Robos are required for the correct targeting of retinal ganglion cell axons in the visual pathway of the brain.

Axonal projections from the retina to the brain are regulated by molecules including the Slit family of ligands [Thompson, H., Barker, D., Camand, O., Erskine, L., 2006a. Slits contribute to the guidance of retinal ganglion cell axons in the mammalian optic tract. Dev. Biol. 296, 476-484, Thompson, H., Camand, O., Barker, D., Erskine, L., 2006b. Slit proteins regulate distinct aspects of retinal ganglion cell axon guidance within dorsal and ventral retina. J. Neurosci. 26, 8082-8091]. However, the roles of Slit receptors in mammals, (termed Robos), have not been investigated in visual system development. Here we examined Robo1 and 2 mutant mice and found that Robos regulate the correct targeting of retinal ganglion cell (RGC) axons along the entire visual projection. We noted aberrant projections of RGC axons into the cerebral cortex, an area not normally targeted by RGC axons. The optic chiasm was expanded along the rostro-caudal axis (similar to Slit mutant mice, Plump, A.S., Erskine, L., Sabatier, C., Brose, K., Epstein, C.J., Goodman, C.S., Mason, C.A., Tessier-Lavigne, M., 2002. Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33, 219-232), with ectopic crossing points, and some axons projecting caudally toward the corticospinal tract. Further, we found that axons exuberantly projected into the diencephalon. These defects were more pronounced in Robo2 than Robo1 knockout animals, implicating Robo2 as the predominant Robo receptor in visual system development.

Evidence for the existence of two Robo3 isoforms with divergent biochemical properties.

Robo3 is a member of the roundabout (Robo) family of proteins that plays a key role in axon guidance and cell migration in the developing nervous system. Recent studies have shown that Robo3 plays a crucial role in controlling axon guidance at the midline of the CNS. Here we describe and compare two human Robo3 isoforms, Robo3A and Robo3B, which differ by the insertion of 26 amino acids at the N-terminus, and these forms appear to be evolutionary conserved. We investigated the bioactivity of these isoforms and show that they have different binding properties to Slit, and that orthologs of these forms are expressed in the mouse embryo. In addition, we show that, like other members of the Robo family, Robo3 can bind homophilically, but it is also capable of binding heterophilically to Robo1 and NCAM. We propose that these properties of Robo3 may contribute to its function at the midline of the CNS.

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

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.

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

The ventral midline of the central nervous system is an important intermediate target where growing commissural axons either cross and project contralaterally or remain on the same side of the body. New studies on mice and humans show that this decision by commissural axons is largely dependent on Slits, extracellular matrix proteins that are widely expressed in the midline of the nervous system, and their receptors, Robos (Long et al. [2004] Neuron 42:213-223; Sabatier et al. [2004] Cell 117:157-169; Jen et al. [2004] Science 304:1509-1513). Here, we show that the Robo family proteins Robo1 and Rig-1 exhibit differential expression patterns on commissural axons as they approach, cross, and leave the midline of the developing mouse spinal cord and demonstrate that Robo1 and Robo2 bind Slit1 and Slit2, but Rig-1 does not. In addition, we show that cultured chick commissural axons are repelled by a source of Slit protein, and the soluble Robo-Fc proteins are capable of neutralizing this repulsion. Finally, we exploit the large size and accessibility of the early chick embryo to analyze the function of Slit/Robo signaling in midline commissural axon guidance, and we demonstrate that the in vivo perturbation of Robo-Slit interaction at the floor plate causes consistent guidance defects of commissural axons during midline crossing. These findings demonstrate the evolutionarily conserved role for Robo-Slit interaction in the control of midline crossing axons in vertebrates.

Rig-1 a new member of Robo family genes exhibits distinct pattern of expression during mouse development.

The Robo genes encode a family of proteins that are the receptors for the midline repellent Slits and play a role in axon guidance. In addition to Robo1 and Robo2, Rig-1 has been recently identified in mouse as a novel member of the Robo family of proteins. As a first step in elucidating the role of Rig-1 during vertebrate development, we characterised the expression of Rig-1 by in situ hybridisation together with Robo1 and Robo2 in the spinal cord and other tissues of the mouse embryo. Our results show that Rig-1 has a dynamic pattern of expression in the developing CNS. In the spinal cord Rig-1 shows overlapping but distinct pattern of expression with Robo1 and Robo2.

Dynamic expression patterns of Robo (Robo1 and Robo2) in the developing murine central nervous system.

The Robo family of molecules is important for axon guidance across the midline during central nervous system (CNS) development in invertebrates and vertebrates. Here we describe the patterns of Robo protein expression in the developing mouse CNS from embryonic day (E) 9.5 to postnatal day (P) 4, as determined by immunohistochemical labeling with an antibody (S3) raised against a common epitope present in the Robo ectodomain of Robos 1 and 2. In the spinal cord, midline-crossing axons are initially (at E11) S3-positive. At later times, midline Robo expression disappears, but is strongly upregulated in longitudinally running postcrossing axons. It is also strongly expressed in noncrossing longitudinal axons. Differential expression of Robo along axons was also found in axons cultured from E14 spinal cord. These findings resemble those from the Drosophila ventral nerve cord and indicate that in vertebrates a low level of Robo expression occurs in the initial crossing of the midline, while a high level of expression in the postcrossing fibers prevents recrossing. Likewise, Robo-positive ipsilateral axons are prevented from crossing at all. However, in the brain different rules appear to apply. Most commissural axons including those of the corpus callosum are strongly S3-positive along their whole length from their time of formation to postnatal life, but some have more complex age-dependent expression patterns. S3 labeling of the optic pathway is also complex, being initially strong in the retinal ganglion cells, optic tract, and chiasma but thereafter being lost except in a proportion of postchiasmal axons. The corticospinal tract is strongly positive throughout its course at all stages examined, including its decussation, formed at about P2 in the central part of the medulla oblongata.