Mechanisms regulating interneuron diversity and maturation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

LOSS OF EZH2 IN MGE PROGENITORS ALTERS INTERNEURON FATE Enhancer of zeste homolog 2 (Ezh2) is the methyltransferase component of the Polycomb Repressive Complex 2 (PRC2), which is critical for trimethylation of histone 3 at lysine 27 (H3K27me3) and results in gene repression. Mutations in EZH2 and dysregulation of H3K27me3 can lead to neurodevelopmental abnormalities such as Weaver Syndrome and ataxia-telangiectasia. During cortical neurogenesis, H3K27me3 is a critical epigenetic modification that regulates cellular maturation rate, and in turn, determines when precursor cells exit the cell cycle. Loss of function studies reveal that Ezh2 plays a critical role in epigenetic regulation of neuronal fate and maturation in some brain regions, but a role for Ezh2 in forebrain GABAergic interneurons has not been explored. Here, we removed Ezh2 from the medial ganglionic eminence (MGE) to study its role in interneuron development. We find that loss of Ezh2 shifts interneuron fate, with an increase in somatostatin-expressing (SST+) and a decrease in parvalbumin-expressing (PV+) interneurons in multiple brain regions. Intrinsic electrophysiological properties are normal, but PV+ interneurons display increased axonal complexity. We also observe fewer MGE-derived interneurons at P5, indicating reduced interneuron production during development. Last, we utilized single cell Multiome and CUT&Tag assays to characterize transcriptional and H3K27me3 differences in the MGE between WT and KO mice. Our results reveal a critical role for Ezh2 in interneuron fate and maturation. Single cell multiome (RNA-seq and ATAC-seq) analysis revealed genetic and epigenetic changes in the embryonic MGE that are consistent with these cell fate changes. Lastly, CUT&Tag analysis revealed differential changes in H3K27me3 levels at specific genomic loci, with some genes displaying a relative increase in H3K27me3 levels. Thus, loss of Ezh2 in the MGE causes significant changes in interneuron fate, morphology, and gene expression and regulation. A manuscript describing these results will be submitted soon and posted on bioRxiv. A ROLE FOR EPIGENETIC REGULATION OF MGE-DERIVED INTERNEURONS Our recent publications established a ground truth of transcriptional and epigenetic states in four distinct embryonic brain regions that give rise to distinct subtypes of forebrain interneurons (RhodesPetros 2022; LeePetros 2022). We are now expanding on these findings to characterize how various mutations alter the transcriptome, epigenetic environment and ultimately cell fate. In one study, we are exploring how disruption of a candidate Nkx2.1 enhancer alters the fate of MGE-derived interneurons. In another study, we are studying how disruption of histone methylation at a specific residue effects interneuron fate and leads to mice with enhanced seizure susceptibility. These studies are ongoing. MECHANISMS REGULATING FATE DETERMINATION OF CGE-DERIVED INTERNEURONS While significant progress has been made characterizing mechanisms regulating initial fate decisions of MGE-derived interneurons, our understanding of CGE-derived interneurons lags significantly behind. This is in part because we lack genetic tools to specifically target and manipulate CGE-derived cells. There is an expansion of CGE-derived interneuron subtypes in humans and primates compared with mice, so a better understanding of the developmental trajectory of these cells is warranted. To this end, we are currently performing several experiments to better understand the developmental logic of CGE-derived cells. First, we previously performed cell transplant assays of postmitotic MGE-derived interneuron precursors to better understand how the brain environment influences cell fate (QuattrocoloPetros, Cell Reports 2017; QuattrocoloPetros JoVE 2018). We are currently performing similar cell transplantation experiments to determine whether specific CGE-derived interneuron subtypes are derived from distinct regions of the CGE. By combining this spatial logic with the scRNA-Seq and scATAC-seq studies we published last year (RhodesPetros Nature Communications 2022; LeePetros eLife 2022), we hope to link early transcription and chromatin accessibility profiles in CGE progenitors with mature interneuron fates. Second, we have developed a Perturb-Seq approach to identify candidate genes that promote CGE-derived interneurons. We are using CRISPR-Cas9 mouse embryonic stem cell (mESC) to activate or repress an array of genes involved in neurodevelopment, using a lentiviral library consisting of numerous gRNAs targeted to genes of interest. We will follow up any intriguing, fate-determining gene candidate hits from this Perturb-Seq screen to explore their possible role in CGE-derived interneurons. Third, based on insights from our scRNA-seq experiments (LeePetros eLife 2022), we have generated new transgenic mouse models with the goal of specifically targeting CGE-derived interneurons during development. We are currently testing the specificity and efficacy of this mouse line to label CGE-derived interneurons (or specific subsets) during neurodevelopment. In combination, this series of experiments should significantly increase our understanding of mechanisms regulating CGE-derived interneurons.