Mechanisms regulating interneuron diversity and maturation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

CHARACTERIZING THE EPIGENETIC LANDSCAPE DURING EMBRYONIC NEUROGENESIS While most studies have focused on genes that regulate initial interneuron fate decisions during embryogenesis, a role for epigenetic mechanisms in this process has not been investigated. There is ample evidence that the epigenetic code plays critical roles during neurodevelopment, notably at cell state changes. In particular, DNA and histone modifications often follow specific rules termed the epigenetic code, similar to the genetic code. Collectively, DNA methylation and histone modifications have been reported to regulate transcription and chromatin (nuclear DNA and associated proteins) structure in many stem cell and developmentally critical processes. This idea is particularly relevant since epigenetic changes are observed in many neurological and psychiatric diseases and most single-nucleotide variants (SNVs) identified in diseases-specific GWAS studies map to non-coding regions, implying epigenetic regulation of gene expression may underlie some disease etiologies. To this end, we have characterized the epigenomic landscape of progenitor cells in distinct embryonic brain regions using single cell ATAC-sequencing. By integrating this data with our own single cell transcriptome analysis, we have established a ground state of chromatin accessibility and gene expression at the single cell level throughout the embryonic forebrain. We have uncovered numerous 'high confidence' promoter-enhancer interactions that may play important roles in fate determination of specific neuronal subtypes from distinct embryonic brain regions. We have begun to integrate these findings with neurological and psychiatric databases of human SNPs to determine if there are specific embryonic brain regions and/or cell types that display preferential accessibility at disease-associated SNPs. Following up on these initial observations, we are currently performing more targeted approaches to understand how perturbation of several genes critical for histone methylation affect chromatin accessibility, gene expression and ultimately cell fate. We hope to perform similar sets of experiments on additional disease-related genes in the future. DEFINING THE TRANSCRIPTIONAL HETEROGENEITY OF VENTRICULAR ZONE RADIAL GLIA CELLS The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression. However, a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the mouse embryonic cortex and ganglionic eminences (GEs). By using a transgenic mouse line to enrich for VZ cells, we detect significant transcriptional heterogeneity within VZ and SVZ progenitors, both between forebrain regions and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of telencephalic VZ cells that will aid our understanding of initial fate decisions in the forebrain. We are currently establishing CRISPR-based strategies in mouse ESCs to manipulate candidate genes and determine their role in interneuron fate determination and maturation. DEVELOPING A NOVEL APPROACH TO IDENTIFY GENETIC CASCADES UNDERLYING INITIAL INTERNEURON FATE DECISIONS The ability to longitudinally track gene expression within defined populations is essential for understanding how changes in expression mediate both development and plasticity. Previous screens that were designed to identify genes and transcription factors specific to SST- or PV-fated interneurons were largely unsuccessful because several issues significantly hinder these types of studies. First, these interneurons originate from the medial ganglionic eminence (MGE), which is a heterogeneous population of progenitors that gives rise to both interneurons and a variety of GABAergic projection neurons, making it difficult to segregate interneuron progenitors from other cell types. Additionally, many markers that define mature interneuron subtypes are not expressed embryonically, and thus these class-defining markers are not helpful for studying MGE progenitors. In an ideal scenario, we would like to identify actively transcribed genes in MGE progenitors undergoing fate decisions while retaining the capacity to identify whether these cells become PV- or Sst-expressing interneurons in the postnatal brain. To this end, we developed a spatially and temporally inducible form of DNA adenine methylase identification (DamID) that will allow us to label the transcriptome of MGE progenitors. Labeled cells can be harvested at maturity when we have the tools to distinguish specific interneuron cell types. Then the methylated genomic DNA will be analyzed, allowing us to retrospectively look back in time to identify candidate fate determining genes expressed in specific interneuron populations. Our initial tests in mESCs were promising, as we observed drug-inducible genomic DNA methylation in the appropriate expected experimental conditions. Based on these promising results, we have since generated mouse lines from these mESCs. We are currently testing the in vivo function of the Dam methylation system to determine if the genetic methylation is functioning in the mouse as it did in the mESCs. We are also pursuing an alternate viral strategy that will allow us to temporally activate Dam after injection into the mouse embryo, then harvest specific interneuron cell types in the adult to retroactively look at actively transcribed genes throughout development.