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, 335 (2), 418-26

Robo2 Is Required for Slit-mediated Intraretinal Axon Guidance

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Robo2 Is Required for Slit-mediated Intraretinal Axon Guidance

Hannah Thompson et al. Dev Biol.

Abstract

The developing optic pathway has proven one of the most informative model systems for studying mechanisms of axon guidance. The first step in this process is the directed extension of retinal ganglion cell (RGC) axons within the optic fibre layer (OFL) of the retina towards their exit point from the eye, the optic disc. Previously, we have shown that the inhibitory guidance molecules, Slit1 and Slit2, regulate two distinct aspects of intraretinal axon guidance in a region-specific manner. Using knockout mice, we have found that both of these guidance activities are mediated via Robo2. Of the four vertebrate Robos, only Robo1 and Robo2 are expressed by RGCs. In mice lacking robo1 intraretinal axon guidance occurs normally. However, in mice lacking robo2 RGC axons make qualitatively and quantitatively identical intraretinal pathfinding errors to those reported previously in Slit mutants. This demonstrates clearly that, as in other regions of the optic pathway, Robo2 is the major receptor required for intraretinal axon guidance. Furthermore, the results suggest strongly that redundancy with other guidance signals rather than different receptor utilisation is the most likely explanation for the regional specificity of Slit function during intraretinal axon pathfinding.

Figures

Fig. 1
Fig. 1
Robo1 and Robo2 are expressed by RGCs. (A–F) Coronal sections of E16.5 wild-type retinas stained by in situ hybridisation for robo1 (A, C), robo2 (B, D), robo3 (rig1; E) or robo4 (F). robo1 and robo2 are expressed in the RGC layer whereas no expression of robo3 and robo4 was detected in the retina. The boxed regions in A and B are shown at higher power in C and D, respectively. (G, H) Coronal sections of E16.5 wild-type retinas stained with antibodies against Robo1 (G) or Robo2 (H). (I, J) Higher power images of the boxed regions in G and H, respectively. Robo1 and Robo2 are expressed by RGC axons in the optic fibre layer (arrows) and optic nerve (arrowheads). RGCL, retinal ganglion cell layer; NL, neuroblastic layer; ON, optic nerve. Scale bar, 200 μm (A, B, E–H) and 40 μm (C, D, I, J).
Fig. 2
Fig. 2
Robo2 helps restrict RGC axons to the optic fibre layer. (A, B) Schematic diagrams illustrating the regions of the retinas imaged. (C–F) Confocal images taken at the level of the neuroblastic layer of flat-mounted E16.5 retinas stained with an anti-β-tubulin antibody. The optic disc is located towards the top of each picture. (G–J) Coronal sections of anti-β-tubulin stained E16.5 retinas. In each image the optic disc is located towards the left-hand side. In wild-type (C, G), robo1/ (D, H) and robo2+/ (E, I) retinas, RGC axons are restricted to the optic fibre layer. In robo2-deficient retinas (F, J), large bundles of axons (arrows) are located within the outer retina (retinal ganglion cell and neuroblastic layers). Inset in J shows the ectopic axons at higher magnification. D, dorsal; NL, neuroblastic layer; OD, optic disc; OFL, optic fibre layer; RGCL, retinal ganglion cell layer; V, ventral. Scale bar, 100 μm (C–J). (K) Mean ± SEM number of axon bundles located within the outer retina of wild-type, robo1+/;robo2+/, robo1/ and robo2/ retinas. ⁎p < 0.001 compared with wild-type. Numbers above bars = numbers analysed. (L) Mean ± SEM number of axons bundles in the outer layers of dorsal (white bars) or ventral (grey bars) retina of wild-type, robo1+/;robo2+/, robo1/ and robo2/ embryos. In robo2/ retinas, significantly more ectopic bundles of axons are found within the outer layers of ventral versus dorsal retina.
Fig. 3
Fig. 3
Ectopic axons in the outer retina of robo2-deficient embryos are not retina–retina axons originating in the contralateral eye. (A) Schematic diagram illustrating retina–retina labelling of RGC axons using DiI. A crystal of DiI is placed on the optic disc of one retina and the contralateral eye is imaged. nr, neural retina. (B–G) Coronal sections of wild-type (B–D) and robo2-deficient (E–G) retinas DiI-labelled from the contralateral eye. (B) In wild-type mice, a small number of axons project to the contralateral retina where they terminate with a higher frequency in ventral (C) than dorsal retina (D). Axons that originate in the imaged eye and project to the contralateral (labelled) eye also are labelled. Arrows indicate the cell bodies of labelled RGCs; arrowheads, labelled RGC axons in the optic fibre layer. (E) In robo2-deficient mice significantly more RGC axons project to the contralateral eye. As in wild-type mice, these retina–retina axons are restricted to the optic fibre layer of the retina and terminate more frequently in ventral (F) than dorsal (G) retina. More labelled cell bodies of axons that originate in the imaged eye and project to the contralateral (labelled) eye also are present. NL, neuroblastic layer; OD, optic disc; OFL, optic fibre layer; RGCL, retinal ganglion cell layer; WT, wild-type. Scale bar, 200 μm (B, E) and 100 μm (C, D, F, G).
Fig. 4
Fig. 4
Robo2 helps control the initial direction of RGC axon outgrowth. (A) Schematic diagram illustrating the regions of the flat-mounted retinas imaged. (B–I) Confocal images taken at the level of the optic fibre layer of E16.5 retinas stained with an anti-β-tubulin antibody. In each image, the direction of the optic disc is towards the top of the picture. In wild-type (B, F), robo1/ (C, G) and robo2+/ (D, H) retinas, RGC axons extend directly towards the optic disc. In robo2/ (E, I) retinas, RGC axons within the peripheral (I) but not central (E) region of the dorsal retina extend aberrantly away from the optic disc (arrows). WT, wild-type. Scale bar, 100 μm (B–I).
Fig. 5
Fig. 5
Slit–Robo signalling is not required for RGC axons to exit the eye. (A) Schematic diagram illustrating the method used to label small subsets of RGC axons with DiI. Small crystals of DiI were placed in the peripheral region of either dorsal or ventral retina. (B–K) Images of labelled ventral (D–G) or dorsal (H–K) RGC axons in the region of the optic disc (circle) in wild-type (B, C, D, H), slit1−/−;;slit2−/− (E, I) robo1−/− (F, J) or robo2/ (G, K) embryos. In retinas of each genotype, the vast majority of axons extend towards the optic disc and exit the eye normally. In only a small number of cases were minor targeting errors observed (B, C). OD, optic disc; WT, wild-type. Scale bar, 50 μm (B–C) and 100 μm (D–K). (L) Quantification of the percentage of wild-type (WT), slit1−/−;slit2−/− robo1−/− and robo2−/− retinas with optic disc targeting errors. Numbers above bars = numbers analysed.
Fig. 6
Fig. 6
Retinal morphology is normal in robo2-deficient retinas. Coronal sections of E16.5 wild-type (A C, E, G) and robo2/ (B, D, F, H) retinas labelled with antibodies against phosphohistone-H3 (red) and Brn3a (green; A, B), Islet 1/2 (C, D), Pax6 (E, F) or AP2α (G, H). In each image the direction of the optic disc is on the left. In robo2-deficient retinas mitotic cells (B) and differentiated RGCs, amacrine cells and bipolar cells (B, D, F, H) are arrayed similar to wild-type (A, C, E, G). INL, inner nuclear layer; NL, neuroblastic layer; OFL, optic fibre layer; RGCL, retinal ganglion cell layer; WT, wild-type. Scale bar, 50 μm.

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