MiR-125/let-7 cluster orchestrates neuronal cell fate determination and cortical layer formation during neurogenesis

MicroRNA (miRNA) clusters, defined as genomically co-localized miRNAs regulated by a shared promoter and processed from polycistronic transcripts, exhibit synergistic regulatory roles in developmental processes. Among these, the evolutionarily conserved miR-125/let-7 cluster has been identified as a key regulator of neural stem cell (NSC) dynamics. In this study, we used Dicer conditional knockout (cKO) mice to confirm the essential role of miRNAs in mouse neocortical layer formation. The miR-125/let-7 cluster is co-expressed in mice and shows significant enrichment in upper-layer (UL) neurons. Using in utero electroporation (IUE), we found that miR-125b or let-7b overexpression partially rescues cortical phenotypes in Dicer-deficient mice, restoring proper UL organization but failing to rescue laminar fate defects in deep-layer cortical neurons. Our findings demonstrate that the miR-125b/let-7b exhibits a specialized function in regulating UL neuronal fate specification in mice and promotes the differentiation of NSC. Notably, miR-125b and let-7b exhibit both overlapping and distinct regulatory functions. Collectively, these results underscore the cooperative mechanisms by which miRNA clusters orchestrate cortical development.