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The receptor Roundabout-1 (Robo1) and its ligand Slit are known to influence axon guidance and central nervous system (CNS) patterning in both vertebrate and nonvertebrate systems. Although Robo-Slit interactions mediate axon guidance in the Drosophila CNS, their role in establishing the early axon scaffold in the embryonic vertebrate brain remains unclear. We report here the identification and expression of a Xenopus Robo1 orthologue that is highly homologous to mammalian Robo1. By using overexpression studies and immunohistochemical and in situ hybridization techniques, we have investigated the role of Robo1 in the development of a subset of neurons and axon tracts in the Xenopus forebrain. Robo1 is expressed in forebrain nuclei and in neuroepithelial cells underlying the main axon tracts. Misexpression of Robo1 led to aberrant development of axon tracts as well as the ectopic differentiation of forebrain neurons. These results implicate Robo1 in both neuronal differentiation and axon guidance in embryonic vertebrate forebrain.
Figure 1. Stage 32 Xenopus embryos microinjected with enhanced green fluorescent protein (EGFP; A,B) and Robo1/EGFP (C-H) RNA. All embryos display strong EGFP expression (B,D,F,H). Control EGFP-injected embryos are morphologically normal as are a proportion of Robo1/EGFP-injected animals (A,B). A range of developmental abnormalities was observed in embryos injected with Robo1/EGFP. The most common included mild curvature and shortening of the body in the rostral-caudal axis (class I,; E,F), through to severe torsion, shortening, and tail abnormalities (class III) (G,H). Scale bar = 500 mu m in A (applies to A-H).
Figure 8. Whole-mount in situ hybridization of Robo1 in stage 32 (St-32) Xenopus embryos (A-C,F,G) and stage 30 (St-30; D,E). Staining is shown in whole embryos (A,B), dissected brains (C-G), and combined in situ staining and immunostaining for NOC-2 is shown in D-G. Both sides of the embryo are shown in D and E (St-30 and St-30c, contralateral) and in E and F St-32 and St-32c) to depict bilateral symmetry of staining. Expression of Robo1 is observed in a restricted pattern throughout the brain and spinal cord. Expression is also seen in a range of non-neuronal tissues, including the somites and the neural crest-derived visceral arches. Expression is noticeably absent in the notochord (A,B). In the dissected brain (C-G), expression corresponds with the position of the nPT and is also observed in a longitudinal band of neuroepithelial cells on the ventrolateral surface of the brain, which underlies the TPOC (D-G; yellow arrows in C). This intersects with a horizontal band of expression, which extends dorsally to the epiphysis and corresponds with the position of the DVDT (white arrowheads, D,F). Immediately caudal, the longitudinal band of expression is expanded dorsally and ventrally. It is in this region that axons turn ventrally into the VC and the axons of the TPC join the TPOC (asterisks in C,D,F). Caudal to this TPOC-VC choice point is a horizontal band of neuroepithelial cells, which do not express Robo1 mRNA (C-G, single black arrowheads). In this region of the brain, the TPOC forms into tightly fasciculated axon bundles (see Fig. 2D). In the hindbrain, Robo1 expression continues in a longitudinal band in the ventrolateral neuroepithelial cells underlying the VLT. In the spinal cord, Robo1 expression is in segmental horizontal bands (black triangles in C and in the insert). Expression is absent in the developing hypothalamic region and the majority of the diencephalon. Expression is also noticeably absent from both the dorsal and ventral midline, with the exception of a restricted patch of Robo1 expression at the presumptive midbrain-hindbrain boundary. For abbreviations, see legend to Figure 2. Scale bar in A = 400 mu m in A, 150 mu m in B, 80 mu m in C, 50 mu m in D-G.
