Mechanisms regulating interneuron diversity and maturation

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

HOW THE ENVIRONMENT SCULPTS INTERNEURON DIVERSITY AND MATURATION The composition of interneuron subtypes varies significantly between different brain regions. Numerous experiments indicate that general interneuron classes (e.g., parvalbumin- (PV) or somatostatin-expressing (SST)) are determined as cells become postmitotic during embryogenesis, the role that the brain environment plays in interneuron fate determination and maturation remains unknown. To explore this issue, we harvested early postnatal interneuron precursors (P0-P2) from specific brain regions and transplanted them into wildtype hosts either homotopically (cortex-to-cortex) or heterotopically (cortex-to-hippocampus). This technique allows us to determine if transplanted interneurons adopt properties of the host environment (indicating a strong role for the environment in regulating interneuron diversity) or if they retain subtype features more consistent with the donor region. Our findings indicate that the environment largely determines the composition of interneuron subtypes in a brain region regardless of donor region. However, some interneuron subtypes seem to be more genetically predefined and resistant to environmental influences. These findings were published in Cell Reports in late 2017. We have continued these studies by harvesting interneurons from the striatum and transplanting them to distinct brain regions (as well as graft interneurons from other regions into the striatum), which has led to additional important insights into how the brain environment sculpts interneuron maturation. Additionally, we have established a single cell sequencing platform in the lab that we are currently adapting for transplanted interneurons. We are reharvesting grafted interneurons 3 weeks post-transplantation so that we can compare the transcriptome of endogenous, homotopic and heterotopically transplanted cells to characterize (in an unbiased manner) how environmental changes influence the transcriptome of transplanted interneurons. IDENTIFYING A ROLE FOR EPIGENETICS IN EARLY INTERNEURON FATE DECISIONS 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, DNAme and histone modification 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 will characterize the epigenomic landscape of progenitor cells in distinct embryonic brain regions and integrate these findings with transcriptome analysis using the recently developed single cell ATAC-seq protocols. In a more targeted approach, we will investigate the role of the histone modification enzyme Ezh2 in interneuron development. These combined approaches will generate a more complete picture of a cells state during initial fate decisions. DEVELOPING A NOVEL APPROACH TO IDENTIFY GENETIC CASCADES UNDERLYING INITIAL INTERNEURON FATE DECISIONS The ability to longitudinally track gene expression within defined populations is essential for understanding how changes in expression mediate both development and plasticity. Previous screens that were designed to identify genes and transcription factors specific to SST- or PV-fated interneurons were largely unsuccessful because several issues significantly hinder these types of studies. First, these interneurons originate from the medial ganglionic eminence (MGE), which is a heterogeneous population of progenitors that gives rise to both interneurons and a variety of GABAergic projection neurons, making it difficult to segregate interneuron progenitors from other cell types. Additionally, many markers that define mature interneuron subtypes are not expressed embryonically, and thus these class-defining markers are not helpful for studying MGE progenitors. In an ideal scenario, we would like to identify actively transcribed genes in MGE progenitors undergoing fate decisions while retaining the capacity to identify whether these cells become PV- or Sst-expressing interneurons in the postnatal brain. To this end, we developed a spatially and temporally inducible form of DNA adenine methylase identification (DamID) that will allow us to label the transcriptome of MGE progenitors. Labeled cells can be harvested at maturity when we have the tools to distinguish specific interneuron cell types. Then the methylated genomic DNA will be analyzed, allowing us to retrospectively look back in time to identify candidate fate determining genes expressed in specific interneuron populations. Our initial tests in mESCs were promising, as we observed drug-inducible genomic DNA methylation in the appropriate expected experimental conditions. Based on these promising results, we have since generated mouse lines from these mESCs. We are currently testing the in vivo function of the Dam methylation system to determine if the genetic methylation is functioning in the mouse as it did in the mESCs. We are also pursuing an alternate viral strategy that will allow us to temporally activate Dam after injection into the mouse embryo, then harvest specific interneuron cell types in the adult to retroactively look at actively transcribed genes throughout development. EXPLORING THE LOGIC OF SYNAPTIC CONNECTIVITY OF CHANDELIER CELLS Understanding synaptic connectivity is one of the most complex questions in neuroscience. Chandelier cells, a subset of PV+ interneurons, form unique synaptic contacts on the axon initial segments (AIS) of excitatory projection neurons, with a small handful of ChCs contacting each AIS. This specific connectivity pattern presents an intriguing situation to explore the logic of this synaptic connectivity. Do chandelier cells consistently synapse on AIS with other similar targeting chandelier cells, or instead are these connections randomly distributed between chandelier cells? We are currently using several strategies involving Brainbow reporters to explore the synaptic connectivity logic of chandelier cells in the mouse cortex.