Cell Type-specific Analysis Research Articles

WebCSEA: web-based cell-type-specific enrichment analysis of genes.

Human complex traits and common diseases show tissue- and cell-type- specificity. Recently, single-cell RNA sequencing (scRNA-seq) technology has successfully depicted cellular heterogeneity in human tissue, providing an unprecedented opportunity to understand the context-specific expression of complex trait-associated genes in human tissue-cell types (TCs). Here, we present the first web-based application to quickly assess the cell-type-specificity of genes, named Web-based Cell-type Specific Enrichment Analysis of Genes (WebCSEA, available at https://bioinfo.uth.edu/webcsea/). Specifically, we curated a total of 111 scRNA-seq panels of human tissues and 1,355 TCs from 61 different general tissues across 11 human organ systems. We adapted our previous decoding tissue-specificity (deTS) algorithm to measure the enrichment for each tissue-cell type (TC). To overcome the potential bias from the number of signature genes between different TCs, we further developed a permutation-based method that accurately estimates the TC-specificity of a given inquiry gene list. WebCSEA also provides an interactive heatmap that displays the cell-type specificity across 1355 human TCs, and other interactive and static visualizations of cell-type specificity by human organ system, developmental stage, and top-ranked tissues and cell types. In short, WebCSEA is a one-click application that provides a comprehensive exploration of the TC-specificity of genes among human major TC map.

Single-Cell RNA-Seq Analysis Reveals Lung Epithelial Cell Type-Specific Responses to HDM and Regulation by Tet1.

Tet1 protects against house dust mite (HDM)-induced lung inflammation in mice and alters the lung methylome and transcriptome. In order to explore the role of Tet1 in individual lung epithelial cell types in HDM-induced inflammation, we established a model of HDM-induced lung inflammation in Tet1 knockout and littermate wild-type mice, then studied EpCAM+ lung epithelial cells using single-cell RNA-seq analysis. We identified eight EpCAM+ lung epithelial cell types, among which AT2 cells were the most abundant. HDM challenge altered the relative abundance of epithelial cell types and resulted in cell type-specific transcriptomic changes. Bulk and cell type-specific analysis also showed that loss of Tet1 led to the altered expression of genes linked to augmented HDM-induced lung inflammation, including alarms, detoxification enzymes, oxidative stress response genes, and tissue repair genes. The transcriptomic regulation was accompanied by alterations in TF activities. Trajectory analysis supports that HDM may enhance the differentiation of AP and BAS cells into AT2 cells, independent of Tet1. Collectively, our data showed that lung epithelial cells had common and unique transcriptomic signatures of allergic lung inflammation. Tet1 deletion altered transcriptomic networks in various lung epithelial cells, which may promote allergen-induced lung inflammation.

Cell type-specific abnormalities of central nervous system in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a multisystem genetic disorder involving the muscle, heart and CNS. It is caused by toxic RNA transcription from expanded CTG repeats in the 3′-untranslated region of DMPK, leading to dysregulated splicing of various genes and multisystemic symptoms. Although aberrant splicing of several genes has been identified as the cause of some muscular symptoms, the pathogenesis of CNS symptoms prevalent in patients with myotonic dystrophy type 1 remains unelucidated, possibly due to a limitation in studying a diverse mixture of different cell types, including neuronal cells and glial cells. Previous studies revealed neuronal loss in the cortex, myelin loss in the white matter and the presence of axonal neuropathy in patients with myotonic dystrophy type 1. To elucidate the CNS pathogenesis, we investigated cell type-specific abnormalities in cortical neurons, white matter glial cells and spinal motor neurons via laser-capture microdissection. We observed that the CTG repeat instability and cytosine–phosphate–guanine (CpG) methylation status varied among the CNS cell lineages; cortical neurons had more unstable and longer repeats with higher CpG methylation than white matter glial cells, and spinal motor neurons had more stable repeats with lower methylation status. We also identified splicing abnormalities in each CNS cell lineage, such as DLGAP1 in white matter glial cells and CAMKK2 in spinal motor neurons. Furthermore, we demonstrated that aberrant splicing of CAMKK2 is associated with abnormal neurite morphology in myotonic dystrophy type 1 motor neurons. Our laser-capture microdissection-based study revealed cell type-dependent genetic, epigenetic and splicing abnormalities in myotonic dystrophy type 1 CNS, indicating the significant potential of cell type-specific analysis in elucidating the CNS pathogenesis.

Cell-Type Specific Methylation Analysis Reveals Multiple Loci Associated With PTSD in African Americans

Multiple studies identified DNA methylation differences in immune-related genes in PTSD. Immunologic studies of leukocytes in individuals with PTSD reported elevated lymphocyte, total T cell, and CD4+T cell counts. Thus, correcting for cell-type composition in epigenomic studies may obscure some PTSD-associated signals. This study leverages a new bioinformatic method to identify novel DNA methylation patterns associated with PTSD in specific cell-types.

Advancing discovery in hearing research via biologist-friendly access to multi-omic data

High-throughput cell type-specific multi-omic analyses have advanced our understanding of inner ear biology in an unprecedented way. The full benefit of these data, however, is reached from their re-use. Successful re-use of data requires identifying the natural users and ensuring proper data democratization and federation for their seamless and meaningful access. Here we discuss universal challenges in access and re-use of multi-omic data, possible solutions, and introduce the gEAR (the gene Expression Analysis Resource, umgear.org)—a tool for multi-omic data visualization, sharing and access for the ear field.

Seeing the Forest and Its Trees Together: Implementing 3D Light Microscopy Pipelines for Cell Type Mapping in the Mouse Brain.

The brain is composed of diverse neuronal and non-neuronal cell types with complex regional connectivity patterns that create the anatomical infrastructure underlying cognition. Remarkable advances in neuroscience techniques enable labeling and imaging of these individual cell types and their interactions throughout intact mammalian brains at a cellular resolution allowing neuroscientists to examine microscopic details in macroscopic brain circuits. Nevertheless, implementing these tools is fraught with many technical and analytical challenges with a need for high-level data analysis. Here we review key technical considerations for implementing a brain mapping pipeline using the mouse brain as a primary model system. Specifically, we provide practical details for choosing methods including cell type specific labeling, sample preparation (e.g., tissue clearing), microscopy modalities, image processing, and data analysis (e.g., image registration to standard atlases). We also highlight the need to develop better 3D atlases with standardized anatomical labels and nomenclature across species and developmental time points to extend the mapping to other species including humans and to facilitate data sharing, confederation, and integrative analysis. In summary, this review provides key elements and currently available resources to consider while developing and implementing high-resolution mapping methods.

Cell type-specific analysis by single-cell profiling identifies a stable mammalian tRNA-mRNA interface and increased translation efficiency in neurons.

The correlation between codon and anticodon pools influences the efficiency of translation, but whether differences exist in these pools across individual cells is unknown. We determined that codon usage and amino acid demand are highly stable across different cell types using available mouse and human single-cell RNA-sequencing atlases. After showing the robustness of ATAC-sequencing measurements for the analysis of tRNA gene usage, we quantified anticodon usage and amino acid supply in both mouse and human single-cell ATAC-seq atlases. We found that tRNA gene usage is overall coordinated across cell types, except in neurons, which clustered separately from other cell types. Integration of these data sets revealed a strong and statistically significant correlation between amino acid supply and demand across almost all cell types. Neurons have an enhanced translation efficiency over other cell types, driven by an increased supply of tRNAAla (AGC) anticodons. This results in faster decoding of the Ala-GCC codon, as determined by cell type–specific ribosome profiling, suggesting that the reduction of tRNAAla (AGC) anticodon pools may be implicated in neurological pathologies. This study, the first such examination of codon usage, anticodon usage, and translation efficiency resolved at the cell-type level with single-cell information, identifies a conserved landscape of translation elongation across mammalian cellular diversity and evolution.

Bottom-up proteomic analysis of human adult cardiac tissue and isolated cardiomyocytes

The heart is composed of multiple cell types, each with a specific function. Cell-type-specific approaches are necessary for defining the intricate molecular mechanisms underlying cardiac development, homeostasis, and pathology. While single-cell RNA-seq studies are beginning to define the chamber-specific cellular composition of the heart, our views of the proteome are more limited because most proteomics studies have utilized homogenized human cardiac tissue. To promote future cell-type specific analyses of the human heart, we describe the first method for cardiomyocyte isolation from cryopreserved human cardiac tissue followed by flow cytometry for purity assessment. We also describe a facile method for preparing isolated cardiomyocytes and whole cardiac tissue homogenate for bottom-up proteomic analyses. Prior experience in dissociating cardiac tissue or proteomics is not required to execute these methods. We compare different sample preparation workflows and analysis methods to demonstrate how these can impact the depth of proteome coverage achieved. We expect this how-to guide will serve as a starting point for investigators interested in general and cell-type-specific views of the cardiac proteome.

Sleep loss drives acetylcholine- and somatostatin interneuron–mediated gating of hippocampal activity to inhibit memory consolidation

Sleep loss disrupts consolidation of hippocampus-dependent memory. To characterize effects of learning and sleep loss, we quantified activity-dependent phosphorylation of ribosomal protein S6 (pS6) across the dorsal hippocampus of mice. We find that pS6 is enhanced in dentate gyrus (DG) following single-trial contextual fear conditioning (CFC) but is reduced throughout the hippocampus after brief sleep deprivation (SD; which disrupts contextual fear memory [CFM] consolidation). To characterize neuronal populations affected by SD, we used translating ribosome affinity purification sequencing to identify cell type-specific transcripts on pS6 ribosomes (pS6-TRAP). Cell type-specific enrichment analysis revealed that SD selectively activated hippocampal somatostatin-expressing (Sst+) interneurons and cholinergic and orexinergic hippocampal inputs. To understand the functional consequences of SD-elevated Sst+ interneuron activity, we used pharmacogenetics to activate or inhibit hippocampal Sst+ interneurons or cholinergic input from the medial septum. The activation of either cell population was sufficient to disrupt sleep-dependent CFM consolidation by gating activity in granule cells. The inhibition of either cell population during sleep promoted CFM consolidation and increased S6 phosphorylation among DG granule cells, suggesting their disinhibition by these manipulations. The inhibition of either population across post-CFC SD was insufficient to fully rescue CFM deficits, suggesting that additional features of sleeping brain activity are required for consolidation. Together, our data suggest that state-dependent gating of DG activity may be mediated by cholinergic input and local Sst+ interneurons. This mechanism could act as a sleep loss-driven inhibitory gate on hippocampal information processing.

Detecting cord blood cell type-specific epigenetic associations with gestational diabetes mellitus and early childhood growth

BackgroundEpigenome-wide association studies (EWAS) have provided opportunities to understand the role of epigenetic mechanisms in development and pathophysiology of many chronic diseases. However, an important limitation of conventional EWAS is that profiles of epigenetic variability are often obtained in samples of mixed cell types. Here, we aim to assess whether changes in cord blood DNA methylation (DNAm) associated with gestational diabetes mellitus (GDM) exposure and early childhood growth markers occur in a cell type-specific manner.ResultsWe analyzed 275 cord blood samples collected at delivery from a prospective pre-birth cohort with genome-wide DNAm profiled by the Illumina MethylationEPIC array. We estimated proportions of seven common cell types in each sample using a cord blood-specific DNAm reference panel. Leveraging a recently developed approach named CellDMC, we performed cell type-specific EWAS to identify CpG loci significantly associated with GDM, or 3-year-old body mass index (BMI) z-score. A total of 1410 CpG loci displayed significant cell type-specific differences in methylation level between 23 GDM cases and 252 controls with a false discovery rate < 0.05. Gene Ontology enrichment analysis indicated that LDL transportation emerged from CpG specifically identified from B-cells DNAm analyses and the mitogen-activated protein kinase pathway emerged from CpG specifically identified from natural killer cells DNAm analyses. In addition, we identified four and six loci associated with 3-year-old BMI z-score that were specific to CD8+ T-cells and monocytes, respectively. By performing genome-wide permutation tests, we validated that most of our detected signals had low false positive rates.ConclusionCompared to conventional EWAS adjusting for the effects of cell type heterogeneity, the proposed approach based on cell type-specific EWAS could provide additional biologically meaningful associations between CpG methylation, prenatal maternal GDM or 3-year-old BMI. With careful validation, these findings may provide new insights into the pathogenesis, programming, and consequences of related childhood metabolic dysregulation. Therefore, we propose that cell type-specific analyses are worth cautious explorations.

Cell type-specific and cross-population polygenic risk score analyses of MIR137 gene pathway in schizophrenia

SummaryCell type-specific pathway-based polygenic risk scores (PRSs) may better inform disease biology and improve the precision of PRS-based clinical prediction. For microRNA-137 (MIR137), a leading neuropsychiatric risk gene and a post-transcriptional master regulator, we conducted a cell type-specific gene set PRS analysis in both European and Han Chinese schizophrenia (SZ) samples. We found that the PRS of neuronal MIR137-target genes better explains SZ risk than PRS derived from MIR137-target genes in iPSC or from the reported gene sets showing MIR137-altered expression. Compared with the PRS derived from the whole genome or the target genes of TCF4, the PRS of neuronal MIR137-target genes explained a disproportionally larger (relative to SNP number) SZ risk in the European sample, but with a more modest advantage in the Han Chinese sample. Our study demonstrated a cell type-specific polygenic contribution of MIR137-target genes to SZ risk, highlighting the value of cell type-specific pathway-based PRS analysis for uncovering disease-relevant biological features.

RNA-seq analysis of laser microdissected Arabidopsis thaliana leaf epidermis, mesophyll and vasculature defines tissue-specific transcriptional responses to multiple stress treatments.

Acclimation of plants to adverse conditions requires the coordination of gene expression and signalling pathways between tissues and cell types. As the energy and carbon capturing organs, leaves are significantly affected by abiotic and biotic stresses. However, tissue- or cell type-specific analyses of stress responses have focussed on the Arabidopsis root. Here, we comparatively explore the transcriptomes of three leaf tissues (epidermis, mesophyll, vasculature) after induction of diverse stress pathways by chemical stimuli (antimycin A, 3-amino-1,2,4-triazole, methyl viologen, salicylic acid) and ultraviolet light in Arabidopsis using laser capture microdissection followed by RNA sequencing. Stimulation of stress pathways caused an overall reduction in the number of genes expressed in a tissue-specific manner, though a small subset gained or changed their tissue specificity. We find no evidence of a common stress response, with only a few genes consistently responsive to two or more treatments in the analysed tissues. However, differentially expressed genes overlap between tissues for individual treatments. A focussed analysis provided evidence for an interaction of auxin and ethylene that mediates retrograde signalling during mitochondrial dysfunction specifically in the epidermis, and a gene regulatory network defined the hierarchy of interactions. Taken together, we have generated an extensive reference dataset that will be valuable for future experiments analysing transcriptional responses on a tissue or single-cell level. Our results will enable the tailoring of the tissue-specific engineering of stress-tolerant plants.

Single-Cell Analysis of the Pan-Cancer Immune Microenvironment and scTIME Portal.

Single-cell sequencing opens a new era for the investigation of tumor immune microenvironments (TIME). However, at single-cell resolution, a pan-cancer analysis that addresses the identity and diversity of TIMEs is lacking. Here, we first built a pan-cancer single-cell reference of TIMEs with refined subcell types and recognized new cell type-specific transcription factors. We then presented a pan-cancer view of the common features of the TIME and compared the variation of each immune cell type across patients and tumor types in the aspects of abundance, cell states, and cell communications. We found that the abundance and the cell states of dysfunctional T cells were most variable, whereas those of regulatory T cells were relatively stable. A subset of tumor-associated macrophages (TAM), PLTP + C1QC + TAMs, may regulate the abundance of dysfunctional T cells through cytokine/chemokine signaling. The ligand-receptor communication network of TIMEs was tumor-type specific and dominated by the tumor-enriched immune cells. We additionally developed the single-cell TIME (scTIME) portal (http://scTIME.sklehabc.com) with the scTIME-specific analysis modules and a unified cell annotation. In addition to the immune cell compositions and correlation analysis using refined cell type classifications, the portal also provides cell-cell interaction and cell type-specific gene signature analysis. Our single-cell pan-cancer analysis and scTIME portal will provide more insights into the features of TIMEs, as well as the molecular and cellular mechanisms underlying immunotherapies.

Epigenetic Regulation of Temperature Responses - Past Successes and Future Challenges.

In contrast to animals, plants cannot avoid unfavorable temperature conditions. Instead, plants have evolved intricate signaling pathways that enable them to perceive and respond to temperature. General acclimation processes that prepare the plant to respond to stressful heat and cold, usually occur throughout the whole plant. More specific temperature responses, however, are limited to certain tissues or cell types. While global responses are amenable to epigenomic analyses, responses which are highly localized are more problematic as the chromatin in question is not easily accessible. Here we review the current knowledge of the epigenetic regulation of FLOWERING LOCUS C and FLOWERING LOCUS T as examples of temperature-responsive flowering time regulators that are expressed broadly throughout the plants and in specific cell types, respectively. While undoubtably extremely successful, we reason that future analyses would benefit from higher spatiotemporal resolution. We conclude by reviewing methods and successful applications of tissue- and cell type-specific epigenomic analyses and provide a brief outlook into the future, single-cell epigenomics.

Diet Quality and Risk of Lung Cancer in the Multiethnic Cohort Study.

Diet quality, assessed by the Healthy Eating Index-2015 (HEI-2015), the Alternative Healthy Eating Index-2010 (AHEI-2010), the alternate Mediterranean Diet (aMED) score, the Dietary Approaches to Stop Hypertension (DASH) score, and the Dietary Inflammatory Index (DII®), was examined in relation to risk of lung cancer in the Multiethnic Cohort Study. The analysis included 179,318 African Americans, Native Hawaiians, Japanese Americans, Latinos, and Whites aged 45–75 years, with 5350 incident lung cancer cases during an average follow-up of 17.5 ± 5.4 years. In multivariable Cox models comprehensively adjusted for cigarette smoking, the hazard ratios (95% confidence intervals) for the highest vs. lowest quality group based on quintiles were as follows: 0.85 (0.77–0.93) for HEI-2015; 0.84 (0.77–0.92) for AHEI-2010; 0.83 (0.76–0.91) for aMED; 0.83 (0.73–0.91) for DASH; and 0.90 (0.82–0.99) for DII. In histological cell type-specific analyses, the inverse association was stronger for squamous cell carcinoma than for adeno-, small cell, and large cell carcinomas for all indexes. There was no indication of differences in associations by sex, race/ethnicity, and smoking status. These findings support that high-quality diets are associated with lower risk of lung cancer, especially squamous cell carcinomas, in a multiethnic population.

Deconvolution of cell type-specific drug responses in human tumor tissue with single-cell RNA-seq

BackgroundPreclinical studies require models that recapitulate the cellular diversity of human tumors and provide insight into the drug sensitivities of specific cellular populations. The ideal platform would enable rapid screening of cell type-specific drug sensitivities directly in patient tumor tissue and reveal strategies to overcome intratumoral heterogeneity.MethodsWe combine multiplexed drug perturbation in acute slice culture from freshly resected tumors with single-cell RNA sequencing (scRNA-seq) to profile transcriptome-wide drug responses in individual patients. We applied this approach to drug perturbations on slices derived from six glioblastoma (GBM) resections to identify conserved drug responses and to one additional GBM resection to identify patient-specific responses.ResultsWe used scRNA-seq to demonstrate that acute slice cultures recapitulate the cellular and molecular features of the originating tumor tissue and the feasibility of drug screening from an individual tumor. Detailed investigation of etoposide, a topoisomerase poison, and the histone deacetylase (HDAC) inhibitor panobinostat in acute slice cultures revealed cell type-specific responses across multiple patients. Etoposide has a conserved impact on proliferating tumor cells, while panobinostat treatment affects both tumor and non-tumor populations, including unexpected effects on the immune microenvironment.ConclusionsAcute slice cultures recapitulate the major cellular and molecular features of GBM at the single-cell level. In combination with scRNA-seq, this approach enables cell type-specific analysis of sensitivity to multiple drugs in individual tumors. We anticipate that this approach will facilitate pre-clinical studies that identify effective therapies for solid tumors.

028 Sleep loss disrupts hippocampal memory consolidation via an acetylcholine- and somatostatin interneuron-mediated inhibitory gate

Abstract Introduction Sleep loss profoundly disrupts consolidation of hippocampus-dependent memory. To better characterize effects of learning and sleep loss on the hippocampal circuit, we quantified activity-dependent phosphorylation of ribosomal subunit S6 (pS6) across the dorsal hippocampus of mice. Methods We first measured pS6 throughout the hippocampus after learning (single trial contextual fear conditioning; CFC), and after subsequent sleep or sleep deprivation (SD). To characterize cell populations with activity affected by SD, we used translating ribosome affinity purification (TRAP)-seq to identify cell type-specific transcripts on pS6 ribosomes after SD vs. sleep. We next used pharmacogenetics to mimic the effects of SD, selectively activating hippocampal Sst+ interneurons or cholinergic inputs to hippocampus from the medial septum (MS) while mice slept in the hours following CFC. We also inhibited these neuronal populations to assess effects on memory consolidation. Results We find that pS6 in enhanced in the dentate gyrus (DG) following single-trial CFC, but is reduced throughout the hippocampus after brief SD – a manipulation which disrupts contextual fear memory (CFM) consolidation. Cell type-specific enrichment analysis (CSEA) of these transcripts revealed that hippocampal somatostatin-expressing (Sst+) interneurons, and cholinergic and orexinergic inputs to hippocampus, are selectively activated after SD. We used TRAP targeted to hippocampal Sst+ interneurons to identify cellular mechanisms mediating SD-driven Sst+ interneuron activation. . We find that activation of Sst+ interneurons is sufficient to disrupt CFM consolidation, by gating activity in surrounding pyramidal neurons, while inhibition of Sst+ interneurons enhances memory consolidation. Similarly, pharmacogenetic activation of cholinergic input to hippocampus from the MS disrupted CFM. Inhibition of MS cholinergic neurons promoted CFM consolidation and disinhibited neurons in the DG, increasing pS6 expression among DG granule cells. Conclusion Our data suggest that state-dependent gating of DG activity during SD is mediated by cholinergic input. Together these data provide evidence for an inhibitory gate on hippocampal information processing, which is activated by sleep loss. Support (if any) R01-NS118440 to SJA from NINDS, DP2-MH104119 to SJA from the NIH Director’s Office, and a Human Frontiers Science Program Young Investigator Award

Network-based analysis on genetic variants reveals the immunological mechanism underlying Alzheimer's disease.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by the impairment of cognitive function and loss of memory. Previous studies indicate an essential role of immune response in AD, but the detailed mechanisms remain unclear. In this study, we obtained 1664 credible risk variants (CRVs) based on the most significant SNP detected by International Genomics of Alzheimer's Project, from which 99 genes (CRVs-related genes) were identified. Function analysis revealed that these genes were mainly involved in immune response and amyloid-β and its precursor metabolisms, indicating a potential role of immune response in regulating neurobiological processes in the etiology of neurodegenerative disease. Pathway crosstalk analysis revealed the complicated connections between immune-related pathways. Further, we found that the CRVs-related genes showed temporal-specific expression in the thalamus in adolescence developmental period. Cell type-specific expression analysis found that CRVs-related genes might be specifically expressed in brain cells such as astrocytes and oligodendrocytes. Protein-protein interaction network analysis identified the highly interconnected 'hub' genes, all of which were susceptible loci of AD. These results indicated that the CRVs may exert a potential influence in AD by regulating immune response, thalamus development, astrocytes activities, and amyloid-β binding. Our results provided hints for further experimental verification of AD pathophysiology.

The Lbx1 lineage differentially contributes to inhibitory cell types of the dorsal cochlear nucleus, a cerebellum-like structure, and the cerebellum.

The dorsal cochlear nucleus (DCN) is a mammalian-specific nucleus of the auditory system. Anatomically, it is classified as a cerebellum-like structure. These structures are proposed to share genetic programs with the cerebellum. Previous analyses demonstrated that inhibitory serial sister cell types (SCTs) of the DCN and cerebellum are derived from the pancreatic transcription factor 1a (Ptf1a) lineage. Postmitotic neurons of the Ptf1a lineage often express the transcription factor Ladybird homeobox protein homolog 1 (Lbx1) which is involved in neuronal cell fate determination. Lbx1 is therefore an attractive candidate for a further component of the genetic program shared between the DCN and cerebellum. Here, we used cell-type specific marker analysis in combination with an Lbx1 reporter mouse line to analyze in both tissues which cell types of the Ptf1a lineage express Lbx1. In the DCN, stellate cells and Purkinje-like cartwheel cells were part of the Lbx1 lineage and Golgi cells were not, as determined by cell counts. In contrast, in the cerebellum, stellate cells and Golgi cells were part of the Lbx1 lineage and Purkinje cells were not. Hence, two out of three phenotypically similar cell types differed with respect to their Lbx1 expression. Our study demonstrates that Lbx1 is differentially recruited to the developmental genetic program of inhibitory neurons both within a given tissue and between the DCN and cerebellum. The differential expression of Lbx1 within the DCN and the cerebellum might contribute to the genetic individuation of the inhibitory SCTs to adapt to circuit specific tasks.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17β-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.