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 performed a series of experiments to characterize the transcriptome (scRNA-seq), chromatin accessibility (snATAC-seq), histone modifications (CUT&Tag) and higher order chromatin structure (Hi-C and Capture-C) in distinct neurogenic regions (LGE, MGE, CGE and cortex) of the embryonic mouse brain. This comprehensive analysis generated an 'Epigenome Atlas' of the embryonic brain at the initial stages of neurogenesis and revealed striking differences in the chromatin landscape between adjacent brain regions (Rhodes...Petros, Nature Communications 2022). Additionally, 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 made all of this data available in a searchable and modifiable format on the UCSC Genome Browser platform (https://www.nichd.nih.gov/research/atNICHD/Investigators/petros/epigenome-atlas). Following up on this initial study, 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 utilized a transgenic mouse line that specifically labels VZ cells with a fluorescent reporter and 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 (LGE, MGE and CGE). We detected significant transcriptional heterogeneity within VZ and SVZ progenitors, both between forebrain regions and within spatial subdomains of specific GEs (Lee...Petros, eLife 2022). 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 (1) manipulate candidate genes and determine their role in interneuron fate determination and maturation, and (2) identify new genes that are critical for generation of CGE-derived interneurons.