UC San Diego

The development of cortical inhibitory interneurons (cINs) has been fine-tuned to produce a highly specialized population that serves as the substrate for cortical circuit formation. Understanding the developmental programs responsible for the precise distribution and diversification of cINs is key to unlocking the principles of building and evolving brain circuits, yet much remains unknown about their key regulators and molecular mechanisms. MicroRNAs (miRNAs) are a class of non-coding RNAs that are necessary for cIN development, as genetic ablation of miRNAs in developing cINs causes a severe decrease in cIN abundance, migration defects, and a failure to express the mature cell type markers parvalbumin (PV) and somatostatin (SST). However, due to the coarse temporal resolution of genetic knockouts over the protracted developmental period of cINs, it is unclear how or when miRNAs directly regulate cIN specification, migration, and maturation. Further, the miRNA-mediated mechanisms necessary for cIN maturation and migration are unknown, in part because the miRNA-target network specific to embryonic cINs is poorly understood.

We engineered an innovative miRNA toolbox with vastly improved temporal resolution and molecular precision to unlock mechanistic insight on miRNA regulation of cIN development. Using a novel method for rapidly and reversibly blocking miRNA function in neurons, we showed that miRNAs are necessary for cIN developmental progression and fate specification. Transiently blocking miRNAs only during late embryonic development abrogated cell death to reveal an irreversible alteration in the ratio of PV and SST cINs, suggesting that there is an embryonic critical period during which cIN fate decisions require miRNA regulation. To identify specific miRNA-mediated mechanisms of cIN development, we profiled miRNA-target interactions in cINs at two timepoints representing neurogenesis and migration, overcoming the technical challenges of targetome profiling in low abundance cell types via complementary use of two variants of AGO2 CLIPseq. We performed an unbiased analysis of gene-level miRNA load, identified putative nodes in the miRNA-target network that we term “miRNA hotspots”, and screened the top miRNA hotspots for roles in cIN migration. By manipulating the hit gene Ist1 via overexpression or whole 3’UTR gene editing to precisely mutate miRNA recognition elements (MREs), we showed that derepression of Ist1 leads to altered cellular morphology during migration, impaired dispersion across cortical regions, and increased cortical invasion, and further discovered a novel mechanism by which miR-125b preferentially represses Ist1 expression within the migratory streams.