Abstract
Proper connectivity of the nervous system requires temporal and spatial control of axon guidance signaling. As commissural axons navigate across the CNS midline, ROBO-mediated repulsion has traditionally been thought to be repressed before crossing, and then to become upregulated after crossing. The regulation of the ROBO receptors involves multiple mechanisms that control protein expression, trafficking, and activity. Here, we report that mammalian ROBO1 and ROBO2 are not uniformly inhibited precrossing and are instead subject to additional temporal control via alternative splicing at a conserved microexon. The NOVA splicing factors regulate the developmental expression of ROBO1 and ROBO2 variants with small sequence differences and distinct guidance activities. As a result, ROBO-mediated axonal repulsion is activated early in development to prevent premature crossing and becomes inhibited later to allow crossing. Postcrossing, the ROBO1 and ROBO2 isoforms are disinhibited to prevent midline reentry and to guide postcrossing commissural axons to distinct mediolateral positions.
Highlights
Temporal and spatial regulation of cell signaling ensures the fidelity of axon pathfinding, which is crucial for nervous system development and function
We reported previously that Nova1/2 double knockout dampens DCC signaling by reducing the full-length Dcclong isoform while increasing the truncated Dccshort isoform, which results in delayed commissural neuron migration and axonal projection toward the midline (Leggere et al, 2016)
At E12.5, we found that approximately half of the axons that had reached the midline failed to cross and turned longitudinally on the ipsilateral side in Nova1/2 double knockout (dKO) embryos (Figure 1)
Summary
Temporal and spatial regulation of cell signaling ensures the fidelity of axon pathfinding, which is crucial for nervous system development and function. Within the spinal commissural axons, Netrin/DCC (Deleted in colorectal carcinoma) signaling is activated to promote midline attraction while SLIT/ROBO (Roundabout) signaling that mediates repulsion is inhibited prior to crossing. SLIT/ROBO signaling becomes upregulated to facilitate midline expulsion and to block midline reentry (Evans and Bashaw, 2010). Dysregulation of the SLIT/ROBO pathway has been implicated in several forms of cancer and neurological disorders (Ballard and Hinck, 2012; Blockus and Chedotal, 2014). In both Drosophila and vertebrates, control over ROBO repulsion is achieved through multiple mechanisms. PRRG4 (Proline-rich and Gla domain 4) represses ROBO1 surface trafficking, whereas USP33 (Ubiquitin-specific peptidase 33), RabGDI/GDI1 (GDP dissociation inhibitor 1), and CLSTN1 (Calsyntenin1) stabilize or promote ROBO1 surface localization
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