Temporal regulation of axonal repulsion by alternative splicing of a conserved microexon in mammalian Robo1 and Robo2

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.

Alternative splicing programming of axon formation

“…Those attracted to the midline either fail to cross the midline or cross the midline and grow in a wrong angle, indicative of enhanced repulsion activity at both sides of the midline (Figure 5d). In support of this idea, the midline crossing phenotype in Nova dKO is rescued when the repulsion signal is attenuated by Robo1 complete knockout (Johnson et al, ).…”

“…For example, previous models assume silencing of ROBO signaling pre‐crossing. However, before most axons cross the midline, ROBO1/2 almost exclusively express the long (and stronger) isoform at E10.5 (Johnson et al, ). Therefore, repulsive activities already exist pre‐crossing and are strengthened by expressing the long isoforms to inhibit premature crossing of commissural axons.…”

Crosstalk between Bone and Nerves within Bone

“…[ 109 ] Slit‐3 axon guidance cues emitting from the skeleton are sensed by nerves through the receptors, Robo1. [ 148–152 ] Ephrin B2, an Eph receptor‐interacting protein which navigates nerve growth, can be identified from osteoclasts and osteoblasts. [ 153 ] Osteoblasts produce Netrin‐1, the receptor of which is well expressed in the nervous system.…”

New insights on epigenetic mechanisms supporting axonal development: histone marks and miRNAs

“…In rodents, the deletion of NOVA proteins, a family of splicing regulators in neurons, impairs cortical migration in the brain, as well as NMJ formation [103,104]. In addition, deletion of NOVA halts the extension of commissural tracts in both the corpus callosum and spinal cord in mice [105][106][107].…”