Conserved Roles for Slit and Robo Proteins in Midline Commissural Axon Guidance

Abstract

In Drosophila, Slit at the midline activates Robo receptors on commissural axons, thereby repelling them out of the midline into distinct longitudinal tracts on the contralateral side of the central nervous system. In the vertebrate spinal cord, Robo1 and Robo2 are expressed by commissural neurons, whereas all three Slit homologs are expressed at the ventral midline. Previous analysis of Slit1;Slit2 double mutant spinal cords failed to reveal a defect in commissural axon guidance. We report here that when all six Slit alleles are removed, many commissural axons fail to leave the midline, while others recross it. In addition, Robo1 and Robo2 single mutants show guidance defects that reveal a role for these two receptors in guiding commissural axons to different positions within the ventral and lateral funiculi. These results demonstrate a key role for Slit/Robo signaling in midline commissural axon guidance in vertebrates.

Robo family of proteins exhibit differential expression in mouse spinal cord and Robo-Slit interaction is required for midline crossing in vertebrate spinal cord

“…We confirm the expression patterns using our antibodies against Robo1 and Rig-1 as has been described previously (Long et al, 2004;Sabatier et al, 2004). Both Robo1 and Rig-1 are expressed on the precrossing fibers, with no Rig-1 protein on the postcrossing fibers, in contrast to Robo1, which is expressed on the postcrossing axons.…”

“…Commissural axons did not extend across the floor plate normally but, instead, turned longitudinally prematurely, suggesting that Slit proteins are required for commissural axons to navigate along the floor-plate region to the contralateral side of the spinal cord, once they have started crossing the floor plate. Similar, but more severe axon pathfinding defects were observed in mice with all six Slit alleles deleted, where many commissural axons fail to leave the midline and others recross it (Long et al, 2004). It is plausible that the concentration of Fc proteins were not sufficient at all times during the development of the commissural axons to bind all floor-plate Slits; therefore, the pathfinding errors were less dramatic than those that were observed in triple Slit mutant mice.…”

“…However, these antibodies require a tyramide amplification step, which prevents an accurate assessment of precise level of protein expression. Long et al (2004) emphasize that both Robo1 and Robo2 are expressed primarily on the postcrossing portion of commissural axons, with only low levels of proteins observed on the precrossing fibers. It is likely, however, that precrossing fibers express a significant level of Robo1 protein, enabling them to respond to midline Slits and preventing axons from entering the floor plate in Rig-1 mutant embryos .…”

“…These interactions keep commissural axons from recrossing the midline and also maintain ipsilateral axons on their own side (Kidd et al, 1998a. Two recent reports describing Rig-1 knockout mice and triple Slit mutant mice (Long et al, 2004) provided a foundation for addressing the role of Slits and Robos at the ventral midline in vertebrates.…”

“…Although studies of mice deficient in Slit1 and Slit2 show an important role for these proteins in retinal axon guidance and in the development of several major forebrain tracts (Bagri et al, 2002), no obvious defects in commissural axon pathfinding within the developing spinal cord were observed. During the preparation of this manuscript, a new report was published on the generation of triple Slit1, Slit2, Slit3 mutants, which have apparent axon guidance defects at the ventral midline: many commissural axons stay at the midline or recross it several times (Long et al, 2004).…”