Genetic and Epigenetic Mechanisms Regulating Fate and Maturation of Forebrain Inhibitory Interneurons

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 responsible for trimethylation of histone 3 at lysine 27 (H3K27me3), resulting in repression of gene expression. Ezh2 variants in humans can lead to Weaver Syndrome, a complex disease with variable degrees of intellectual disability, and dysregulation of H3K27me3 may be the primary driver in ataxia-telangiectasia. Here, we explore the role of Ezh2 in forebrain GABAergic interneuron development. We removed Ezh2 in the medial ganglionic eminence (MGE) by generating Nkx2-1Cre;Ezh2 conditional knockout mice. Loss of Ezh2 increases somatostatin-expressing (SST+) and decreases parvalbumin-expressing (PV+) interneurons in the forebrain. We observe fewer MGE-derived interneurons in the first postnatal week, indicating reduced interneuron production. Intrinsic electrophysiological properties in SST+ and PV+ interneurons are normal, but PV+ interneurons display increased axonal complexity in Ezh2 mutant mice. Single nuclei multiome analysis revealed differential gene expression patterns in the embryonic MGE that are predictive of these cell fate changes. Lastly, CUT&Tag analysis revealed that some genomic loci are particularly resistant or susceptible to shifts in H3K27me3 levels in the absence of Ezh2, indicating differential selectivity to epigenetic perturbation. Thus, loss of Ezh2 in the MGE alters interneuron fate, morphology, and gene expression and regulation. These findings have important implications for both normal development and potentially in disease etiologies. This study was published in Frontiers in Cellular Neuroscience in 2024 (PMID 38419656). REDUCING METHYLATION OF HISTONE 3.3 LYSINE 4 IN THE MEDIAL GANGLIONIC EMINENCE AND HYPOTHALAMUS RECAPITULATES NEURODEVELOPMENTAL DISORDER PHENOTYPES Methylation of lysine 4 on histone H3 (H3K4) is enriched on active promoters and enhancers and correlates with gene activation. Disruption of H3K4 methylation is associated with numerous neurodevelopmental diseases (NDDs) that display intellectual disability and abnormal body growth. Here, we perturb H3K4 methylation in the medial ganglionic eminence (MGE) and the hypothalamus, two brain regions associated with these disease phenotypes. These mutant mice have fewer forebrain interneurons, deficient network rhythmogenesis, and increased spontaneous seizures and seizure susceptibility. Mutant mice are significantly smaller than control littermates, but they eventually became obese due to striking changes in the genetic and cellular hypothalamus environment in these mice. Perturbation of H3K4 methylation in these cells produces deficits in numerous NDD-associated behaviors, with a bias for more severe phenotypes in female mice. Single cell sequencing reveals transcriptional changes in the embryonic and adult brain that underlie many of these phenotypes. In sum, our findings highlight the critical role of H3K4 methylation in regulating survival and cell-specific gene regulatory mechanisms in forebrain GABAergic and hypothalamic cells during neurodevelopment to control network excitability and body size homoeostasis. A preprint detailing these findings was recently posted on bioRxiv (PMID 40568105). 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. The MGE and CGE generate distinct, non-overlapping cohorts of interneurons that can be defined by their transcriptomic, morphological, and electrophysiological characteristics. In the MGE, somatostatin-expressing (SST+) cells arise predominantly from the dorsal-posterior MGE from E12-E16 whereas parvalbumin-expressing (PV+) cells are born in the ventral-anterior MGE throughout embryogenesis. This relationship between spatiotemporal origin and mature interneuron subtypes has led to genetic insights regarding fate and maturation of these MGE-derived cells. A similar organization has never been explored in the CGE, despite the significant increase in CGE-derived interneurons in primates compared to rodents. Here we harvested fluorescent cells from distinct CGE subdomains at E13.5 and E15.5 and grafted them into WT neonatal mice cortices. One month post-transplantation, brains were immunostained for interneuron markers to relate mature CGE-derived interneurons with spatiotemporal origins within the CGE. Our results indicate that there are significant spatial biases in the CGE, with specific interneuron subtypes preferentially arising from distinct CGE subdomains. These biases are relatively stable over time, implying a minimal relationship between temporal birthdate and interneuron subtype. In the future, combining these insights with spatial transcriptome profiles will generate critical insights into gene regulation of CGE-derived interneurons. A preprint detailing these findings was recently posted on bioRxiv (https://doi.org/10.1101/2025.07.28.667082). By combining this spatial logic with the scRNA-Seq and scATAC-seq studies we published previously (PMIDs 35175194 & 35858915), we hope to link early transcription and chromatin accessibility profiles in CGE progenitors with mature interneuron fates. GENE REGULATORY MECHANISMS IN NEURODEVELOPMENT In a collaboration with Dr. Pedro Rocha's lab (NICHD), we have explored how perturbation CTCF sites disrupt normal chromatin boundary organization and can lead to changes in gene expression, cell fate, and in some cases significant phenotypes. In this study, we have shown how disruption of CTCF sites near several Fgf ligands leads to their strong upregulation in the midbrain during embryogenesis due to aberrant interaction with an adjacent enhancer of Ano1, resulting in increased cellular proliferation and encephalocele. This study was published in Developmental Cell in 2025 (PMID 40015278). ONGOING STUDIES 1. We have developed a Perturb-Seq approach to identify candidate genes that promote CGE-derived interneurons. We are using CRISPR-Cas9 mouse embryonic stem cells (mESCs) to activate or repress genes expressed in the embryonic mouse brain. 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. 2. The transcription factor Arx is critical for interneuron development, and mutations in ARX in humans is one of the leading causes of X-linked intellectual disabilities (XLID). We are currently exploring how loss of Arx in the MGE induces genetic, cellular and behavioral phenotypes in both male knockout and female heterozygous mice. 3. Based on brain region specific chromatin interactions identified in our previous study (PMID 35858915), we are exploring how perturbation of promoter-enhancer interactions at the Nkx2.1 locus (a ‘master regulator’ of the MGE) alters gene expression, chromatin organization and ultimately cell fate of MGE-derived interneurons. 4. Individual transcription factors are often expressed in multiple cell and tissue types during development, but with distinct genomic targets. This in part explains why human variants in transcription factors can lead to complex, multi-organ phenotypes that are challenging to treat. Mechanisms that guide differential transcription factor activity in distinct tissues are not well understood. The transcription factor Nkx2.1 is expressed in the embryonic thyroid, lung, hypothalamus, and medial ganglionic eminence (MGE), where it plays critical roles in development of these tissues. We are currently comparing the transcriptome, chromatin accessibility & Nkx2.1 binding between these tissues to understand how Nkx2.1 regulates distinct gene programs in these different tissues.