Temporal control of midline crossing via incoherent feedforward and feedback loops regulating Robo3

Spinal commissural neurons play a fundamental role in motor control and sensory perception. Robo3, a receptor expressed on pre-crossing commissural axons, is required for midline crossing. Its downregulation in post-crossing axons is essential for forming synapses with contralateral CNS targets. We demonstrate that, at the transcriptional level, the dynamic expression of Robo3 in pre-crossing neurons is controlled by incoherent feedforward loops (iFFLs) and negative feedback loops (NFLs) that precisely regulate its transience and thereby determine midline crossing. Lhx2 and Lhx9 activate Robo3 while also inducing its repressor, Barhl2. Additionally, NFLs fine-tune the transience of Robo3 expression by adjusting the relative strength of the activation and repression modules. Our genomic analysis reveals that this regulatory circuitry converges on an enhancer element of the Robo3 gene. These findings imply that diverse iFFLs, together with NFLs, are essential for regulating the extent of midline crossing across different spinal commissural subtypes.