Discovering miRNA-mediated mechanisms of interneuron development

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Project Summary Inhibitory interneuron (IN) development requires successful completion of a series of processes, which include long-range migration, lamination, molecular specification, circuit integration, and functional maturation. IN dysfunction is highly implicated in neurodevelopmental and psychiatric disorders, so understanding how INs achieve their mature distribution, number, and identity is critical to understanding disease. Cell-intrinsic gene expression programs, involving the precise regulation of huge sets of genes, interact with environmental cues to control development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression known to be necessary for multiple aspects of IN development, yet the specific mechanisms by which miRNAs do this remain unknown, in part because IN-specific patterns of miRNA activity were unknown. We bridged this gap by performing Ago cross-linking and immunoprecipitation followed by sequencing (Ago CLIPseq) to comprehensively profile the IN targetome at developmental timepoints. However, identifying developmental pathways regulated by individual miRNAs has been challenging because the targetome is extremely complex—miRNAs simultaneously regulate many genes and can compensate for each other through co-targeting relationships. We propose an alternative approach of investigating miRNA regulation on a gene-by-gene level. Through bioinformatic analysis of the CLIPseq data, I identified miRNA hotspots, or genes co-targeted by multiple miRNAs, which we hypothesize is a signature of strong miRNA regulation shared by many key developmental genes. With these candidate genes, I am now poised to untangle how miRNA regulation of specific genes controls particular aspects of IN development. I will use new CRISPR-based methods for disrupting miRNA regulation of candidate genes in a cell type-specific manner and screen for developmental defects, first using a set of in vitro and in vivo readouts for IN migration, survival, and subtype specification. Then, I will assess perturbations of overall developmental trajectory using targeted Perturb-seq, which harnesses the transcriptome as a complex readout of maturation state in single cells. Ultimately, with this experimental pipeline I will discover genes and miRNA mechanisms that control IN development.