MicroRNA mechanisms instructing Purkinje cell specification.

MicroRNAs (miRNAs) are critical for brain development; however, if, when, and how miRNAs drive neuronal subtype specification remains poorly understood. To address this, we engineered technologies with vastly improved spatiotemporal resolution that allow the dissection of cell-type-specific miRNA-target networks. Fast and reversible miRNA loss of function showed that miRNAs are necessary for Purkinje cell (PC) differentiation, which previously appeared to be miRNA independent, and identified distinct critical miRNA windows for dendritogenesis and climbing fiber synaptogenesis, structural features defining PC identity. Using new mouse models that enable miRNA-target network mapping in rare cell types, we uncovered PC-specific post-transcriptional programs. Manipulation of these programs revealed that the PC-enriched miR-206 and targets Shank3, Prag1, En2, and Vash1, which are uniquely repressed in PCs, are critical regulators of PC-specific dendritogenesis and synaptogenesis, with miR-206 knockdown and target overexpression partially phenocopying miRNA loss of function. Our results suggest that gene expression regulation by miRNAs, beyond transcription, is critical for neuronal subtype specification.