Mechanisms Regulating Fate and Maturation of Forebrain GABAergic Interneurons

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), autism spectrum disorder, schizophrenia, and substance-related disorders (Figure 2A). Two common symptoms of many H3K4–related disorders are intellectual disability and abnormal body growth. Studying H3K4 methylation has been challenging because H3K4 can be methylated by six different proteins, and experimentally deleting a single methyltransferase can lead to confounding compensations by other proteins. To overcome this challenge, we utilized a Cre–dependent transgenic mouse in which the H3K4 lysine is mutated to a methionine (H3K4M), thus blocking methylation at this residue. The homozygous H3K4M mouse contains two wild-type (WT) H3K4 alleles and two mutant H3K4M alleles, acting as a functional heterozygote, and is thus a good model for exploring the role of altered H3K4 methylation in human disease. We generated Nkx2.1-Cre;H3K4M mice to target this mutation to the medial ganglionic eminence (MGE) and the hypothalamus, two brain regions associated with intellectual disability and regulation of body growth (Figure 2B). Such mutant mice have fewer forebrain interneurons, deficient network rhythmogenesis, and increased spontaneous seizures and seizure susceptibility (Figure 2C). Mutant mice are significantly smaller than control littermates, but they eventually became obese because of striking changes in the genetic and cellular hypothalamus environment in these mice (Figure 2D). Perturbation of H3K4 methylation in these cells produces deficits in numerous NDD–associated behaviors, such as increased anxiety and altered memory and sensorimotor responses (Figure 2E). We observed a striking cellular disorganization in the hypothalamus of H3K4M mutant mice, likely compromising the normal blood-brain behavior and leading to numerous hypothalamus-related deficits (Figure 2F). Our single-cell sequencing experiments reveal many transcriptional changes in both the MGE and hypothalamus, which underlie cell-fate and behavioral changes in these mutant mice (Figure 2F). taken together, 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. Our article on this study is currently under revision, and a preprint is available [Reference 2].

Forebrain inhibitory interneurons are born from transient structures during embryogenesis known as the medial and caudal ganglionic eminences (MGE and CGE, respectively). The MGE and CGE generate distinct, non-overlapping cohorts of interneurons that can be defined by their transcriptomic, morphological, and electrophysiological characteristics. Previous studies transplanted fluorescent embryonic precursors from MGE subdomains into postnatal brains to determine the spatial and temporal origin of mature interneuron subtypes. These studies identified a spatial and temporal organization relating MGE progenitors to mature interneuron subtypes. Combining these insights with gene-expression patterns has generated important insights into MGE development. Surprisingly, whether a similar organization occurs in the CGE has not been explored. The CGE contributes about 30% of interneurons in mice, but humans and primates have a larger proportion of CGE–derived interneurons. Additionally, CGE progenitors are the primary source of tumors and cortical lesions in the tuberous sclerosis complex (TSC), underscoring the importance of understanding development of CGE cells. We harvested fluorescent cells from the anterior, posterior, ventral, and dorsal CGE cells (aCGE, pCGE, vCGE, dCGE) at E13.5 and E15.5 and grafted them into WT neonatal mice cortices (Figure 3A). Brains are harvested 30–35 days post-transplantation, sectioned, and immunostained for CGE–specific interneuron markers (VIP, CCK, Reln, CR, etc.) to correlate spatial and temporal origin with mature CGE–derived interneuron subtypes (Figure 3B). We identified significant spatial biases for most CGE–derived interneuron subtypes to preferentially arise from distinct CGE subdomains (Figure 3C). These biases are relatively stable over time, implying a minimal relationship between temporal birthdate and interneuron subtype (Figure 3D), which differs significantly from the MGE. Our spatiotemporal map of CGE interneuron development (Figure 3E) can now be integrated with datasets showing differential gene expression throughout the CGE to identify candidate genes that regulate fate and maturation of specific CGE–derived interneuron subtypes. This manuscript is currently in revision and a preprint is available at bioRxiv [Reference 3].

Characterizing how chromatin architecture regulates proper promoter-enhancer interactions and gene expression is critical to an understanding of normal development and disease etiologies. Many promoter-enhancer interactions are tissue- or cell-type–specific, so that it is critical to study these interactions in vivo. Our previous study identified a direct chromatin interaction between Nkx2.1 (encodes the homeobox protein Nkx-2.1, which regulates genes that play a role in the development and movement of interneurons) and a gene upstream called MAP3K12–binding inhibitory protein 1 (MBIP). Like Nkx2.1, MBIP is also only expressed in the MGE (Figure 4A). Given that Nkx2.1 is a ‘master regulator’ of MGE–derived interneurons, Nkx2.1 represents an ideal locus to explore how brain region–specific chromatin interactions regulate gene expression and cell fate.

We used a cutting-edge genetic strategy to generate a mouse line that disrupts the MGE–specific Nkx2.1-Mbip interaction, as confirmed by Micro-C (Nkx2.1–CTCF mice, Figure 4B). There is a strong reduction of Mbip and a moderate downregulation of Nkx2.1 in the MGE in these mice (Figure 4C). We are currently quantifying the number of SST⁺ (somatostatin-expressing) and PV⁺ (fast-spiking parvalbumin-positive) interneurons in the cortex to determine whether there are any changes in the density and/or proportion of MGE–derived interneurons in the Nkx2.1–CTCF mutant mice. Success with this project will motivate us to apply these genetic perturbations to other genomic loci at critical fate-determining genes that have brain region–specific chromatin interactions. We have already generated another mouse line using similar technology to disrupt a different brain region–specific chromatin interaction in order to study its role in interneuron development.