Studies of the spatiotemporal, transcriptomic, and morphological diversity of radial glia (RG) have spurred our current models of human corticogenesis. In the developing cortex, neural intermediate ...progenitor cells (nIPCs) are a neuron-producing transit-amplifying cell type born in the germinal zones of the cortex from RG. The potential diversity of the nIPC population, that produces a significant portion of excitatory cortical neurons, is understudied, particularly in the developing human brain. Here we explore the spatiotemporal, transcriptomic, and morphological variation that exists within the human nIPC population and provide a resource for future studies. We observe that the spatial distribution of nIPCs in the cortex changes abruptly around gestational week (GW) 19/20, marking a distinct shift in cellular distribution and organization during late neurogenesis. We also identify five transcriptomic subtypes, one of which appears at this spatiotemporal transition. Finally, we observe a diversity of nIPC morphologies that do not correlate with specific transcriptomic subtypes. These results provide an analysis of the spatiotemporal, transcriptional, and morphological diversity of nIPCs in developing brain tissue and provide an atlas of nIPC subtypes in the developing human cortex that can benchmark in vitro models of human development such as cerebral organoids and help inform future studies of how nIPCs contribute to cortical neurogenesis.
Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using ...single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone
. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.
Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter ...spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration.
Accurate RNA quantification at the single-cell level is critical for understanding the dynamics of gene expression and regulation across space and time. Single molecule FISH (smFISH), such as ...RNAscope, provides spatial and quantitative measurements of individual transcripts, therefore, can be used to explore differential gene expression among a heterogeneous cell population if combined with cell identify information. However, such analysis is not straightforward, and existing image analysis pipelines cannot integrate both RNA transcripts and cellular staining information to automatically output cell type-specific gene expression. We developed an efficient and customizable analysis method, Single-Molecule Automatic RNA Transcription Quantification (SMART-Q), to enable the analysis of gene transcripts in a cell type-specific manner. SMART-Q efficiently infers cell identity information from multiplexed immuno-staining and quantifies cell type-specific transcripts using a 3D Gaussian fitting algorithm. Furthermore, we have optimized SMART-Q for user experiences, such as flexible parameters specification, batch data outputs, and visualization of analysis results. SMART-Q meets the demands for efficient quantification of single-molecule RNA and can be widely used for cell type-specific RNA transcript analysis.
Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) is being increasingly used to study gene regulation. However, major analytical gaps limit its utility in studying ...gene regulatory programs in complex diseases. In response, MOCHA (Model-based single cell Open CHromatin Analysis) presents major advances over existing analysis tools, including: 1) improving identification of sample-specific open chromatin, 2) statistical modeling of technical drop-out with zero-inflated methods, 3) mitigation of false positives in single cell analysis, 4) identification of alternative transcription-starting-site regulation, and 5) modules for inferring temporal gene regulatory networks from longitudinal data. These advances, in addition to open chromatin analyses, provide a robust framework after quality control and cell labeling to study gene regulatory programs in human disease. We benchmark MOCHA with four state-of-the-art tools to demonstrate its advances. We also construct cross-sectional and longitudinal gene regulatory networks, identifying potential mechanisms of COVID-19 response. MOCHA provides researchers with a robust analytical tool for functional genomic inference from scATAC-seq data.Analytical gaps limit the utility of scATAC-seq for studying gene regulatory programs in human disease. Here, authors describe MOCHA, a robust analytical tool with advanced statistical modelling that enables functional genomic inference in large cross-sectional and longitudinal human studies.
Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and ...CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4
T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα
T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.
The human cerebral cortex is comprised of a diverse set of neurons that are essential to its functions in perception, judgment, and sensory integration. The complexity of this organ emerges during ...development with the aid of tightly coordinated gene expression. Modern techniques now allow us to map the transcriptional diversity of cell types in the developing brain and the epigenetic alterations that generate them. Here, we explore two projects that advance our understanding of transcriptional diversity in the developing human cortex. The first maps cell-type-specific enhancers that might drive transcriptional diversity, and the second identifies subtypes of neuronal progenitors in the dorsal cortex. To map enhancers in part 1, we ran a PLAC-Seq, ATAC-seq, and RNA-seq on four FACS-sorted populations of human neural cell types and validated our cell-type specific results using a CRISPRi system. For part 2, the identification of neuronal progenitors relied on single cell RNA-seq results, paired with histology and viral labeling to identify 5 subpopulations. One of these populations stained for DLX5 and was related to a late neurogenic shift in neuronal progenitor location, while the other four populations dominated early neurogenesis through the germinal zone of the developing cortex. Interestingly, we did not see a clear relationship between morphology and cell type identity.
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