Post-transcriptional mechanisms of interneuron development - ABSTRACT Cortical inhibitory interneurons (cINs) are critical for circuit function. Despite tremendous advances in single cell technologies that have painted high-resolution transcriptomic maps of their developmental trajectories, we still don’t fully understand the mechanisms driving cIN migration and specification, suggesting that post-transcriptional mechanisms might be at play. Indeed, microRNAs (miRNAs) are necessary for cIN development, as demonstrated by defects in migration and specification after knockout of the miRNA biogenesis enzyme Dicer. However, miRNA mechanisms underlying these phe- notypes remain to be discovered because current tools are inadequate to address the complexity of cIN development. We engineered new tools to fill this gap. Using a peptide that rapidly and reversibly blocks the miRNA machinery, we find that during miRNA LoF during late embryonic development leads to impaired cIN migration at E18 and an altered ratio of parvalbumin (PV) to somatostatin (SST) cINs at P21, without altering overall cIN abundance. Mapping of miRNA- target interactions in developing cINs identified ‘miRNA hotspots’, the genes most heavily regulated by miRNAs. Through a CRISPR activation screen, we found that hotspots play critical roles in cIN migration. In particular, de-repression of Ist1 increases neuronal branching and alters migration dynamics in vitro and in vivo. In this proposal, we will identify mecha- nisms by which miRNA hotspot de-repression leads to cIN migration defects. For example, we will elucidate how elevated Ist1 expression leads to aberrant activity in the endosomal sorting complexes required for transport (ESCRT) pathway and how Armcx2 de-repression perturbs mitochondrial motility to affect cIN migration. Further, will investigate how transient miRNA LoF during late embryonic stages or miRNA hotspot de-repression shifts the PV to SST ratio, either by selective apoptosis or by class fate plasticity. Successful completion of this proposal will define, for the first time, the mechanistic links between miRNA-mediated post-transcriptional regulation and the essential processes of cIN migration and the determination of PV to SST ratio. This is particularly significant because defects in cIN development have been linked to multiple psychiatric disorders, but the mechanisms instructing these processes are still largely unknown. This proposal will yield critical advancement, providing evidence that key aspects of cIN development are regulated post-transcriptionally. It will also provide a new toolbox for interrogating miRNA-target networks in specific neuronal cell types, and innovative methodologies to dissect miRNA mechanisms. The combination of technical and conceptual innovations will be transformative for the field, and we predict that the approach outlined here will be widely adopted.
