Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

Abstract

The floor plate is known to be a source of repellent signals for cranial motor axons, preventing them from crossing the midline of the hindbrain. However, it is unknown which molecules mediate this effect in vivo. We show that Slit and Robo proteins are candidate motor axon guidance molecules, as Robo proteins are expressed by cranial motoneurons, and Slit proteins are expressed by the tissues that delimit motor axon trajectories, i.e. the floor plate and the rhombic lip. We present in vitro evidence showing that Slit1 and Slit2 proteins are selective inhibitors and repellents for dorsally projecting, but not for ventrally projecting, cranial motor axons. Analysis of mice deficient in Slit and Robo function shows that cranial motor axons aberrantly enter the midline, while ectopic expression of Slit1 in chick embryos leads to specific motor axon projection errors. Expression of dominant-negative Robo receptors within cranial motoneurons in chick embryos strikingly perturbs their projections, causing some motor axons to enter the midline, and preventing dorsally projecting motor axons from exiting the hindbrain. These data suggest that Slit proteins play a key role in guiding dorsally projecting cranial motoneurons and in facilitating their neural tube exit.

The Slit receptor Robo1 is predominantly expressed via the Dutt1 alternative promoter in pioneer neurons in the embryonic mouse brain and spinal cord

“…The neuroepithelial labeling in the brain and spinal cord is consistent with previous expression studies of Robo1 mRNA (Connor and Key, 2002;Hammond et al, 2005;Sundaresan et al, 1998). However, two independent Robo1 antibodies, neither isoform-specific, have been used to label spinal cord sections, with labeling in post-crossing commissural axons and not in neuroepithelium (Long et al, 2004;Sundaresan et al, 2004).…”

“…The best studied commissural neurons arise in dorsal spinal cord near the roof plate and project their axons ventrally, crossing the midline under Robo guidance (Long, et al, 2004). In dorsal spinal cord neurons, Dutt1 expression was much more extensive than Robo1, indicating that the Dutt1 isoform is the predominant isoform in commissural axons.The neuroepithelial labeling in the brain and spinal cord is consistent with previous expression studies of Robo1 mRNA (Connor and Key, 2002;Hammond et al, 2005;Sundaresan et al, 1998). However, two independent Robo1 antibodies, neither isoform-specific, have been used to label spinal cord sections, with labeling in post-crossing commissural axons and not in neuroepithelium (Long et al, 2004;Sundaresan et al, 2004).…”

“…For example, mice with Robo mutations have defects in midline crossing of spinal cord commissural axons (Long et al, 2004). Other axon populations dependent on Slit/Robo signaling include major forebrain tracts and commissures (Andrews et al, 2006; Bagri et al, 2002), retinal axons (Plump et al, 2002Thompson et al, 2006), and motor axons (Hammond et al, 2005), as well as others. …”

Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin

“…Chicken embryos were obtained (26) and then used for immunostaining, in situ hybridization, immunohistochemistry (17), or in ovo electroporation as previously described (27). All experiments were approved by the ethical review committee of King's College London.…”

Moving away from the midline: new developments for Slit and Robo

“…Slits are expressed in the floor plate, the rhombic lip and in several cranial motor nuclei, such as the facial nucleus (Geisen et al, 2008;Gilthorpe et al, 2002;Hammond et al, 2005). In vitro assays had previously shown that Slits can repel migrating precerebellar neurons (Causeret et al, 2002; Causeret et al, 2004;Gilthorpe et al, 2002).…”