses hundreds of transcription factors. Transcriptome-instructed genetic analyses revealed that (1) one broadly expressed transcription factor (Acj6) only regulates olfactory receptor expression in one ORN type and only wiring specificity in another type, (2) one type-restricted transcription factor (Forkhead) only regulates receptor expression, and (3) another type-restricted transcription factor (Unplugged) regulates both events. Thus, ORNs utilize diverse strategies and complex regulatory networks to coordinate their physiology and connectivity.

Neurons undergo substantial morphological and functional changes during development to form precise synaptic connections and acquire specific physiological properties. What are the underlying transcriptomic bases? Here, we obtained the single-cell transcriptomes of Drosophila olfactory projection neurons (PNs) at four developmental stages. We decoded the identity of 21 transcriptomic clusters corresponding to 20 PN types and developed methods to match transcriptomic clusters representing the same PN type across development. We discovered that PN transcriptomes reflect unique biological processes unfolding at each stage-neurite growth and pruning during metamorphosis at an early pupal stage; peaked transcriptomic diversity during olfactory circuit assembly at mid-pupal stages; and neuronal signaling in adults. At early developmental stages, PN types with adjacent birth order share similar transcriptomes. Together, our work reveals principles of cellular diversity during brain development and provides a resource for future studies of neural development in PNs and other neuronal types.

Single-cell RNA sequencing (scRNA-seq) has been used extensively to study cell-specific gene expression in animals, but it has not been widely applied to plants. Here, we describe the use of a commercially available droplet-based microfluidics platform for high-throughput scRNA-seq to obtain single-cell transcriptomes from protoplasts of more than 10,000 Arabidopsis (Arabidopsis thaliana) root cells. We find that all major tissues and developmental stages are represented in this single-cell transcriptome population. Further, distinct subpopulations and rare cell types, including putative quiescent center cells, were identified. A focused analysis of root epidermal cell transcriptomes defined developmental trajectories for individual cells progressing from meristematic through mature stages of root-hair and nonhair cell differentiation. In addition, single-cell transcriptomes were obtained from root epidermis mutants, enabling a comparative analysis of gene expression at single-cell resolution and providing an unprecedented view of the impact of the mutated genes. Overall, this study demonstrates the feasibility and utility of scRNA-seq in plants and provides a first-generation gene expression map of the Arabidopsis root at single-cell resolution.

Sperm cells of seed plants have lost their motility and are transported by the vegetative pollen tube cell for fertilization, but the extent to which they regulate their own transportation is a long-standing debate. Here we show that Arabidopsis lacking two bHLH transcription factors produces pollen without sperm cells. This abnormal pollen mostly behaves like the wild type and demonstrates that sperm cells are dispensable for normal pollen tube development.

Background

Atopic dermatitis (AD) is a prevalent inflammatory skin disease with a complex pathogenesis involving immune cell and epidermal abnormalities. Despite whole tissue biopsy studies that have advanced the mechanistic understanding of AD, single cell-based molecular alterations are largely unknown.

Objective

Our aims were to construct a detailed, high-resolution atlas of cell populations and assess variability in cell composition and cell-specific gene expression in the skin of patients with AD versus in controls.

Methods

We performed single-cell RNA sequencing on skin biopsy specimens from 5 patients with AD (4 lesional samples and 5 nonlesional samples) and 7 healthy control subjects, using 10× Genomics.

Results

We created transcriptomic profiles for 39,042 AD (lesional and nonlesional) and healthy skin cells. Fibroblasts demonstrated a novel COL6A5⁺COL18A1⁺ subpopulation that was unique to lesional AD and expressed CCL2 and CCL19 cytokines. A corresponding LAMP3⁺ dendritic cell (DC) population that expressed the CCL19 receptor CCR7 was also unique to AD lesions, illustrating a potential role for fibroblast signaling to immune cells. The lesional AD samples were characterized by expansion of inflammatory DCs (CD1A⁺FCER1A⁺) and tissue-resident memory T cells (CD69⁺CD103⁺). The frequencies of type 2 (IL13⁺)/type 22 (IL22⁺) T cells were higher than those of type 1 (IFNG⁺) in lesional AD, whereas this ratio was slightly diminished in nonlesional AD and further diminished in controls.

Conclusion

AD lesions were characterized by expanded type 2/type 22 T cells and inflammatory DCs, and by a unique inflammatory fibroblast that may interact with immune cells to regulate lymphoid cell organization and type 2 inflammation.

Developmental processes underlying normal tissue regeneration have been implicated in cancer, but the degree of their enactment during tumor progression and under the selective pressures of immune surveillance, remain unknown. Here we show that human primary lung adenocarcinomas are characterized by the emergence of regenerative cell types, typically seen in response to lung injury, and by striking infidelity among transcription factors specifying most alveolar and bronchial epithelial lineages. In contrast, metastases are enriched for key endoderm and lung-specifying transcription factors, SOX2 and SOX9, and recapitulate more primitive transcriptional programs spanning stem-like to regenerative pulmonary epithelial progenitor states. This developmental continuum mirrors the progressive stages of spontaneous outbreak from metastatic dormancy in a mouse model and exhibits SOX9-dependent resistance to natural killer cells. Loss of developmental stage-specific constraint in macrometastases triggered by natural killer cell depletion suggests a dynamic interplay between developmental plasticity and immune-mediated pruning during metastasis.

Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8⁺ T cells and an interferon-response state for CD4⁺ T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8⁺ compared to CD4⁺ T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.

There is currently little information available about how individual cell types contribute to Alzheimer's disease. Here we applied single-nucleus RNA sequencing to entorhinal cortex samples from control and Alzheimer's disease brains (n = 6 per group), yielding a total of 13,214 high-quality nuclei. We detail cell-type-specific gene expression patterns, unveiling how transcriptional changes in specific cell subpopulations are associated with Alzheimer's disease. We report that the Alzheimer's disease risk gene APOE is specifically repressed in Alzheimer's disease oligodendrocyte progenitor cells and astrocyte subpopulations and upregulated in an Alzheimer's disease-specific microglial subopulation. Integrating transcription factor regulatory modules with Alzheimer's disease risk loci revealed drivers of cell-type-specific state transitions towards Alzheimer's disease. For example, transcription factor EB, a master regulator of lysosomal function, regulates multiple disease genes in a specific Alzheimer's disease astrocyte subpopulation. These results provide insights into the coordinated control of Alzheimer's disease risk genes and their cell-type-specific contribution to disease susceptibility. These results are available at http://adsn.ddnetbio.com.

Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases.

Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single-nucleus RNA sequencing (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side-by-side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na⁺/K⁺-ATPase, WNK1, mineralocorticoid receptor, and NEDD4L expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule, and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.

Lymphatic vessels form a critical component in the regulation of human health and disease. While their functional significance is increasingly being recognized, the comprehensive heterogeneity of lymphatics remains uncharacterized. Here, we report the profiling of 33,000 lymphatic endothelial cells (LECs) in human lymph nodes (LNs) by single-cell RNA sequencing. Unbiased clustering revealed six major types of human LECs. LECs lining the subcapsular sinus (SCS) of LNs abundantly expressed neutrophil chemoattractants, whereas LECs lining the medullary sinus (MS) expressed a C-type lectin CD209. Binding of a carbohydrate Lewis X (CD15) to CD209 mediated neutrophil binding to the MS. The neutrophil-selective homing by MS LECs may retain neutrophils in the LN medulla and allow lymph-borne pathogens to clear, preventing their spread through LNs in humans. Our study provides a comprehensive characterization of LEC heterogeneity and unveils a previously undefined role for medullary LECs in human immunity.

Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.

Skeletal muscle is a heterogeneous tissue comprised of muscle fiber and mononuclear cell types that, in addition to movement, influences immunity, metabolism and cognition. We investigated the gene expression patterns of skeletal muscle cells using RNA-seq of subtype-pooled single human muscle fibers and single cell RNA-seq of mononuclear cells from human vastus lateralis, mouse quadriceps, and mouse diaphragm. We identified 11 human skeletal muscle mononuclear cell types, including two fibro-adipogenic progenitor (FAP) cell subtypes. The human FBN1+ FAP cell subtype is novel and a corresponding FBN1+ FAP cell type was also found in single cell RNA-seq analysis in mouse. Transcriptome exercise studies using bulk tissue analysis do not resolve changes in individual cell-type proportion or gene expression. The cell-type gene signatures provide the means to use computational methods to identify cell-type level changes in bulk studies. As an example, we analyzed public transcriptome data from an exercise training study and revealed significant changes in specific mononuclear cell-type proportions related to age, sex, acute exercise and training. Our single-cell expression map of skeletal muscle cell types will further the understanding of the diverse effects of exercise and the pathophysiology of muscle disease.

Recently our group demonstrated that acellular tissue engineered vessels (A-TEVs) comprised of small intestinal submucosa (SIS) immobilized with heparin and vascular endothelial growth factor (VEGF) could be implanted into the arterial system of a pre-clinical ovine animal model, where they endothelialized within one month and remained patent. Here we report that immobilized VEGF captures blood circulating monocytes (MC) with high specificity under a range of shear stresses. Adherent MC differentiate into a mixed endothelial (EC) and macrophage (Mφ) phenotype and further develop into mature EC that align in the direction of flow and produce nitric oxide under high shear stress. In-vivo, newly recruited cells on the vascular lumen express MC markers and at later times they co-express MC and EC-specific proteins and maintain graft patency. This novel finding indicates that the highly prevalent circulating MC contribute directly to the endothelialization of acellular vascular grafts under the right chemical and biomechanical cues.

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air-liquid interface culture to cerebral organoids, leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies, including long-range projection within and away from the organoid, growth-cone turning, and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.

Aberrant neuronal development and the persistence of mitotic cellular populations have been implicated in a multitude of neurological disorders, including Huntington's disease (HD). However, the mechanism underlying this potential pathology remains unclear. We used a modified protocol to differentiate induced pluripotent stem cells (iPSCs) from HD patients and unaffected controls into neuronal cultures enriched for medium spiny neurons, the cell type most affected in HD. We performed single-cell and bulk transcriptomic and epigenomic analyses and demonstrated that a persistent cyclin D1⁺ neural stem cell (NSC) population is observed selectively in adult-onset HD iPSCs during differentiation. Treatment with a WNT inhibitor abrogates this NSC population while preserving neurons. Taken together, our findings identify a mechanism that may promote aberrant neurodevelopment and adult neurogenesis in adult-onset HD striatal neurons with the potential for therapeutic compensation.

Genomic analysis of a patient's tumor is the cornerstone of precision oncology, but it does not address whether metastases should be treated differently. Here we tested whether comparative single-cell RNA sequencing (scRNA-seq) of a primary small intestinal neuroendocrine tumor to a matched liver metastasis could guide the treatment of a patient's metastatic disease. Following surgery, the patient was put on maintenance treatment with a somatostatin analog. However, the scRNA-seq analysis revealed that the neuroendocrine epithelial cells in the liver metastasis were less differentiated and expressed relatively little SSTR2, the predominant somatostatin receptor. There were also differences in the tumor microenvironments. RNA expression of vascular endothelial growth factors was higher in the primary tumor cells, reflected by an increased number of endothelial cells. Interestingly, vascular expression of the major VEGF receptors was considerably higher in the liver metastasis, indicating that the metastatic vasculature may be primed for expansion and susceptible to treatment with angiogenesis inhibitors. The patient eventually progressed on Sandostatin, and although consideration was given to adding an angiogenesis inhibitor to her regimen, her disease progression involved non-liver metastases that had not been characterized. Although in this specific case comparative scRNA-seq did not alter treatment, its potential to help guide therapy of metastatic disease was clearly demonstrated.

Early human embryonic development involves extensive lineage diversification, cell-fate specification and tissue patterning¹. Despite its basic and clinical importance, early human embryonic development remains relatively unexplained owing to interspecies divergence^2,3 and limited accessibility to human embryo samples. Here we report that human pluripotent stem cells (hPSCs) in a microfluidic device recapitulate, in a highly controllable and scalable fashion, landmarks of the development of the epiblast and amniotic ectoderm parts of the conceptus, including lumenogenesis of the epiblast and the resultant pro-amniotic cavity, formation of a bipolar embryonic sac, and specification of primordial germ cells and primitive streak cells. We further show that amniotic ectoderm-like cells function as a signalling centre to trigger the onset of gastrulation-like events in hPSCs. Given its controllability and scalability, the microfluidic model provides a powerful experimental system to advance knowledge of human embryology and reproduction. This model could assist in the rational design of differentiation protocols of hPSCs for disease modelling and cell therapy, and in high-throughput drug and toxicity screens to prevent pregnancy failure and birth defects.

Human primary prostate epithelial (PrE) cells represent patient-derived in vitro models and are traditionally grown as a monolayer in two-dimensional culture. It has been recently demonstrated that expansion of primary cells into three-dimensional prostatic organoids better mimics prostate epithelial glands by recapitulating epithelial differentiation and cell polarity. Here, we sought to identify cell populations present in monolayer PrE cells and organoid culture, grown from the same patient, using single-cell RNA-sequencing. Single-cell RNA-sequencing is a powerful tool to analyze transcriptome profiles of thousands of individual cells simultaneously, creating an in-depth atlas of cell populations within a sample. Organoids consisted of six distinct cell clusters (populations) of intermediate differentiation compared to only three clusters in the monolayer prostate epithelial cells. Integrated analysis of the datasets allowed for direct comparison of the monolayer and organoid samples and identified 10 clusters, including a distinct putative prostate stem cell population that was high in Keratin 13 (KRT13), Lymphocyte Antigen 6D (LY6D), and Prostate Stem Cell Antigen (PSCA). Many of the genes within the clusters were validated through RT-qPCR and immunofluorescence in PrE samples from 5 additional patients. KRT13+ cells were observed in discrete areas of the parent tissue and organoids. Pathway analyses and lack of EdU incorporation corroborated a stem-like phenotype based on the gene expression and quiescent state of the KRT13+ cluster. Other clusters within the samples were similar to epithelial populations reported within patient prostate tissues. In summary, these data show that the epithelial stem population is preserved in PrE cultures, with organoids uniquely expanding intermediate cell types not present in monolayer culture.

Complex interactions between different host immune cell types can determine the outcome of pathogen infections. Advances in single cell RNA-sequencing (scRNA-seq) allow probing of these immune interactions, such as cell-type compositions, which are then interpreted by deconvolution algorithms using bulk RNA-seq measurements. However, not all aspects of immune surveillance are represented by current algorithms. Here, using scRNA-seq of human peripheral blood cells infected with Salmonella, we develop a deconvolution algorithm for inferring cell-type specific infection responses from bulk measurements. We apply our dynamic deconvolution algorithm to a cohort of healthy individuals challenged ex vivo with Salmonella, and to three cohorts of tuberculosis patients during different stages of disease. We reveal cell-type specific immune responses associated not only with ex vivo infection phenotype but also with clinical disease stage. We propose that our approach provides a predictive power to identify risk for disease, and human infection outcomes.

Human organoid systems recapitulate in vivo organ architecture yet fail to capture complex pathologies such as inflammation and fibrosis. Here, using 11 different healthy and diseased pluripotent stem cell lines, we developed a reproducible method to derive multi-cellular human liver organoids composed of hepatocyte-, stellate-, and Kupffer-like cells that exhibit transcriptomic resemblance to in vivo-derived tissues. Under free fatty acid treatment, organoids, but not reaggregated cocultured spheroids, recapitulated key features of steatohepatitis, including steatosis, inflammation, and fibrosis phenotypes in a successive manner. Interestingly, an organoid-level biophysical readout with atomic force microscopy demonstrated that organoid stiffening reflects the fibrosis severity. Furthermore, organoids from patients with genetic dysfunction of lysosomal acid lipase phenocopied severe steatohepatitis, rescued by FXR agonism-mediated reactive oxygen species suppression. The presented key methodology and preliminary results offer a new approach for studying a personalized basis for inflammation and fibrosis in humans, thus facilitating the discovery of effective treatments.

Natural killer (NK) cells are critical to both innate and adaptive immunity. However, the development and heterogeneity of human NK cells are yet to be fully defined. Using single-cell RNA-sequencing technology, here we identify distinct NK populations in human bone marrow and blood, including one population expressing higher levels of immediate early genes indicative of a homeostatic activation. Functionally matured NK cells with high expression of CX3CR1, HAVCR2 (TIM-3), and ZEB2 represents terminally differentiated status with the unique transcriptional profile. Transcriptomic and pseudotime analyses identify a transitional population between CD56^bright and CD56^dim NK cells. Finally, a donor with GATA2^T354M mutation exhibits reduced percentage of CD56^bright NK cells with altered transcriptome and elevated cell death. These data expand our understanding of the heterogeneity and development of human NK cells.

Alcoholism remains a prevalent health concern throughout the world. Previous studies have identified transcriptomic patterns in the brain associated with alcohol dependence in both humans and animal models. But none of these studies have systematically investigated expression within the unique cell types present in the brain. We utilized single nucleus RNA sequencing (snRNA-seq) to examine the transcriptomes of over 16 000 nuclei isolated from the prefrontal cortex of alcoholic and control individuals. Each nucleus was assigned to one of seven major cell types by unsupervised clustering. Cell type enrichment patterns varied greatly among neuroinflammatory-related genes, which are known to play roles in alcohol dependence and neurodegeneration. Differential expression analysis identified cell type-specific genes with altered expression in alcoholics. The largest number of differentially expressed genes (DEGs), including both protein-coding and non-coding, were detected in astrocytes, oligodendrocytes and microglia. To our knowledge, this is the first single cell transcriptome analysis of alcohol-associated gene expression in any species and the first such analysis in humans for any addictive substance. These findings greatly advance the understanding of transcriptomic changes in the brain of alcohol-dependent individuals.

The human testis undergoes dramatic developmental and structural changes during puberty, including proliferation and maturation of somatic niche cells, and the onset of spermatogenesis. To characterize this understudied process, we profiled and analyzed single-cell transcriptomes of ∼10,000 testicular cells from four boys spanning puberty and compared them to those of infants and adults. During puberty, undifferentiated spermatogonia sequentially expand and differentiate prior to the initiation of gametogenesis. Notably, we identify a common pre-pubertal progenitor for Leydig and myoid cells and delineate candidate factors controlling pubertal differentiation. Furthermore, pre-pubertal Sertoli cells exhibit two distinct transcriptional states differing in metabolic profiles before converging to an alternative single mature population during puberty. Roles for testosterone in Sertoli cell maturation, antimicrobial peptide secretion, and spermatogonial differentiation are further highlighted through single-cell analysis of testosterone-suppressed transfemale testes. Taken together, our transcriptional atlas of the developing human testis provides multiple insights into developmental changes and key factors accompanying male puberty.

The natural history of small cell lung cancer (SCLC) includes rapid evolution from chemosensitivity to chemoresistance, although mechanisms underlying this evolution remain obscure due to scarcity of post-relapse tissue samples. We generated circulating tumor cell (CTC)-derived xenografts (CDXs) from SCLC patients to study intratumoral heterogeneity (ITH) via single-cell RNAseq of chemo-sensitive and -resistant CDXs and patient CTCs. We found globally increased ITH including heterogeneous expression of therapeutic targets and potential resistance pathways, such as EMT, between cellular subpopulations following treatment-resistance. Similarly, serial profiling of patient CTCs directly from blood confirmed increased ITH post-relapse. These data suggest that treatment-resistance in SCLC is characterized by coexisting subpopulations of cells with heterogeneous gene expression leading to multiple, concurrent resistance mechanisms. These findings emphasize the need for clinical efforts to focus on rational combination therapies for treatment-naïve SCLC tumors to maximize initial responses and counteract the emergence of ITH and diverse resistance mechanisms.

Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2-activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon-responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO⁺ T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4⁺ T cell compartment in the human fetal intestine. We identified 22 CD4⁺ T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4⁺ T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4⁺ T cells. Imaging mass cytometry indicated that memory-like CD4⁺ T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4⁺ T cells in the human fetal intestine that is consistent with exposure to foreign antigens.

Background

Human kidney organoids hold promise for studying development, disease modelling and drug screening. However, the utility of stem cell-derived kidney tissues will depend on how faithfully these replicate normal fetal development at the level of cellular identity and complexity.

Methods

Here, we present an integrated analysis of single cell datasets from human kidney organoids and human fetal kidney to assess similarities and differences between the component cell types.

Results

Clusters in the combined dataset contained cells from both organoid and fetal kidney with transcriptional congruence for key stromal, endothelial and nephron cell type-specific markers. Organoid enriched neural, glial and muscle progenitor populations were also evident. Major transcriptional differences between organoid and human tissue were likely related to technical artefacts. Cell type-specific comparisons revealed differences in stromal, endothelial and nephron progenitor cell types including expression of WNT2B in the human fetal kidney stroma.

Conclusions

This study supports the fidelity of kidney organoids as models of the developing kidney and affirms their potential in disease modelling and drug screening.

The development of single-cell RNA sequencing (scRNA-seq) has allowed high-resolution analysis of cell-type diversity and transcriptional networks controlling cell-fate specification. To identify the transcriptional networks governing human retinal development, we performed scRNA-seq analysis on 16 time points from developing retina as well as four early stages of retinal organoid differentiation. We identified evolutionarily conserved patterns of gene expression during retinal progenitor maturation and specification of all seven major retinal cell types. Furthermore, we identified gene-expression differences between developing macula and periphery and between distinct populations of horizontal cells. We also identified species-specific patterns of gene expression during human and mouse retinal development. Finally, we identified an unexpected role for ATOH7 expression in regulation of photoreceptor specification during late retinogenesis. These results provide a roadmap to future studies of human retinal development and may help guide the design of cell-based therapies for treating retinal dystrophies.

Defining the ontogeny of the human adaptive immune system during embryogenesis has implications for understanding childhood diseases including leukaemias and autoimmune conditions. Using RAG1:GFP human pluripotent stem cell reporter lines, we examined human T-cell genesis from pluripotent-stem-cell-derived haematopoietic organoids. Under conditions favouring T-cell development, RAG1+ cells progressively upregulated a cohort of recognized T-cell-associated genes, arresting development at the CD4+CD8+ stage. Sort and re-culture experiments showed that early RAG1+ cells also possessed B-cell, myeloid and erythroid potential. Flow cytometry and single-cell-RNA-sequencing data showed that early RAG1+ cells co-expressed the endothelial/haematopoietic progenitor markers CD34, VECAD and CD90, whereas imaging studies identified RAG1+ cells within CD31+ endothelial structures that co-expressed SOX17+ or the endothelial marker CAV1. Collectively, these observations provide evidence for a wave of human T-cell development that originates directly from haemogenic endothelium via a RAG1+ intermediate with multilineage potential.

As single-cell RNA sequencing (scRNA-seq) data becomes widely available, cell-to-cell variability in gene expression, or single-cell expression variability (scEV), has been increasingly appreciated. However, it remains unclear whether this variability is functionally important and, if so, what are its implications for multi-cellular organisms. Here, we analyzed multiple scRNA-seq data sets from lymphoblastoid cell lines (LCLs), lung airway epithelial cells (LAECs), and dermal fibroblasts (DFs) and, for each cell type, selected a group of homogenous cells with highly similar expression profiles. We estimated the scEV levels for genes after correcting the mean-variance dependency in that data and identified 465, 466, and 364 highly variable genes (HVGs) in LCLs, LAECs, and DFs, respectively. Functions of these HVGs were found to be enriched with those biological processes precisely relevant to the corresponding cell type's function, from which the scRNA-seq data used to identify HVGs were generated-e.g., cytokine signaling pathways were enriched in HVGs identified in LCLs, collagen formation in LAECs, and keratinization in DFs. We repeated the same analysis with scRNA-seq data from induced pluripotent stem cells (iPSCs) and identified only 79 HVGs with no statistically significant enriched functions; the overall scEV in iPSCs was of negligible magnitude. Our results support the "variation is function" hypothesis, arguing that scEV is required for cell type-specific, higher-level system function. Thus, quantifying and characterizing scEV are of importance for our understating of normal and pathological cellular processes.

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.

The liver plays a critical role in both immune defense and tolerance in the body. The liver-resident immune cells (LrICs) determine the immune properties, but the unique composition and heterogeneity of these cells are incompletely understood. Here, we dissect the diversity of LrICs by a comprehensive transcriptomic profiling using the unbiased single-cell RNA-sequencing (scRNA-seq). A total of 70, 706 of CD45+ immune cells from the paired liver perfusion, spleen and peripheral blood as references were profiled. We identified more than 30 discrete cell populations comprising 13 of T and NK cell, 7 of B cell, 4 of plasma cell, and 8 of myeloid cell subsets in human liver and donor-paired spleen and blood, and characterized their tissue distribution, gene expression and functional modules. Especially, four of CXCR6+ T and NK cell subsets were found to be present preferentially in the liver, where they manifested heterogeneity, distinct function and prominent homeostatic proliferation. We propose a universal category system of T and NK cells based on distinct chemokine receptors, confirmed subsequently by phenotype, transcriptional factors and functionality. We also identified adaptive changes by the spleen and liver-derived monocyte and macrophage populations. Finally, we give a global glimpse on B cell and plasma cell subsets in human spleen and liver. We, therefore, reveal the heterogeneity and functional diversity of LrICs in human. This study presents comprehensively the landscape of LrICs and will enable further study on their roles in various human diseases.

Intestinal-type gastric cancer is preceded by premalignant lesions, including chronic atrophic gastritis and intestinal metaplasia. In this study, we constructed a single-cell atlas for 32,332 high-quality cells from gastric antral mucosa biopsies of patients spanning a cascade of gastric premalignant lesions and early gastric cancer (EGC) using single-cell RNA sequencing. We then constructed a single-cell network underlying cellular and molecular characteristics of gastric epithelial cells across different lesions. We found that gland mucous cells tended to acquire an intestinal-like stem cell phenotype during metaplasia, and we identified OR51E1 as a marker for unique endocrine cells in the early-malignant lesion. We also found that HES6 might mark the pre-goblet cell cluster, potentially aiding identification of metaplasia at the early stage. Finally, we identified a panel of EGC-specific signatures, with clinical implications for the precise diagnosis of EGC. Our study offers unparalleled insights into the human gastric cellulome in premalignant and early-malignant lesions.

As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.

Naive pluripotency exists in epiblast cells of mouse pre-implantation embryos. However, whether the naive pluripotency is transient or nonexistent in primate embryos remains unclear. Using RNA-seq in single blastomeres from 16-cell embryos through to hatched blastocysts of rhesus monkey, we constructed the lineage segregation roadmap in which the specification of trophectoderm, epiblast, and primitive endoderm is initiated simultaneously at the early blastocyst stage. Importantly, we uncovered the existence of distinct pluripotent states in monkey pre-implantation embryos. At the early- and middle-blastocyst stages, the epiblast cells have the transcriptome features of naive pluripotency, whereas they display a continuum of primed pluripotency characteristics at the late and hatched blastocyst stages. Moreover, we identified potential regulators that might play roles in the transition from naive to primed pluripotency. Thus, our study suggests the transient existence of naive pluripotency in primates and proposes an ideal time window for derivation of primate embryonic stem cells with naive pluripotency.

Atopic dermatitis (AD) is an allergic skin disease that causes significant morbidity and affects multiple species. AD is highly prevalent in companion dogs, and the clinical management of the disease remains challenging. An improved understanding of the immunologic and genetic pathways that lead to disease could inform the development of novel treatments. In allergic humans and mouse models of AD, the disease is associated with Th2 and group 2 innate lymphoid cell (ILC2) activation that drives type 2 inflammation. Type 2 inflammation also appears to be associated with AD in dogs, but gaps remain in our understanding of how key type 2-associated cell types such as canine Th2 cells and ILC2s contribute to the pathogenesis of canine AD. Here, we describe previously uncharacterized canine ILC2-like cells and Th2 cells ex vivo that produced type 2 cytokines and expressed the transcription factor Gata3. Increased circulating Th2 cells were associated with chronic canine AD. Single-cell RNA sequencing revealed a unique gene expression signature in T cells in dogs with AD. These findings underline the importance of pro-allergic Th2 cells in orchestrating AD and provide new methods and pathways that can inform the development of improved therapies.

Spermatogenesis is an important biological process in male reproduction. The interaction between male germ cells and somatic cells during spermatogenesis, is necessary for male reproductive activities. This cellular heterogeneity has made it difficult to profile distinct cell types at different stages of development. Here, we present the first comprehensive, unbiased single-cell transcriptomic study of sheep spermatogenesis using 10× genomics single cell sequencing (scRNA-seq). We collected scRNA-seq data from 11 772 cells from the adult sheep testis and identified all known germ cells (including early primary spermatocytes, late primary spermatocytes, round spermatids, elongated spermatids, and sperm), and somatic cells (Sertoli cells and Leydig cells), as well as one somatic cell that unexpectedly contained leukocytes. The functional enrichment analysis indicated that several pathways of cell cycle, gamete generation, protein processing, and mRNA surveillance pathways were significantly enriched in testicular germ cell types, and ribosome pathway was significantly enriched in testicular somatic cell types. Further analysis identified several stage-specific marker genes of sheep germ cells, such as EZH2, SOX18, SCP2, PCNA, and PRKCD. Our research explored for the first time of the changes in the transcription level of various cell types during the process of sheep spermatogenesis, providing new insights for sheep spermatogenesis and spermatogenic cell development.

Although the genetic triggers for gonadal sex differentiation vary across species, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using single-cell sequencing in the embryonic chicken gonad during sexual differentiation. The data show that chicken embryonic-supporting cells do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, they derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes and derivation of embryonic steroidogenic cells from a differentiated supporting-cell lineage. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.

During vertebrate development, progenitor cells give rise to tissues and organs through a complex choreography that commences at gastrulation. A hallmark event of gastrulation is the formation of the primitive streak, a linear assembly of cells along the anterior-posterior (AP) axis of the developing organism. To examine the primitive streak at a single-cell resolution, we measured the transcriptomes of individual chick cells from the streak or the surrounding tissue (the rest of the area pellucida) in Hamburger-Hamilton stage 4 embryos. The single-cell transcriptomes were then ordered by the statistical method Wave-Crest to deduce both the relative position along the AP axis and the prospective lineage of single cells. The ordered transcriptomes reveal intricate patterns of gene expression along the primitive streak.

BACKGROUND:Through precise implementation of distinct cell type specification programs, differentially regulated in both space and time, complex patterns emerge during organogenesis. Thanks to its easy experimental accessibility, the developing chicken limb has long served as a paradigm to study vertebrate pattern formation. Through decades' worth of research, we now have a firm grasp on the molecular mechanisms driving limb formation at the tissue-level. However, to elucidate the dynamic interplay between transcriptional cell type specification programs and pattern formation at its relevant cellular scale, we lack appropriately resolved molecular data at the genome-wide level. Here, making use of droplet-based single-cell RNA-sequencing, we catalogue the developmental emergence of distinct tissue types and their transcriptome dynamics in the distal chicken limb, the so-called autopod, at cellular resolution. RESULTS:Using single-cell RNA-sequencing technology, we sequenced a total of 17,628 cells coming from three key developmental stages of chicken autopod patterning. Overall, we identified 23 cell populations with distinct transcriptional profiles. Amongst them were small, albeit essential populations like the apical ectodermal ridge, demonstrating the ability to detect even rare cell types. Moreover, we uncovered the existence of molecularly distinct sub-populations within previously defined compartments of the developing limb, some of which have important signaling functions during autopod pattern formation. Finally, we inferred gene co-expression modules that coincide with distinct tissue types across developmental time, and used them to track patterning-relevant cell populations of the forming digits. CONCLUSIONS:We provide a comprehensive functional genomics resource to study the molecular effectors of chicken limb patterning at cellular resolution. Our single-cell transcriptomic atlas captures all major cell populations of the developing autopod, and highlights the transcriptional complexity in many of its components. Finally, integrating our data-set with other single-cell transcriptomics resources will enable researchers to assess molecular similarities in orthologous cell types across the major tetrapod clades, and provide an extensive candidate gene list to functionally test cell-type-specific drivers of limb morphological diversification.

Spinal cord pMN progenitors sequentially produce motor neurons and oligodendrocyte precursor cells (OPCs). Some OPCs differentiate rapidly as myelinating oligodendrocytes, whereas others remain into adulthood. How pMN progenitors switch from producing motor neurons to OPCs with distinct fates is poorly understood. pMN progenitors express prdm8, which encodes a transcriptional repressor, during motor neuron and OPC formation. To determine whether prdm8 controls pMN cell fate specification, we used zebrafish as a model system to investigate prdm8 function. Our analysis revealed that prdm8 mutant embryos have fewer motor neurons resulting from a premature switch from motor neuron to OPC production. Additionally, prdm8 mutant larvae have excess oligodendrocytes and a concomitant deficit of OPCs. Notably, pMN cells of mutant embryos have elevated Shh signaling, coincident with the motor neuron to OPC switch. Inhibition of Shh signaling restored the number of motor neurons to normal but did not rescue the proportion of oligodendrocytes. These data suggest that Prdm8 regulates the motor neuron-OPC switch by controlling the level of Shh activity in pMN progenitors, and also regulates the allocation of oligodendrocyte lineage cell fates.This article has an associated 'The people behind the papers' interview.

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

Time series of single-cell transcriptome measurements can reveal dynamic features of cell differentiation pathways. From measurements of whole frog embryos spanning zygotic genome activation through early organogenesis, we derived a detailed catalog of cell states in vertebrate development and a map of differentiation across all lineages over time. The inferred map recapitulates most if not all developmental relationships and associates new regulators and marker genes with each cell state. We find that many embryonic cell states appear earlier than previously appreciated. We also assess conflicting models of neural crest development. Incorporating a matched time series of zebrafish development from a companion paper, we reveal conserved and divergent features of vertebrate early developmental gene expression programs.

Caenorhabditis elegans is an animal with few cells but a wide diversity of cell types. In this study, we characterize the molecular basis for their specification by profiling the transcriptomes of 86,024 single embryonic cells. We identify 502 terminal and preterminal cell types, mapping most single-cell transcriptomes to their exact position in C. elegans' invariant lineage. Using these annotations, we find that (i) the correlation between a cell's lineage and its transcriptome increases from middle to late gastrulation, then falls substantially as cells in the nervous system and pharynx adopt their terminal fates; (ii) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and (iii) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state.

Schistosomiasis is a neglected tropical disease that infects 240 million people. With no vaccines and only one drug available, new therapeutic targets are needed. The causative agents, schistosomes, are intravascular flatworm parasites that feed on blood and lay eggs, resulting in pathology. The function of the parasite's various tissues in successful parasitism are poorly understood, hindering identification of therapeutic targets. Using single-cell RNA sequencing (RNA-seq), we characterize 43,642 cells from the adult schistosome and identify 68 distinct cell populations, including specialized stem cells that maintain the parasite's blood-digesting gut. These stem cells express the gene hnf4, which is required for gut maintenance, blood feeding, and pathology in vivo. Together, these data provide molecular insights into the organ systems of this important pathogen and identify potential therapeutic targets.

The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocytic developmental cycle (IDC). Unbiased analyses of single-cell data grouped the cells into eight transcriptional states during IDC. Interestingly, we uncovered a gene signature from the single iRBC analyses that can successfully discriminate between developing asexual and sexual stage parasites at cellular resolution, and we verify five, previously undefined, gametocyte stage specific genes. Moreover, we show the capacity of detecting expressed genes from the variable gene families in single parasites, despite the sparse nature of data. In total, the single parasite transcriptomics holds promise for molecular dissection of rare parasite phenotypes throughout the malaria lifecycle.

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics' droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.

Dedifferentiation is a critical response to tissue damage, yet is not well understood, even at a basic phenomenological level. Developing Dictyostelium cells undergo highly efficient dedifferentiation, completed by most cells within 24 hr. We use this rapid response to investigate the control features of dedifferentiation, combining single cell imaging with high temporal resolution transcriptomics. Gene expression during dedifferentiation was predominantly a simple reversal of developmental changes, with expression changes not following this pattern primarily associated with ribosome biogenesis. Mutation of genes induced early in dedifferentiation did not strongly perturb the reversal of development. This apparent robustness may arise from adaptability of cells: the relative temporal ordering of cell and molecular events was not absolute, suggesting cell programmes reach the same end using different mechanisms. In addition, although cells start from different fates, they rapidly converged on a single expression trajectory. These regulatory features may contribute to dedifferentiation responses during regeneration.

Rice, an important food resource, is highly sensitive to salt stress, which is directly related to food security. Although many studies have identified physiological mechanisms that confer tolerance to the osmotic effects of salinity, the link between rice genotype and salt tolerance is not very clear yet. Association of gene co-expression network and rice phenotypic data under stress has penitential to identify stress-responsive genes, but there is no standard method to associate stress phenotype with gene co-expression network. A novel method for integration of gene co-expression network and stress phenotype data was developed to conduct a system analysis to link genotype to phenotype. We applied a LASSO-based method to the gene co-expression network of rice with salt stress to discover key genes and their interactions for salt tolerance-related phenotypes. Submodules in gene modules identified from the co-expression network were selected by the LASSO regression, which establishes a linear relationship between gene expression profiles and physiological responses, that is, sodium/potassium condenses under salt stress. Genes in these submodules have functions related to ion transport, osmotic adjustment, and oxidative tolerance. We argued that these genes in submodules are biologically meaningful and useful for studies on rice salt tolerance. This method can be applied to other studies to efficiently and reliably integrate co-expression network and phenotypic data.

Background

Cultivated rice consists of two subspecies, Indica and Japonica, that exhibit well-characterized differences at the morphological and genetic levels. However, the differences between these subspecies at the transcriptome level remains largely unexamined. Here, we provide a comprehensive characterization of transcriptome divergence and cis-regulatory variation within rice using transcriptome data from 91 accessions from a rice diversity panel (RDP1).

Results

The transcriptomes of the two subspecies of rice are highly divergent. Japonica have significantly lower expression and genetic diversity relative to Indica, which is likely a consequence of a population bottleneck during Japonica domestication. We leveraged high-density genotypic data and transcript levels to identify cis-regulatory variants that may explain the genetic divergence between the subspecies. We identified significantly more eQTL that were specific to the Indica subspecies compared to Japonica, suggesting that the observed differences in expression and genetic variability also extends to cis-regulatory variation.

Conclusions

Using RNA sequencing data for 91diverse rice accessions and high-density genotypic data, we show that the two species are highly divergent with respect to gene expression levels, as well as the genetic regulation of expression. The data generated by this study provide, to date, the largest collection of genome-wide transcriptional levels for rice, and provides a community resource to accelerate functional genomic studies in rice.

Detailed molecular profiling of Oryza sativa (rice) was carried out to uncover the features that are essential for germination and early seedling growth under anoxic conditions. Temporal analysis of the transcriptome and methylome from germination to young seedlings under aerobic and anaerobic conditions revealed 82% similarity in the transcriptome and no differences in the epigenome up to 24 h. Following germination, significant changes in the transcriptome and DNA methylation were observed between 4-day aerobically and anaerobically grown coleoptiles. A link between the epigenomic state and cell division versus cell elongation is suggested, as no differences in DNA methylation were observed between 24-h aerobically and anaerobically germinating embryos, when there is little cell division. After that, epigenetic changes appear to correlate with differences between cell elongation (anaerobic conditions) versus cell division (aerobic conditions) in the coleoptiles. Re-oxygenation of 3-day anaerobically grown seedlings resulted in rapid transcriptomic changes in DNA methylation in these coleoptiles. Unlike the transcriptome, changes in DNA methylation upon re-oxygenation did not reflect those seen in aerobic coleoptiles, but instead, reverted to a pattern similar to dry seeds. Reversion to the 'dry seed' state of DNA methylation upon re-oxygenation may act to 'reset the clock' for the rapid molecular changes and cell division that result upon re-oxygenation.

Rice bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) implies substantial yield loss to rice. In times of climate change, increasing temperatures are observed and further acceleration is expected worldwide. Increasing temperature often turns into inhibition of host plant defense to pathogens. Recently, a reduced resistance in rice IRBB4 carrying Xa4, but an increase in resistance in IRBB7 carrying Xa7 resistance by increasing temperature has been reported. Influence of high temperature on both R genes (Xa4+Xa7) combined in IRBB67 was analyzed under growth chamber conditions and transcriptomic analysis performed. The pyramided line IRBB67 showed no differences in lesion length between both temperature regimes, demonstrating that non-effectiveness of Xa4 at high temperature did not affect IRBB67 resistance. Moreover, Xa4 complements Xa7 resistance with no Xoo spread in planta beyond the symptomatic area under both temperature regimes in IRBB67. Time course transcriptomic analysis revealed that temperature enhanced IRBB67 resistance to combined heat and Xoo. Our findings highlight altered cellular compartments and point at a role of the cell wall involved in Xoo resistance and heat stress tolerance in both susceptible (IR24) and the resistant (IRBB67) NILs. Interestingly, up-regulation of trehalose-6-phosphatase gene and low affinity cation transporter in IRBB67 suggest that IRBB67 maintained a certain homeostasis under high temperature which may have enhanced its resistance. The interplay of both heat stress and Xoo responses as determined by up-regulated and down-regulated genes demonstrates how resistant plants cope with combined biotic and abiotic stresses.

Background

Histone lysine acylations by short-chain fatty acids are distinct from the widely studied histone lysine acetylation in chromatin, although both modifications are regulated by primary metabolism in mammalian cells. It remains unknown whether and how histone acylation and acetylation interact to regulate gene expression in plants that have distinct regulatory pathways of primary metabolism.

Results

We identify 4 lysine butyrylation (Kbu) sites (H3K14, H4K12, H2BK42, and H2BK134) and 45 crotonylation (Kcr) sites on rice histones by mass spectrometry. Comparative analysis of genome-wide Kbu and Kcr and H3K9ac in combination with RNA sequencing reveals 25,306 genes marked by Kbu and Kcr in rice and more than 95% of H3K9ac-marked genes are marked by both. Kbu and Kcr are enriched at the 5' region of expressed genes. In rice under starvation and submergence, Kbu and Kcr appear to be less dynamic and display changes in different sets of genes compared to H3K9ac. Furthermore, Kbu seems to preferentially poise gene activation by external stresses, rather than internal circadian rhythm which has been shown to be tightly associated with H3K9ac. In addition, we show that rice sirtuin histone deacetylase (SRT2) is involved in the removal of Kcr.

Conclusion

Kbu, Kcr, and H3K9ac redundantly mark a large number of active genes but display different responses to external and internal signals. Thus, the proportion of rice histone lysine acetylation and acylation is dynamically regulated by environmental and metabolic cues, which may represent an epigenetic mechanism to fine-tune gene expression for plant adaptation.

Recently, long non-coding RNAs (lncRNAs) have been demonstrated to be involved in many biological processes of plants; however, a systematic study on transcriptional and, in particular, post-transcriptional regulation of stress-responsive lncRNAs in Oryza sativa (rice) is lacking. We sequenced three types of RNA libraries (poly(A)+, poly(A)- and nuclear RNAs) under four abiotic stresses (cold, heat, drought and salt). Based on an integrative bioinformatics approach and ~200 high-throughput data sets, ~170 of which have been published, we revealed over 7000 lncRNAs, nearly half of which were identified for the first time. Notably, we found that the majority of the ~500 poly(A) lncRNAs that were differentially expressed under stress were significantly downregulated, but approximately 25% were found to have upregulated non-poly(A) forms. Moreover, hundreds of lncRNAs with downregulated polyadenylation (DPA) tend to be highly conserved, show significant nuclear retention and are co-expressed with protein-coding genes that function under stress. Remarkably, these DPA lncRNAs are significantly enriched in quantitative trait loci (QTLs) for stress tolerance or development, suggesting their potential important roles in rice growth under various stresses. In particular, we observed substantially accumulated DPA lncRNAs in plants exposed to drought and salt, which is consistent with the severe reduction of RNA 3'-end processing factors under these conditions. Taken together, the results of this study reveal that polyadenylation and subcellular localization of many rice lncRNAs are likely to be regulated at the post-transcriptional level. Our findings strongly suggest that many upregulated/downregulated lncRNAs previously identified by traditional RNA-seq analyses need to be carefully reviewed to assess the influence of post-transcriptional modification.

The circadian clock enables organisms to rapidly adapt to the ever-changing environmental conditions that are caused by daily light/dark cycles. Circadian clock genes universally affect key agricultural traits, particularly flowering time. Here, we show that OsPRR37, a circadian clock gene, delays rice flowering time in an expression level-dependent manner. Using high-throughput mRNA sequencing on an OsPRR37 overexpressing transgenic line (OsPRR37-OE5) and the recipient parent Guangluai4 that contains the loss-of-function Osprr37, we identify 14,992 genes that display diurnal rhythms, which account for 52.9% of the transcriptome. Overexpressing OsPRR37 weakens the transcriptomic rhythms and alters the phases of rhythmic genes. In total, 3,210 differentially expressed genes (DEGs) are identified, among which 1,863 rhythmic DEGs show a correlation between the change of absolute amplitudes and the mean expression levels. We further reveal that OsPRR37 functions as a transcriptional repressor to repress the expression levels and amplitudes of day-phased clock genes. More importantly, OsPRR37 confers expanded regulation on the evening-phased rhythmic DEGs by repressing the morning-phased rhythmic DEGs. Further study shows that OsPRR37 expands its regulation on flowering pathways by repressing Ehd1. Thus, our results demonstrate an expanded regulation mechanism of the circadian clock on the diurnal rhythms of the transcriptome.

Flag leaves (FL) and second leaves (SL) in rice show differential aging patterns during monocarpic senescence. Coordination of aging programs between FL and SL is important for grain yield and quality. However, the molecular bases for differential aging programs between FL and SL have not been systematically explored in rice. Here, we performed mRNA-sequencing of FL and SL at six time points during grain-filling and identified four molecular bases for differential aging programs between FL and SL: phenylpropanoid biosynthesis, photosynthesis, amino acid (AA) transport, and hormone response. Of them, photosynthesis (carbon assimilation) and AA transport (nitrogen remobilization) predominantly occurred in FL and SL, respectively, during grain-filling. Unlike other molecular bases, AA transport showed consistent differential expression patterns between FL and SL in independent samples. Moreover, long-distance AA transporters showed invariant differential expression patterns between FL and SL after panicle removal, which was consistent to invariant differential nitrogen contents between FL and SL after panicle removal. Therefore, our results suggest that the supplies of carbon and nitrogen to seeds is functionally segregated between FL and SL and that long-distance AA transport is an invariant core program for high nitrogen remobilization in SL.

The rice (Oryza sativa L.) ethylene-responsive transcription factor gene SUB1A-1 confers tolerance to prolonged, complete submergence by limiting underwater elongation growth. Upon desubmergence, SUB1A-1 genotypes rapidly recover photosynthetic function and recommence development towards flowering. The underpinnings of the transition from stress amelioration to the return to homeostasis are not well known. Here, transcriptomic and metabolomic analyses were conducted to identify mechanisms by which SUB1A improves physiological function over the 24 hr following a sublethal submergence event. Evaluation of near-isogenic genotypes after submergence and over a day of reaeration demonstrated that SUB1A transiently constrains the remodelling of cellular activities associated with growth. SUB1A influenced the abundance of ca. 1,400 transcripts and had a continued impact on metabolite content, particularly free amino acids, glucose, and sucrose, throughout the recovery period. SUB1A promoted recovery of metabolic homeostasis but had limited influence on mRNAs associated with growth processes and photosynthesis. The involvement of low energy sensing during submergence and recovery was supported by dynamics in trehalose-6-phosphate and mRNAs encoding key enzymes and signalling proteins, which were modulated by SUB1A. This study provides new evidence of convergent signalling pathways critical to the rapidly reversible management of carbon and nitrogen metabolism in submergence resilient rice.

Key message

Our study systematically explored potential genes and molecular pathways as candidates for differences in seed weight resulting from soybean domestication. In addition, potential contributions of lncRNAs to seed weight were also investigated. Soybeans have a long history of domestication in China, and there are several significant phenotypic differences between cultivated and wild soybeans, for example, seeds of cultivars are generally larger and heavier than those from wild accessions. We analyzed seed transcriptomes from thirteen soybean samples, including six landraces and seven wild accessions using strand-specific RNA sequencing. Differentially expressed genes related to seed weight were identified, and some of their homologs were associated with seed development in Arabidopsis. We also identified 1251 long intergenic noncoding RNAs (lincRNAs), 243 intronic RNAs and 81 antisense lncRNAs de novo from these soybean transcriptomes. We then profiled the expression patterns of lncRNAs in cultivated and wild soybean seeds, and found that transcript levels of a number of lncRNAs were sample-specific. Moreover, gene transcript and lincRNA co-expression network analysis showed that some soybean lincRNAs might have functional roles as they were hubs of co-expression modules. In conclusion, this study systematically explored potential genes and molecular pathways as candidates for differences in seed weight resulting from soybean domestication, and will provide a useful future resource for molecular breeding of soybeans.

Background

Variation in seed oil composition and content among soybean varieties is largely attributed to differences in transcript sequences and/or transcript accumulation of oil production related genes in seeds. Discovery and analysis of sequence and expression variations in these genes will accelerate soybean oil quality improvement.

Results

In an effort to identify these variations, we sequenced the transcriptomes of soybean seeds from nine lines varying in oil composition and/or total oil content. Our results showed that 69,338 distinct transcripts from 32,885 annotated genes were expressed in seeds. A total of 8,037 transcript expression polymorphisms and 50,485 transcript sequence polymorphisms (48,792 SNPs and 1,693 small Indels) were identified among the lines. Effects of the transcript polymorphisms on their encoded protein sequences and functions were predicted. The studies also provided independent evidence that the lack of FAD2-1A gene activity and a non-synonymous SNP in the coding sequence of FAB2C caused elevated oleic acid and stearic acid levels in soybean lines M23 and FAM94-41, respectively.

Conclusions

As a proof-of-concept, we developed an integrated RNA-seq and bioinformatics approach to identify and functionally annotate transcript polymorphisms, and demonstrated its high effectiveness for discovery of genetic and transcript variations that result in altered oil quality traits. The collection of transcript polymorphisms coupled with their predicted functional effects will be a valuable asset for further discovery of genes, gene variants, and functional markers to improve soybean oil quality.

Background

Low phytic acid (lpa) crops are potentially eco-friendly alternative to conventional normal phytic acid (PA) crops, improving mineral bioavailability in monogastric animals as well as decreasing phosphate pollution. The lpa crops developed to date carry mutations that are directly or indirectly associated with PA biosynthesis and accumulation during seed development. These lpa crops typically exhibit altered carbohydrate profiles, increased free phosphate, and lower seedling emergence, the latter of which reduces overall crop yield, hence limiting their large-scale cultivation. Improving lpa crop yield requires an understanding of the downstream effects of the lpa genotype on seed development. Towards that end, we present a comprehensive comparison of gene-expression profiles between lpa and normal PA soybean lines (Glycine max) at five stages of seed development using RNA-Seq approaches. The lpa line used in this study carries single point mutations in a myo-inositol phosphate synthase gene along with two multidrug-resistance protein ABC transporter genes.

Results

RNA sequencing data of lpa and normal PA soybean lines from five seed-developmental stages (total of 30 libraries) were used for differential expression and functional enrichment analyses. A total of 4235 differentially expressed genes, including 512-transcription factor genes were identified. Eighteen biological processes such as apoptosis, glucan metabolism, cellular transport, photosynthesis and 9 transcription factor families including WRKY, CAMTA3 and SNF2 were enriched during seed development. Genes associated with apoptosis, glucan metabolism, and cellular transport showed enhanced expression in early stages of lpa seed development, while those associated with photosynthesis showed decreased expression in late developmental stages. The results suggest that lpa-causing mutations play a role in inducing and suppressing plant defense responses during early and late stages of seed development, respectively.

Conclusions

This study provides a global perspective of transcriptomal changes during soybean seed development in an lpa mutant. The mutants are characterized by earlier expression of genes associated with cell wall biosynthesis and a decrease in photosynthetic genes in late stages. The biological processes and transcription factors identified in this study are signatures of lpa-causing mutations.

During early seedling growth, the cotyledons transition from a storage tissue to become effectively the first leaf-like structures of the plant. In this programmed developmental process, they likely undergo a massive change in gene expression to redirect their metabolism and physiological processes. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks that set this transition in motion, we carried out high-throughput RNA sequencing of cotyledons from seven developmental stages of soybean seedlings. We identified 154 gene models with high expression exclusively in the early seedling stages. A significant number (about 25 %) of those genes with known annotations were involved in carbohydrate metabolism. A detailed examination of glyoxylate cycle genes revealed the upregulation of their expression in the early stages of development. A total of approximately 50 % of the highly expressed genes whose expression peaked in the mid-developmental stages encoded ribosomal family proteins. Our analysis also identified 219 gene models with high expression at late developmental stages. The majority of these genes are involved in photosynthesis, including photosystem I- and II-associated genes. Additionally, the advantage of RNA-Seq to detect genes expressed at low levels revealed approximately 460 transcription factors with notable expression in at least one stage of the developing soybean seedling. Relatively over-represented transcription factor genes encode AP2, zinc finger, NAC, WRKY, and MYB families. These transcription factor genes may lead to the transcriptional reprogramming during the transition of seedling cotyledons from storage tissue to metabolically active organs that serve as the first functional leaves of the plant.

To understand gene expression networks leading to functional properties and compositional traits of the soybean seed, we have undertaken a detailed examination of soybean seed development from a few days post-fertilization to the mature seed using Illumina high-throughput transcriptome sequencing (RNA-Seq). RNA was sequenced from seven different stages of seed development, yielding between 12 million and 78 million sequenced transcripts. These have been aligned to the 79,000 gene models predicted from the soybean genome recently sequenced by the Department of Energy Joint Genome Institute. Over one hundred gene models were identified with high expression exclusively in young seed stages, starting at just four days after fertilization. These were annotated as being related to many basic components and processes such as histones and proline-rich proteins. Genes encoding storage proteins such as glycinin and beta-conglycinin had their highest expression levels at the stages of largest fresh weight, confirming previous knowledge that these storage products are being rapidly accumulated before the seed begins the desiccation process. Other gene models showed high expression in the dry, mature seeds, perhaps indicating the preparation of pathways needed later, in the early stages of imbibition. Many highly-expressed gene models at the dry seed stage are, as expected, annotated as hydrophilic proteins associated with low water conditions, such as late embryogenesis abundant (LEA) proteins and dehydrins, which help preserve the cellular structures and nutrients within the seed during desiccation. More significantly, the power of RNA-Seq to detect genes expressed at low levels revealed hundreds of transcription factors with notable expression in at least one stage of seed development. Results from a second biological replicate demonstrate high reproducibility of these data revealing a comprehensive view of the transciptome of seed development in the cultivar Williams, the reference cultivar for the first soybean genome sequence.

Transcription factors control important gene networks, altering the expression of a wide variety of genes, including those of agronomic importance, despite often being expressed at low levels. Detecting transcription factor proteins is difficult, because current high-throughput methods may not be sensitive enough. One-dimensional, silicon-substrate photonic crystal (PC) arrays provide an alternative substrate for printing multiplexed protein microarrays that have greater sensitivity through an increased signal-to-noise ratio of the fluorescent signal compared with performing the same assay upon a traditional aminosilanized glass surface. As a model system to test proof of concept of the silicon-substrate PC arrays to directly detect rare proteins in crude plant extracts, we selected representatives of four different transcription factor families (zinc finger GATA, basic helix-loop-helix, BTF3/NAC [for basic transcription factor of the NAC family], and YABBY) that have increasing transcript levels during the stages of seedling cotyledon development. Antibodies to synthetic peptides representing the transcription factors were printed on both glass slides and silicon-substrate PC slides along with antibodies to abundant cotyledon proteins, seed lectin, and Kunitz trypsin inhibitor. The silicon-substrate PC arrays proved more sensitive than those performed on glass slides, detecting rare proteins that were below background on the glass slides. The zinc finger transcription factor was detected on the PC arrays in crude extracts of all stages of the seedling cotyledons, whereas YABBY seemed to be at the lower limit of their sensitivity. Interestingly, the basic helix-loop-helix and NAC proteins showed developmental profiles consistent with their transcript patterns, indicating proof of concept for detecting these low-abundance proteins in crude extracts.

Soybean is an economically important leguminous crop, and pod dehiscence of soybean could cause huge yield loss. In this study, we measured fruit-cracking forces and percentages of dehisced pods for ten soybean accessions, then separated them into two groups as shattering-sensitive (SS) and shattering-resistant (SR) soybeans. Pod transcriptomes from these two groups were analyzed, and 225 differentially expressed genes (DEGs) were identified between SS and SR soybeans. Some of these DEGs have been previously reported to be associated with pod dehiscence in soybean. The expression patterns of selected DEGs were validated by real-time quantitative reverse transcription PCR, which confirmed the expression changes found in RNA-seq analysis. We also de novo identified 246 soybean pod-long intergenic ncRNAs (lincRNAs), 401 intronic lncRNAs, and 23 antisense lncRNAs from these transcriptomes. Furthermore, genes and lincRNAs co-expression network analysis showed that there are distinct expression patterns between SS and SR soybeans in some co-expression modules. In conclusion, we systematically investigated potential genes and molecular pathways as candidates for differences in soybean pod dehiscence and will provide a useful resource for molecular breeding of soybeans.

Glycerol-3-phosphate (G3P) is a well-known mobile regulator of systemic acquired resistance (SAR), which provides broad spectrum systemic immunity in response to localized foliar pathogenic infections. We show that G3P-derived foliar immunity is also activated in response to genetically-regulated incompatible interactions with nitrogen-fixing bacteria. Using gene knock-down we show that G3P is essential for strain-specific exclusion of non-desirable root-nodulating bacteria and the associated foliar pathogen immunity in soybean. Grafting studies show that while recognition of rhizobium incompatibility is root driven, bacterial exclusion requires G3P biosynthesis in the shoot. Biochemical analyses support shoot-to-root transport of G3P during incompatible rhizobia interaction. We describe a root-shoot-root signaling mechanism which simultaneously enables the plant to exclude non-desirable nitrogen-fixing rhizobia in the root and pathogenic microbes in the shoot.

Background

Fusarium oxysporum is one of the most common fungal pathogens causing soybean root rot and seedling blight in U.S.A. In a recent study, significant variation in aggressiveness was observed among isolates of F. oxysporum collected from roots in Iowa, ranging from highly pathogenic to weakly or non-pathogenic isolates.

Results

We used RNA-seq analysis to investigate the molecular aspects of the interactions of a partially resistant soybean genotype with non-pathogenic/pathogenic isolates of F. oxysporum at 72 and 96 h post inoculation (hpi). Markedly different gene expression profiles were observed in response to the two isolates. A peak of highly differentially expressed genes (HDEGs) was triggered at 72 hpi in soybean roots and the number of HDEGs was about eight times higher in response to the pathogenic isolate compared to the non-pathogenic one (1,659 vs. 203 HDEGs, respectively). Furthermore, the magnitude of induction was much greater in response to the pathogenic isolate. This response included a stronger activation of defense-related genes, transcription factors, and genes involved in ethylene biosynthesis, secondary and sugar metabolism.

Conclusions

The obtained data provide an important insight into the transcriptional responses of soybean-F. oxysporum interactions and illustrate the more drastic changes in the host transcriptome in response to the pathogenic isolate. These results may be useful in the developing new methods of broadening resistance of soybean to F. oxysporum, including the over-expression of key soybean genes.

Soybean aphid (Aphis glycines; SBA) and soybean cyst nematode (Heterodera glycines; SCN) are two major pests of soybean (Glycine max) in the United States of America. This study aims to characterize three-way interactions among soybean, SBA, and SCN using both demographic and genetic datasets. SCN-resistant and SCN-susceptible soybean cultivars with a combination of soybean aphids (biotype 1) and SCN (HG type 0) in a randomized complete block design (RCBD) with six blocks were used to evaluate the three-way interactions in a greenhouse setup. Treatments receiving SCN were infested at planting with 2000 nematode eggs, and the treatments with soybean aphids were infested at second trifoliate growth stage (V2) with 15 soybean aphids. The whole roots were sampled from plants at 5 and 30 days post SBA infestation for RNA sequencing using Illumina Hiseq. 3000. The data comprises of 47 libraries that are useful for further analyses of important genes, which are involved in interaction effects of SBA and SCN on soybean.

Soybean aphid is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins, of which 396 were differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNA-seq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes upregulated in the Rag2 line were annotated as being involved in cell wall, secondary, and hormone metabolism as well as in stress, signaling, and transcriptional responses. Genes downregulated in the Rag2 line were annotated as being involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance.

Objectives

Soybean aphid (Aphis glycines Matsumura; SBA) is the most economically damaging insect of soybean (Glycine max) in the United States. One previous study demonstrated that avirulent (biotype 1) and virulent (biotype 2) biotypes could co-occur and interact on resistant (i.e., Rag1) and susceptible soybean resulting in induced susceptibility after 11 days of feeding. The main objective of this research was to employ RNA sequencing (RNA-seq) technique to compare the induced susceptibility effect of biotype 2 on susceptible and resistant soybean at day 1 and day 11 (i.e., both susceptible and resistant soybean were initially challenged by biotype 2 and the effect was monitored through biotype 1 populations).

Data description

We investigated susceptible and Rag1 transcriptome response to SBA feeding in soybean plants colonized by biotype 1 in the presence or absence of an inducer population (i.e., biotype 2). Ten RNA datasets are reported with 266,535,654 sequence reads (55.2 GB) obtained from pooled samples derived from the leaves collected at day 1 and day 11 post SBA infestation. A comprehensive understanding of these transcriptome data will enhance our understanding of interactions among soybean and two different biotypes of soybean aphids at the molecular level.

Low phosphate (Pi) availability is an important limiting factor affecting soybean production. However, the underlying molecular mechanisms responsible for low Pi stress response and tolerance remain largely unknown, especially for the early signaling events under low Pi stress. Here, a genome-wide transcriptomic analysis in soybean leaves treated with a short-term Pi-deprivation (24 h) was performed through high-throughput RNA sequencing (RNA-seq) technology. A total of 533 loci were found to be differentially expressed in response to Pi deprivation, including 36 mis-annotated loci and 32 novel loci. Among the differentially expressed genes (DEGs), 303 were induced and 230 were repressed by Pi deprivation. To validate the reliability of the RNA-seq data, 18 DEGs were randomly selected and analyzed by quantitative RT-PCR (reverse transcription polymerase chain reaction), which exhibited similar fold changes with RNA-seq. Enrichment analyses showed that 29 GO (Gene Ontology) terms and 8 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were significantly enriched in the up-regulated DEGs and 25 GO terms and 16 KEGG pathways were significantly enriched in the down-regulated DEGs. Some DEGs potentially involved in Pi sensing and signaling were up-regulated by short-term Pi deprivation, including five SPX-containing genes. Some DEGs possibly associated with water and nutrient uptake, hormonal and calcium signaling, protein phosphorylation and dephosphorylation and cell wall modification were affected at the early stage of Pi deprivation. The cis-elements of PHO (phosphatase) element, PHO-like element and P responsive element were present more frequently in promoter regions of up-regulated DEGs compared to that of randomly-selected genes in the soybean genome. Our transcriptomic data showed an intricate network containing transporters, transcription factors, kinases and phosphatases, hormone and calcium signaling components is involved in plant responses to early Pi deprivation.

Phosphorus (P) deficiency is a major limitation for legume crop production. Although overall adaptations of plant roots to P deficiency have been extensively studied, only fragmentary information is available in regard to root nodule responses to P deficiency. In this study, genome wide transcriptome analysis was conducted using RNA-seq analysis in soybean nodules grown under P-sufficient (500 μM KH₂PO₄) and P-deficient (25 μM KH₂PO₄) conditions to investigate molecular mechanisms underlying soybean (Glycine max) nodule adaptation to phosphate (Pi) starvation. Phosphorus deficiency significantly decreased soybean nodule growth and nitrogenase activity. Nodule Pi concentrations declined by 49% in response to P deficiency, but this was well below the 87% and 88% decreases observed in shoots and roots, respectively. Nodule transcript profiling revealed that a total of 2055 genes exhibited differential expression patterns between Pi sufficient and deficient conditions. A set of (differentially expressed genes) DEGs appeared to be involved in maintaining Pi homeostasis in soybean nodules, including eight Pi transporters (PTs), eight genes coding proteins containing the SYG1/PHO81/XPR1 domain (SPXs), and 16 purple acid phosphatases (PAPs). The results suggest that a complex transcriptional regulatory network participates in soybean nodule adaption to Pi starvation, most notable a Pi signaling pathway, are involved in maintaining Pi homeostasis in nodules.

Sorghum is a self-pollinated crop with multiple economic uses as cereal, forage, and biofuel feedstock. Hybrid breeding is a cornerstone for sorghum improvement strategies that currently relies on cytoplasmic male sterile lines. To engineer genic male sterility, it is imperative to examine the genetic components regulating anther/pollen development in sorghum. To this end, we have performed transcriptomic analysis from three temporal stages of developing anthers that correspond to meiotic, microspore and mature pollen stages. A total of 5286 genes were differentially regulated among the three anther stages with 890 of them exhibiting anther-preferential expression. Differentially expressed genes could be clubbed into seven distinct developmental trajectories using K-means clustering. Pathway mapping revealed that genes involved in cell cycle, DNA repair, regulation of transcription, brassinosteroid and auxin biosynthesis/signalling exhibit peak expression in meiotic anthers, while those regulating abiotic stress, carbohydrate metabolism, and transport were enriched in microspore stage. Conversely, genes associated with protein degradation, post-translational modifications, cell wall biosynthesis/modifications, abscisic acid, ethylene, cytokinin and jasmonic acid biosynthesis/signalling were highly expressed in mature pollen stage. High concurrence in transcriptional dynamics and cis-regulatory elements of differentially expressed genes in rice and sorghum confirmed conserved developmental pathways regulating anther development across species. Comprehensive literature survey in conjunction with orthology analysis and anther-preferential accumulation enabled shortlisting of 21 prospective candidates for in-depth characterization and engineering male fertility in sorghum.

Basal branching in grasses, or tillering, is an important trait determining both form and function of crops. Although similarities exist between eudicot and grass branching programs, one notable difference is that the tiller buds of grasses are covered by the subtending leaf, whereas eudicot buds are typically unconstrained. The current study shows that contact with the leaf sheath represses sorghum bud growth by providing a mechanical signal that cues the bud to refrain from rapid growth. Leaf removal resulted in massive reprogramming of the bud transcriptome that included signatures of epigenetic modifications and also implicated several hormones in the response. Bud abscisic acid transiently increased, then decreased following leaf removal relative to controls, and abscisic acid was necessary to repress bud growth in the presence of the leaf. Jasmonic acid (JA) levels and signalling increased in buds following leaf removal. Remarkably, application of JA to buds in situ promoted growth. The repression of bud growth by leaf contact shares characteristics of thigmomorphogenic responses in other systems, including the involvement of JA, though the JA effect is opposite. The repression of bud growth by leaf contact may represent a mechanism to time tillering to an appropriate developmental stage of the plant.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Background

Sorghum is an important cereal crop, which requires large quantities of nitrogen fertilizer for achieving commercial yields. Identification of the genes responsible for low-N tolerance in sorghum will facilitate understanding of the molecular mechanisms of low-N tolerance, and also facilitate the genetic improvement of sorghum through marker-assisted selection or gene transformation. In this study we compared the transcriptomes of root tissues from seven sorghum genotypes having differential response to low-N stress.

Results

Illumina RNA-sequencing detected several common differentially expressed genes (DEGs) between four low-N tolerant sorghum genotypes (San Chi San, China17, KS78 and high-NUE bulk) and three sensitive genotypes (CK60, BTx623 and low-NUE bulk). In sensitive genotypes, N-stress increased the abundance of DEG transcripts associated with stress responses including oxidative stress and stimuli were abundant. The tolerant genotypes adapt to N deficiency by producing greater root mass for efficient uptake of nutrients. In tolerant genotypes, higher abundance of transcripts related to high affinity nitrate transporters (NRT2.2, NRT2.3, NRT2.5, and NRT2.6) and lysine histidine transporter 1 (LHT1), may suggest an improved uptake efficiency of inorganic and organic forms of nitrogen. Higher abundance of SEC14 cytosolic factor family protein transcript in tolerant genotypes could lead to increased membrane stability and tolerance to N-stress.

Conclusions

Comparison of transcriptomes between N-stress tolerant and sensitive genotypes revealed several common DEG transcripts. Some of these DEGs were evaluated further by comparing the transcriptomes of genotypes grown under full N. The DEG transcripts showed higher expression in tolerant genotypes could be used for transgenic over-expression in sensitive genotypes of sorghum and related crops for increased tolerance to N-stress, which results in increased nitrogen use efficiency for sustainable agriculture.

The Reduced Height (Rht) genes formed the basis for the green revolution in wheat by decreasing plant height and increasing productive tillers. There are two current widely used Rht mutant alleles, Rht-B1b and Rht-D1b. Both reduce plant height by 20% and increase seed yield by 5-10%. They are also associated with decreased seed size and protein content. Here, we tested the degree to which Rht-B1b impacts flag leaf photosynthetic rates and carbon and nitrogen partitioning to the flag leaf and grain during grain fill under field conditions using near isogenic lines (NILs) that were either standard height (Rht-B1a) or semi-dwarf (Rht-B1b). The results demonstrate that at anthesis, Rht-B1b reduces flag leaf photosynthetic rate per unit area by 18% and chlorophyll A content by 23%. Rht-B1b significantly reduced grain protein beginning at 14 days post anthesis (DPA) with the greatest difference seen at 21 DPA (12%). Rht-B1b also significantly decreased individual seed weight beginning at 21 DPA and by 15.2% at 28 DPA. Global expression analysis using RNA extracted from developing leaves and stems demonstrated that genes associated with carbon and nitrogen metabolism are not substantially altered by Rht-B1b. From this study, we conclude that Rht-B1b reduces flag leaf photosynthetic rate at flowering while changes in grain composition begin shortly after anthesis.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Fusarium graminearum is a destructive wheat pathogen. No fully resistant cultivars are available. Knowledge concerning the molecular weapons of F. graminearum to achieve infection remains limited. Here, we report that deletion of the putative secondary metabolite biosynthesis gene cluster fg3_54 compromises the pathogen's ability to infect wheat through cell-to-cell penetration. Ectopic expression of fgm4, a pathway-specific bANK-like regulatory gene, activates the transcription of the fg3_54 cluster in vitro. We identify a linear, C- terminally reduced and D-amino acid residue-rich octapeptide, fusaoctaxin A, as the product of the two nonribosomal peptide synthetases encoded by fg3_54. Chemically-synthesized fusaoctaxin A restores cell-to-cell invasiveness in fg3_54-deleted F. graminearum, and enables colonization of wheat coleoptiles by two Fusarium strains that lack the fg3_54 homolog and are nonpathogenic to wheat. In conclusion, our results identify fusaoctaxin A as a virulence factor required for cell-to-cell invasion of wheat by F. graminearum.

Using rice (Oryza sativa) as a model crop species, we performed an in-depth temporal transcriptome analysis, covering the early and late stages of Pi deprivation as well as Pi recovery in roots and shoots, using next-generation sequencing. Analyses of 126 paired-end RNA sequencing libraries, spanning nine time points, provided a comprehensive overview of the dynamic responses of rice to Pi stress. Differentially expressed genes were grouped into eight sets based on their responses to Pi starvation and recovery, enabling the complex signaling pathways involved in Pi homeostasis to be untangled. A reference annotation-based transcript assembly was also generated, identifying 438 unannotated loci that were differentially expressed under Pi starvation. Several genes also showed induction of unannotated splice isoforms under Pi starvation. Among these, PHOSPHATE2 (PHO2), a key regulator of Pi homeostasis, displayed a Pi starvation-induced isoform, which was associated with increased translation activity. In addition, microRNA (miRNA) expression profiles after long-term Pi starvation in roots and shoots were assessed, identifying 20 miRNA families that were not previously associated with Pi starvation, such as miR6250. In this article, we present a comprehensive spatio-temporal transcriptome analysis of plant responses to Pi stress, revealing a large number of potential key regulators of Pi homeostasis in plants.

Background

Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes in mammals and plants. However, the systematic examination of lncRNAs in plants lags behind that in mammals. Recently, lncRNAs have been identified in Arabidopsis and wheat; however, no systematic screening of potential lncRNAs has been reported for the rice genome.

Results

In this study, we perform whole transcriptome strand-specific RNA sequencing (ssRNA-seq) of samples from rice anthers, pistils, and seeds 5 days after pollination and from shoots 14 days after germination. Using these data, together with 40 available rice RNA-seq datasets, we systematically analyze rice lncRNAs and definitively identify lncRNAs that are involved in the reproductive process. The results show that rice lncRNAs have some different characteristics compared to those of Arabidopsis and mammals and are expressed in a highly tissue-specific or stage-specific manner. We further verify the functions of a set of lncRNAs that are preferentially expressed in reproductive stages and identify several lncRNAs as competing endogenous RNAs (ceRNAs), which sequester miR160 or miR164 in a type of target mimicry. More importantly, one lncRNA, XLOC_057324, is demonstrated to play a role in panicle development and fertility. We also develop a source of rice lncRNA-associated insertional mutants.

Conclusions

Genome-wide screening and functional analysis enabled the identification of a set of lncRNAs that are involved in the sexual reproduction of rice. The results also provide a source of lncRNAs and associated insertional mutants in rice.

Plant growth is severely affected by toxic concentrations of the non-essential heavy metal cadmium (Cd). Comprehensive transcriptome analysis by RNA-Seq following cadmium exposure is required to further understand plant responses to Cd and facilitate future systems-based analyses of the underlying regulatory networks. In this study, rice plants were hydroponically treated with 50 µM Cd for 24 hours and ∼60,000 expressed transcripts, including transcripts that could not be characterized by microarray-based approaches, were evaluated. Upregulation of various ROS-scavenging enzymes, chelators and metal transporters demonstrated the appropriate expression profiles to Cd exposure. Gene Ontology enrichment analysis of the responsive transcripts indicated the upregulation of many drought stress-related genes under Cd exposure. Further investigation into the expression of drought stress marker genes such as DREB suggested that expression of genes in several drought stress signal pathways was activated under Cd exposure. Furthermore, qRT-PCR analyses of randomly selected Cd-responsive metal transporter transcripts under various metal ion stresses suggested that the expression of Cd-responsive transcripts might be easily affected by other ions. Our transcriptome analysis demonstrated a new transcriptional network linking Cd and drought stresses in rice. Considering our data and that Cd is a non-essential metal, the network underlying Cd stress responses and tolerance, which plants have developed to adapt to other stresses, could help to acclimate to Cd exposure. Our examination of this transcriptional network provides useful information for further studies of the molecular mechanisms of plant adaptation to Cd exposure and the improvement of tolerance in crop species.

Hybridization and polyploidization are prominent processes in plant evolution. Hybrids and allopolyploids typically exhibit radically altered gene expression patterns relative to their parents, a phenomenon termed "transcriptomic shock." To distinguish the effects of hybridization from polyploidization on coregulation of divergent alleles, we analyzed expression of parental copies (homoeologs) of 11,608 genes using RNA-seq-based transcriptome profiling in reciprocal hybrids and tetraploids constructed from subspecies japonica and indica of Asian rice (Oryza sativa L.). The diploid hybrids and their derived allopolyploids differ dramatically in morphology, despite having the same suite of genes and genic proportions. Allelic and homoeolog-specific transcripts were unequivocally diagnosed in the hybrids and tetraploids based on parent-specific SNPs. Compared with the in silico hybrid (parental mix), the range of progenitor expression divergence was significantly reduced in both reciprocally generated F1 hybrids, presumably due to the ameliorating effects of a common trans environment on divergent cis-factors. In contrast, parental expression differences were greatly elaborated at the polyploid level, which we propose is a consequence of stoichiometric disruptions associated with the numerous chromosomal packaging and volumetric changes accompanying nascent polyploidy. We speculate that the emergent property of "whole genome doubling" has repercussions that reverberate throughout the transcriptome and downstream, ultimately generating altered phenotypes. This perspective may yield insight into the nature of adaptation and the origin of evolutionary novelty accompanying polyploidy.

Cytosine methylation at CG sites ((m)CG) plays critical roles in development, epigenetic inheritance, and genome stability in mammals and plants. In the dicot model plant Arabidopsis thaliana, methyltransferase 1 (MET1), a principal CG methylase, functions to maintain (m)CG during DNA replication, with its null mutation resulting in global hypomethylation and pleiotropic developmental defects. Null mutation of a critical CG methylase has not been characterized at a whole-genome level in other higher eukaryotes, leaving the generality of the Arabidopsis findings largely speculative. Rice is a model plant of monocots, to which many of our important crops belong. Here we have characterized a null mutant of OsMet1-2, the major CG methylase in rice. We found that seeds homozygous for OsMet1-2 gene mutation (OsMET1-2(-/-)), which directly segregated from normal heterozygote plants (OsMET1-2(+/-)), were seriously maldeveloped, and all germinated seedlings underwent swift necrotic death. Compared with wild type, genome-wide loss of (m)CG occurred in the mutant methylome, which was accompanied by a plethora of quantitative molecular phenotypes including dysregulated expression of diverse protein-coding genes, activation and repression of transposable elements, and altered small RNA profiles. Our results have revealed conservation but also distinct functional differences in CG methylases between rice and Arabidopsis.

Small RNAs that interact with Argonaute (AGO) proteins play central roles in RNA-mediated silencing. MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice AGO, has specific functions in the development of pre-meiotic germ cells and the progression of meiosis. Here, we show that MEL1, which is located mostly in the cytoplasm of germ cells, associates preferentially with 21-nucleotide phased small interfering RNAs (phasiRNAs) that bear a 5'-terminal cytosine. Most phasiRNAs are derived from 1171 intergenic clusters distributed on all rice chromosomes. From these clusters, over 700 large intergenic, non-coding RNAs (lincRNAs) that contain the consensus sequence complementary to miR2118 are transcribed specifically in inflorescences, and cleaved within the miR2118 site. Cleaved lincRNAs are processed via DICER-LIKE4 (DCL4) protein, resulting in production of phasiRNAs. This study provides the evidence that the miR2118-dependent and the DCL4-dependent pathways are both required for biogenesis of 21-nt phasiRNAs associated with germline-specific MEL1 AGO in rice, and over 700 lincRNAs are key factors for induction of this biogenesis during reproductive-specific stages.

Gene duplication occurs by either DNA- or RNA-based processes; the latter duplicates single genes via retroposition of messenger RNA. The expression of a retroposed gene copy (retrocopy) is expected to be uncorrelated with its source gene because upstream promoter regions are usually not part of the retroposition process. In contrast, DNA-based duplication often encompasses both the coding and the intergenic (promoter) regions; hence, expression is often correlated, at least initially, between DNA-based duplicates. In this study, we identified 150 retrocopies in rice (Oryza sativa L. ssp japonica), most of which represent ancient retroposition events. We measured their expression from high-throughput RNA sequencing (RNAseq) data generated from seven tissues. At least 66% of the retrocopies were expressed but at lower levels than their source genes. However, the tissue specificity of retrogenes was similar to their source genes, and expression between retrocopies and source genes was correlated across tissues. The level of correlation was similar between RNA- and DNA-based duplicates, and they decreased over time at statistically indistinguishable rates. We extended these observations to previously identified retrocopies in Arabidopsis thaliana, suggesting they may be general features of the process of retention of plant retrogenes.

Rice has developed several morphological and physiological strategies to adapt to phosphate starvation in the soil. In order to elucidate the molecular basis of response to phosphate starvation, we performed mRNA sequencing of 4 rice cultivars with variation in growth response to Pi starvation as indicated by the shoot/root dry weight ratio. Approximately 254 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence and an average of about 5,000 transcripts from each cultivar were found to be responsive under phosphate starvation. Comparative analysis of the RNA-Seq profiles of the 4 cultivars revealed similarities as well as distinct differences in expression of these responsive transcripts. We elucidated a set of core responsive transcripts including annotated and unannotated transcripts commonly expressed in the 4 cultivars but with different levels of expression. De novo assembly of unmapped reads to the Nipponbare genome generated a set of sequence contigs representing potential new transcripts that may be involved in tolerance to phosphate starvation. This study can be used for identification of genes and gene networks associated with environmental stress and the development of novel strategies for improving tolerance to phosphate starvation in rice and other cereal crops.

• Arsenic (As) contamination of rice (Oryza sativa) is a worldwide concern and elucidating the molecular mechanisms of As accumulation in rice may provide promising solutions to the problem. Previous studies using microarray techniques to investigate transcriptional regulation of plant responses to As stress have identified numerous differentially expressed genes. However, little is known about the metabolic and regulatory network remodelings, or their interactions with microRNA (miRNA) in plants upon As(III) exposure. • We used Illumina sequencing to acquire global transcriptome alterations and miRNA regulation in rice under As(III) treatments of varying lengths of time and dosages. • We found that the response of roots was more distinct when the dosage was varied, whereas that of shoots was more distinct when the treatment time was varied. In particular, the genes involved in heavy metal transportation, jasmonate (JA) biosynthesis and signaling, and lipid metabolism were closely related to responses of rice under As(III) stress. Furthermore, we discovered 36 new As(III)-responsive miRNAs, 14 of which were likely involved in regulating gene expression in transportation, signaling, and metabolism. • Our findings highlight the significance of JA signaling and lipid metabolism in response to As(III) stress and their regulation by miRNA, which provides a foundation for subsequent functional research.

Methylation patterns of plants are unique as, in addition to the methylation at CG dinucleotides that occurs in mammals, methylation also occurs at non-CG sites. Genes are methylated at CG sites, but transposable elements (TEs) are methylated at both CG and non-CG sites. The role of non-CG methylation in transcriptional silencing of TEs is being extensively studied at this time, but only very rare transpositions have been reported when non-CG methylation machineries have been compromised. To understand the role of non-CG methylation in TE suppression and in plant development, we characterized rice mutants with changes in the chromomethylase gene, OsCMT3a. oscmt3a mutants exhibited a dramatic decrease in CHG methylation, changes in the expression of some genes and TEs, and pleiotropic developmental abnormalities. Genome resequencing identified eight TE families mobilized in oscmt3a during normal propagation. These TEs included tissue culture-activated copia retrotransposons Tos17 and Tos19 (Lullaby), a pericentromeric clustered high-copy-number non-autonomous gypsy retrotransposon Dasheng, two copia retrotransposons Osr4 and Osr13, a hAT-tip100 transposon DaiZ, a MITE transposon mPing, and a LINE element LINE1-6_OS. We confirmed the transposition of these TEs by polymerase chain reaction (PCR) and/or Southern blot analysis, and showed that transposition was dependent on the oscmt3a mutation. These results demonstrated that OsCMT3a-mediated non-CG DNA methylation plays a critical role in development and in the suppression of a wide spectrum of TEs. These in planta mobile TEs are important for studying the interaction between TEs and the host genome, and for rice functional genomics.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to a novel, activation domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets.

Cytosine DNA methylation (mC) is a genome modification that can regulate the expression of coding and non-coding genetic elements. However, little is known about the involvement of mC in response to environmental cues. Using whole genome bisulfite sequencing to assess the spatio-temporal dynamics of mC in rice grown under phosphate starvation and recovery conditions, we identified widespread phosphate starvation-induced changes in mC, preferentially localized in transposable elements (TEs) close to highly induced genes. These changes in mC occurred after changes in nearby gene transcription, were mostly DCL3a-independent, and could partially be propagated through mitosis, however no evidence of meiotic transmission was observed. Similar analyses performed in Arabidopsis revealed a very limited effect of phosphate starvation on mC, suggesting a species-specific mechanism. Overall, this suggests that TEs in proximity to environmentally induced genes are silenced via hypermethylation, and establishes the temporal hierarchy of transcriptional and epigenomic changes in response to stress.

Soybean is one of the most important vegetable oil and protein feed crops. To capture the entire genomic diversity, it is needed to construct a complete high-quality pan-genome from diverse soybean accessions. In this study, we performed individual de novo genome assemblies for 26 representative soybeans that were selected from 2,898 deeply sequenced accessions. Using these assembled genomes together with three previously reported genomes, we constructed a graph-based genome and performed pan-genome analysis, which identified numerous genetic variations that cannot be detected by direct mapping of short sequence reads onto a single reference genome. The structural variations from the 2,898 accessions that were genotyped based on the graph-based genome and the RNA sequencing (RNA-seq) data from the representative 26 accessions helped to link genetic variations to candidate genes that are responsible for important traits. This pan-genome resource will promote evolutionary and functional genomics studies in soybean.

Respiratory immune characteristics associated with Coronavirus Disease 2019 (COVID-19) severity are currently unclear. We characterized bronchoalveolar lavage fluid immune cells from patients with varying severity of COVID-19 and from healthy people by using single-cell RNA sequencing. Proinflammatory monocyte-derived macrophages were abundant in the bronchoalveolar lavage fluid from patients with severe COVID-9. Moderate cases were characterized by the presence of highly clonally expanded CD8⁺ T cells. This atlas of the bronchoalveolar immune microenvironment suggests potential mechanisms underlying pathogenesis and recovery in COVID-19.

Tiller angle in cereals is a key shoot architecture trait that strongly influences grain yield. Studies in rice (Oryza sativa) have implicated shoot gravitropism in the regulation of tiller angle. However, the functional link between shoot gravitropism and tiller angle is unknown. Here, we conducted a large-scale transcriptome analysis of rice shoots in response to gravistimulation and identified two new nodes of a shoot gravitropism regulatory gene network that also controls rice tiller angle. We demonstrate that HEAT STRESS TRANSCRIPTION FACTOR 2D (HSFA2D) is an upstream positive regulator of the LAZY1-mediated asymmetric auxin distribution pathway. We also show that two functionally redundant transcription factor genes, WUSCHEL RELATED HOMEOBOX6 (WOX6) and WOX11, are expressed asymmetrically in response to auxin to connect gravitropism responses with the control of rice tiller angle. These findings define upstream and downstream genetic components that link shoot gravitropism, asymmetric auxin distribution, and rice tiller angle. The results highlight the power of the high-temporal-resolution RNA-seq data set and its use to explore further genetic components controlling tiller angle. Collectively, these approaches will identify genes to improve grain yields by facilitating the optimization of plant architecture.

Groucho/Thymidine uptake 1 (Gro/Tup1) family proteins are evolutionarily conserved transcriptional coregulators in eukaryotic cells. Despite their prominent function in transcriptional repression, little is known about their role in transcriptional activation and the underlying mechanism. Here, we report that the plant Gro/Tup1 family protein LEUNIG_HOMOLOG (LUH) activates MYELOCYTOMATOSIS2 (MYC2)-directed transcription of JAZ2 and LOX2 via the Mediator complex coactivator and the histone acetyltransferase HAC1. We show that the Mediator subunit MED25 physically recruits LUH to MYC2 target promoters that then links MYC2 with HAC1-dependent acetylation of Lys-9 of histone H3 (H3K9ac) to activate JAZ2 and LOX2 Moreover, LUH promotes hormone-dependent enhancement of protein interactions between MYC2 and its coactivators MED25 and HAC1. Our results demonstrate that LUH interacts with MED25 and HAC1 through its distinct domains, thus imposing a selective advantage by acting as a scaffold for MYC2 activation. Therefore, the function of LUH in regulating jasmonate signaling is distinct from the function of TOPLESS, another member of the Gro/Tup1 family that represses MYC2-dependent gene expression in the resting stage.

The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.

Significantly increasing crop yield is a major and worldwide challenge for food supply and security. It is well-known that rice cultivated at Taoyuan in Yunnan of China can produce the highest yield worldwide. Yet, the gene regulatory mechanism underpinning this ultrahigh yield has been a mystery. Here, we systematically collected the transcriptome data for seven key tissues at different developmental stages using rice cultivated both at Taoyuan as the case group and at another regular rice planting place Jinghong as the control group. We identified the top 24 candidate high-yield genes with their network modules from these well-designed datasets by developing a novel computational systems biology method, i.e., dynamic cross-tissue (DCT) network analysis. We used one of the candidate genes, OsSPL4, whose function was previously unknown, for gene editing experimental validation of the high yield, and confirmed that OsSPL4 significantly affects panicle branching and increases the rice yield. This study, which included extensive field phenotyping, cross-tissue systems biology analyses, and functional validation, uncovered the key genes and gene regulatory networks underpinning the ultrahigh yield of rice. The DCT method could be applied to other plant or animal systems if different phenotypes under various environments with the common genome sequences of the examined sample. DCT can be downloaded from https://github.com/ztpub/DCT.

Grain number per panicle (GNP) is an important agronomic trait that contributes to rice grain yield. Despite its importance in rice breeding, the molecular mechanism underlying GNP regulation remains largely unknown. In this study, we identified a previously unrecognized regulatory gene that controls GNP in rice, Oryza sativa REPRODUCTIVE MERISTEM 20 (OsREM20), which encodes a B3 domain transcription factor. Through genetic analysis and transgenic validation we found that genetic variation in the CArG box-containing inverted repeat (IR) sequence of the OsREM20 promoter alters its expression level and contributes to GNP variation among rice varieties. Furthermore, we revealed that the IR sequence regulates OsREM20 expression by affecting the direct binding of OsMADS34 to the CArG box within the IR sequence. Interestingly, the divergent pOsREM20^IR and pOsREM20^ΔIR alleles were found to originate from different Oryza rufipogon accessions, and were independently inherited into the japonica and indica subspecies, respectively, during domestication. Importantly, we demonstrated that IR sequence variations in the OsREM20 promoter can be utilized for germplasm improvement through either genome editing or traditional breeding. Taken together, our study characterizes novel genetic variations responsible for GNP diversity in rice, reveals the underlying molecular mechanism in the regulation of agronomically important gene expression, and provides a promising strategy for improving rice production by manipulating the cis-regulatory element-containing IR sequence.