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Mechanisms Regulating Fate and Maturation of Forebrain GABAergic Interneurons

• Timothy J. Petros, PhD, Head, Unit on Cellular and Molecular Neurodevelopment
• Yajun Zhang, BM, Biologist
• Dongjin Lee, PhD, Postdoctoral Fellow
• Jianing Li, PhD, Postdoctoral Fellow
• Matthew Manion, PhD, Postdoctoral Fellow
• Samra Beyene, MS, Postbaccalaureate Fellow
• Anthony Tanzillo, BS, Postbaccalaureate Fellow
• Allison Tucker, BS, Postbaccalaureate Fellow
• William Emrick, Summer Internship Program Student

Proper brain function requires a balance between excitatory projection neurons and GABAergic inhibitory interneurons. Although interneurons constitute the minority (about 20%) of neurons in the brain, they are the primary source of inhibition and are critical components in the modulation and refinement of the flow of information throughout the nervous system. Interneurons are an extremely heterogeneous cell population, with distinct morphologies, connectivity, neurochemical markers, and electrophysiological properties. In fact, the incredible diversity and heterogeneity of interneurons was observed over a century ago, with Ramón y Cajal hypothesizing in Recollections of My Life that “The functional superiority of the human brain is intimately linked up with the prodigious abundance and unaccustomed wealth of the so-called neurons with short axons.” Abnormal development and function of interneurons has been linked to the pathobiology of numerous neurological and psychiatric diseases, such as epilepsy, schizophrenia, and autism. Many genes implicated in brain disorders are enriched in young interneurons, and thus a thorough description of the cellular and molecular mechanisms regulating this diverse cell population is necessary to understand both normal development and disease models.

The lab focuses on understanding the how intrinsic genetic and epigenetic programs interact with the local brain environmental to generate this incredible diversity of interneuron subtypes. We take a multifaceted approach to this issue, utilizing both in vitro and in vivo approaches to identify candidate mechanisms that regulate interneuron fate decisions. We strive to develop cutting-edge techniques that will overcome the many challenges of studying interneuron development. Our ultimate goal is to discover genetic cascades and signaling mechanisms that direct interneuron differentiation and maturation during normal development and in disease states.

Characterization of transcriptome diversity in radial glia cells throughout the embryonic forebrain

Radial glia cells (RGCs), located throughout the ventricular zone (VZ) of the developing nervous system, give rise to all neurons and glia. Early studies supported the hypothesis that RGCs are relatively homogenous and become progressively restricted over future cell divisions. Initial scRNA-seq studies on the GEs (ganglionic eminences) found that the first genetic signatures of mature interneuron subtypes appear in post-mitotic precursors, whereas very little transcriptional diversity was detected in VZ and SVZ (subventricular zone) progenitors. However, these studies were significantly underpowered for detecting potential transcriptional diversity in VZ neural progenitors because the GEs comprise primarily SVZ and mantle zone cells at these mid-embryonic ages. Transcriptionally heterogeneous VZ cell populations have been reported in other portions of the central nervous system, so we hypothesized that RGCs in the forebrain are more genetically heterogeneous than previously appreciated.

We performed scRNA-seq on the LGE, MGE, CGE (lateral, medial and caudal ganglionic eminences) and cortex from E12.5 and E14.5 mice to identify spatial and temporal genetic heterogeneity of VZ and SVZ neural progenitors, utilizing a mouse line with a destabilized VenusGFP protein driven by the Nestin promoter to enrich for RGCs. We identified significant transcriptional heterogeneity in VZ cells, with many genes specifically enriched in RGCs in the MGE, LGE or CGE. We verified many of these expression patterns using fluorescent in situ hybridization (FISH) and found that some genes were enriched in specific subdomains (e.g., dorsal vs. ventral) within each GE. Given that specific subdomains of the MGE preferentially generate distinct mature interneurons subtypes, our observation indicates that VZ cells already express spatially restricted genes that pattern the GE and likely are critical for future cell-fate decisions [Reference 1]. Also, the field currently lacks a strong Cre–driver to label and manipulate CGE–derived interneurons. We found that the Igfbp5 gene (encoding insulin-like growth-factor binding protein 5) was specifically enriched in the CGE VZ, and we generated an Igfbp5-Cre mouse to explore whether this can be used to target CGE cells.

‘Epigenome Atlas’ of the embryonic mouse brain

While scRNA-seq experiments have greatly increased our understanding of the mature and developing brain, much remains to be discovered. Genome-wide association studies (GWAS) indicate that over 90% of disease-associated single-nucleotide polymorphisms (SNPs) are located outside coding regions, and many neurological disorders have been linked directly to SNPs in enhancer regions. Thus, a thorough characterization of the epigenomic landscape during neurogenesis is critical to understand normal brain development and potential disease etiologies. To this end, we performed the following set of experiments on the MGE, LGE, CGE, and cortex from E12.5 embryonic mouse brain: scRNA-seq, snATAC-seq to define chromatin accessibility at the single-cell level; CUT&Tag on histone modifications H3K4me3, H3K27ac, and H3K27me3 to identify active promoters, active enhancers, and closed chromatin, respectively; and Hi-C (a high-throughput genomic and epigenomic technique) and Capture-C to characterize higher-order chromatin structure and to increase confidence in promoter-enhancer interactions.

Our snATAC-seq data revealed significant differences in chromatin accessibility between brain regions. We used SnapATAC in combination with Cicero to identify candidate promoter-enhancer interactions, many of which were supported by H3K4me3 and H3K27ac data. Combining this with the scRNA data allowed us to follow the trajectory of promoter accessibility, enhancer accessibility, and RNA levels over time. Surprisingly, we found that RNA levels were downregulated prior to closing of promoters and enhancers, which has intriguing implications for how genes are regulated during development. The Hi-C data also revealed distinct chromatin organization between different embryonic brain regions. For example, the transcription factor Nkx2.1 is expressed in the MGE and is critical for all MGE–derived cells. There is a direct interaction between the Nkx2.1 promoter and the Mbip locus in the MGE that is not present in the LGE, CGE, or cortex. Conversely, in these non–MGE regions, the Nkx2.1 promoter interacts directly with the Nkx2.9 and Pax9 locus, whereas this interaction is not observed in the MGE. To our knowledge, this was the first study to explore differential epigenetic landscape in distinct regions of the embryonic mouse brain, and it highlights the importance of characterizing multiple epigenetic mechanisms in vivo because many of these differences would not have been observed using in vitro cell cultures [Reference 2].

We believe that this dataset represents a critical resource for the field, and to make this dataset publicly available in an easily searchable platform, we used the UCSC Genome Browser platform. Any investigator can search for a gene or loci of interest and view the RNA, ATAC, histone modification, Cicero interactions, and Hi-C data in four regions of the embryonic mouse brain.

We spend significant time developing efficient protocols to generate single-cell and single-nuclei suspensions from both embryonic and adult mouse brains. In addition to being critical for many of our own studies, our success with these procedures generated significant interest from other labs who were interested in performing single-cell sequencing experiments. This motivated us to publish an all-encompassing methods paper describing our protocols to generate single cell/nuclei suspensions from embryonic or adult mouse brains for numerous downstream applications [Reference 5].

Loss of Ezh2 in the MGE alters interneuron fate and function.

We were interested in exploring how genetic perturbations alter normal processes and regulate interneuron fate. There is growing evidence that mutations in epigenetic machinery can lead to neuro-development disorders. One gene of interest was Enhancer of zeste homolog 2 (Ezh2), the primary methyltransferase of the polycomb repressive complex 2 (PRC2), which is critical for trimethylation of histone 3 lysine 27 (H3K27me3), resulting in gene repression. In humans, EZH2 variants lead to the Weaver syndrome, a complex disease with varying degrees of intellectual disability. Loss of Ezh2 in mice can lead to premature neuronal differentiation, migration defects, and changes in neuronal fate, but the role of Ezh2 in forebrain interneurons had not been explored. To this end, we generated Nkx2.1-Cre;Ezh2 conditional knockout (KO) mice to remove Ezh2 from the MGE.

Loss of Ezh2 reduced the overall number of MGE–derived interneurons in the cortex and caused a shift in fate with an increase in SST+ (somatostatin-positive) and a decrease in PV+ (parvalbumin-positive) interneurons. Similar shifts in cell fate were observed in the hippocampus and striatum. We did not observe altered cell fate when Ezh2 was removed in postmitotic GE (ganglionic eminence) cells using Dlx6a-Cre, indicating that the critical function of Ezh2 occurs in cycling neural progenitors. In collaboration with Soohyun Lee, we characterized the electrophysiological (e-phys) properties of mutant cells in the cortex. We found no changes in the intrinsic e-phys properties of PV+ or SST+ cells in the cortex of KO mice. However, PV+ cells in the KO displayed more complex axonal arbors, with greater axonal length and arborization than in wild-type (WT) PV+ cells (Figure 4B). We believe this could be a form of compensation, given that one way to circumvent the loss of PV+ cells in the mutant would be for the surviving cells to have increased synaptic outputs and increased inhibition.

We performed the 10x Genomics Multiome assay (snRNA-seq and snATAC-seq) on the MGE of WT and KO mice. We found an increase in genes expressed by SST+ cells (SST, Pde1a) in the KO MGE, whereas genes predictive of PV–fated interneurons (Mef2c, Maf) were enriched in WT MGE. We also observed concurrent shifts in the presence of Mef2c– and Maf–binding motifs in accessible regions in the snATAC-seq data. Thus, loss of Ezh2 induces transcriptional and epigenetic changes in the embryonic MGE, which lead to shifts in mature interneuron fate. We also quantified changes in H3K27me3 levels at specific genomic loci via CUT&Tag. Despite a global reduction in H3K27me3 in Ezh2 KO mice, we observed relative changes in H3K27me3 at specific loci. For example, H3K27me3 signal was nearly entirely depleted at the Foxp4 locus, indicating that this region is extremely susceptible to loss of Ezh2. Conversely, the Nkx2.1 locus displayed a relative increase in H3K27me3, meaning that it was significantly resistance to this epigenetic perturbation. The finding implies that genes critical for specific aspects of development (such as Nkx2.1 in the MGE) may be evolutionarily resistant to epigenetic perturbations. In support of this concept, a similar finding was observed at the Sox2 locus in our collaboration with Pedro Rocha, warranting further investigation of this concept [Reference 3].

Mechanisms regulating fate determination of CGE–derived interneurons

Research over the last 20 or so years has led to many insights into MGE development. However, our knowledge of mechanisms regulating CGE–derived interneurons lags significantly behind. 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 tuberous sclerosis complex (TSC), underscoring the importance of understanding development of CGE cells.

In previous studies, we transplanted fluorescent embryonic precursors from MGE subdomains into postnatal brains to determine the spatial and temporal origin of mature interneuron subtypes. The 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. We are harvesting anterior, posterior, ventral, and dorsal CGE cells (aCGE, pCGE, vCGE, dCGE) from Dlx6aCre;Ai9 mice and transplanting these cells into the cortex of WT pups. We are harvesting CGE subdomains from two different ages (E13.5 and E15.5) to identify both temporal and spatial relationships between CGE progenitors and mature interneuron subtypes, as both location and birthdate play a role in fate determination within the MGE. In total, this constitutes eight different conditions (four CGE subdomains at two different time points). Brains are harvested at P30–35, sectioned and immunostained for CGE–specific interneuron markers (e.g., VIP, CCK, reelin, and calretinin) to correlate spatial and temporal origin with mature CGE–derived interneuron subtypes. Our preliminary cell counts from aCGE and pCGE transplants indicate that a spatial bias does exist in the CGE, with distinct CGE–derived interneuron subtypes preferentially arising from specific CGE subdomains. Our CGE spatio-temporal map will be integrated with our scRNA-seq datasets described above to identify candidate genes expressed in CGE subdomains related to mature subtypes.

Additional Funding

• NICHD Scientific Director's Award

Publications

1. Lee DR, Rhodes C, Mitra A, Zhang Y, Maric D, Dale RK, Petros TJ. Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain. eLife 2022 https://doi.org/10.7554/eLife.71864.
2. Rhodes CT, Thompson JJ, Mitra A, Asokumar D, Lee DR, Lee DJ, Zhang Y, Jason E, Dale RK, Rocha PP, Petros TJ. An epigenome atlas of neural progenitors within the embryonic mouse forebrain. Nat Commun 2022 13(1):4196.
3. Rhodes CT, Asokumar D, Sohn M, Naskar S, Elisha L, Stevenson P, Lee DR, Zhang Y, Rocha PP, Dale RK, Lee S, Petros TJ. Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles. bioRxiv 2023 doi: https://doi.org/10.1101/2023.09.06.556544.
4. Chakraborty S, Kopitchinski N, Zuo Z, Eraso A, Awasthi P, Chari P, Mitra A, Tobias IC, Moorthy SD, Dale RK, Mitchell JA, Petros TJ, Rocha PP. Enhancers that activate target genes across CTCF boundaries increase phenotypic robustness. Nat Genet 2023 55(2):280–290.
5. Lee DR, Zhang Y, Rhodes CT, Petros TJ. Generation of single cell and single nuclei suspensions from embryonic and adult mouse brains. STAR Protocols 2023 4(1):1–22.

Collaborators

• Susan Amara, PhD, Laboratory of Molecular and Cellular Neurobiology, NIMH, Bethesda, MD
• Ryan Dale, PhD, Bioinformatics Core, NICHD, Bethesda, MD
• Anthony LaMantia, PhD, Virginia Tech School of Medicine, Roanoke, VA
• Soohyun Lee, PhD, Unit on Functional Neural Circuits, NIMH, Bethesda, MD
• Chris McBain, PhD, Section on Cellular and Synaptic Physiology, NICHD, Bethesda, MD
• Pedro Rocha, PhD, Unit on Genome Structure and Regulation, NICHD, Bethesda, MD

Contact

For more information, email tim.petros@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/petros.

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