miR-219a-Robo1 axis acts as a conserved switch for slit-mediated commissural axon guidance.

Vertebrate commissural axon (CA) projection and midline crossing require suppression of Slit responsiveness in precrossing axons and its upregulation in postcrossing axons. Differential expression of the Slit receptor Robo1-low in precrossing and high in postcrossing axons-modulates Slit sensitivity and regulates midline crossing. However, the underlying mechanisms controlling this remain unclear. This study demonstrates that the 3' untranslated region (UTR) of Robo1 mRNA is regulated by the conserved miR-219a. In the developing spinal cord, gga-miR-219a and cRobo1 exhibit distinct, trajectory-specific expression patterns. miR-219a suppresses cRobo1 protein translation in commissural neurons (CNs) without affecting mRNA levels. Disruption of the miR-219a-cRobo1 3' UTR axis in precrossing CNs triggers premature Slit repulsion in vitro and causes spinal CA defects in vivo. Conditional knockout of mmu-miR-219a in E11.5 mouse embryos elevates mRobo1 protein and causes CA guidance defects. These findings reveal a conserved miR-219a-dependent mechanism that regulates Robo1 expression and Slit-mediated axon guidance.