During embryonic development, growing axons are guided toward their targets by molecular cues that they interpret as either attractive or repulsive. Among these cues, the Sonic hedgehog (Shh) protein holds a particular position: depending on the neuronal type and its developmental stage, it can either attract or repel axons. In the developing spinal cord, Shh first attracts commissural axons toward the floor plate, then repels them along the anteroposterior axis once they have crossed the midline. In the visual system, Shh acts as a repulsive cue for the axons of retinal ganglion cells projecting to the same side of the brain (iRGCs), contributing to their segregation at the optic chiasm. Despite this dual role, the molecular mechanisms underlying the switch between attraction and repulsion remained poorly understood.
The authors had previously investigated the attraction of commissural axons and had shown that it depends on Shh-triggered endocytosis of the Boc receptor, a process relying on Numb, a membrane-associated protein involved in endocytosis. Numb and its homolog Numb-like (Nbl), broadly expressed in the developing nervous system and functionally redundant, also play a role in maintaining neural progenitors and in the polarized trafficking of several receptors. The question addressed here was whether the function of Numb in Shh signaling extends beyond attraction, into non-canonical repulsive responses.
To address this, the team studied two distinct systems in which Shh acts as a repulsive cue: the segregation of iRGC axons at the optic chiasm and the anteroposterior repulsion of commissural axons after midline crossing. Using transgenic mouse models that allow specific labeling of iRGCs, axon tracing techniques (DiI, cholera toxin subunit B), conditional knockouts of Numb and Nbl, and retinal explant cultures subjected to collapse assays in the presence of Shh, the authors establish that Numb is expressed by iRGCs and that it is indispensable for both repulsive processes. Numb thus appears to be required for both Shh-mediated attraction and repulsion.
These findings place Numb at the heart of the non-canonical Shh signaling pathway involved in axonal repulsion. The authors nonetheless emphasize that their study did not elucidate the molecular mechanism explaining why some axons are attracted and others repelled by the same cue, and that Numb/Nbl may regulate the trafficking of other guidance receptors—open questions that remain to be explored.