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First published online 2 October 2008
doi: 10.1242/dev.023663

Development 135, 3531-3541 (2008)
Published by The Company of Biologists 2008
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Autonomous and non-autonomous Shh signalling mediate the in vivo growth and guidance of mouse retinal ganglion cell axons

Cristina Sánchez-Camacho and Paola Bovolenta*

Departamento de Neurobiología Molecular Celular y del Desarrollo, Instituto Cajal, CSIC and CIBER de Enfermedades Raras (CIBERER), Avda. Dr Arce 37, Madrid 28002, Spain.


Figure 1
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  		Fig. 1. Components of the Shh signalling pathway are differentially expressed in I-RGCs and C-RGCs. (A,B,F-K) In situ hybridisation analysis of coronal sections from E14.5 mouse embryos as indicated. (C-E) Similar sections from Shh::GFPCre/+ E14.5 embryos double labelled for GFP (green) and Zic2 (red); overlay in yellow. Shh mRNA localises to most of the RGC layer but is absent from the ventrotemporal crescent (VTC), where only rare Zic2/GFP+ cells are observed (arrows in A,C-E). Ptc2 (F,H) and Boc (G,I) are expressed in the VTC (thick dotted line) but not in C-RGCs (thin dashed line). Ptc1 (B) and Hip1 (J) are distributed throughout the neuroepithelium and Gli2 (K) localises to the RGC layer. cRGC, contralateral projecting RGCs. Scale bar: 200 µm in A,B,F,G,J,K; 30 µm in C-E; 35 µm in H,I.

 

Figure 2
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  		Fig. 2. Shh promotes neurite outgrowth from VT explants but inhibits neurite outgrowth from DN explants. (A) Ventrotemporal (VT) and dorsonasal (DN) explants grown in the presence of different Shh concentrations. Outgrowth was visualised by staining with phalloidin. (B,C) Quantification of neurite outgrowth from DN (B) and VT (C) explants. Bar graphs show the area covered by neurites (in pixels). The number of explants analysed is indicated above each bar. *P<0.05, **P<0.01 (one-way ANOVA). Scale bar: 500 µm.

 

Figure 3
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  		Fig. 3. Shh neutralisation alters the distribution of RGC axons at the optic chiasm. (A-C) Ventral views of the proximal visual pathway from E18.5 non-injected (A) and from anti-Shh (B) and control IgG (C) hybridoma-treated mouse embryos visualised by complete DiI filling. Anterior is to the top. In anti-Shh-treated embryos, axons spread at the chiasm (large white arrows in B), and ipsilateral tract and contralateral nerve (small white arrow, compare with A) projections appear increased; growth cones accumulate along the path (yellow arrows). (D) Quantification of the width occupied by DiI-labelled fibres at the chiasm in untreated (n=6), control-treated (n=7) and anti-Shh-treated (n=11) embryos (*P<0.05, **P<0.01, one-way ANOVA). (E) Schematic ventral view of the proximal visual system depicting fibre trajectory and Shh and SSEA-1 distribution in control (left) and anti-Shh-treated (right) embryos. (F,G) Confocal images of flat-mounted E18.5 ipsilateral retinas showing DiI retrograde labelling from the optic tract in control and anti-Shh-treated embryos. Arrows in G indicate the presence of abnormal retrograde labelling in quadrants other than VT. cot, contralateral optic tract; iot, ipsilateral optic tract; oc, optic chiasm; od, optic disc; on, optic nerve; ot, optic tract. Scale bar: 500 µm in A-C; 215 µm in F,G.

 

Figure 4
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  		Fig. 4. Modulation of RGC-derived Shh signalling causes transcriptional-dependent changes in growth cone morphology. (A,B) Axons and growth cones from DN (A) and VT (B) explants immunostained with antibodies against Shh (green) and counterstained with phalloidin (red). Individual channels in A',A'',B',B'' with overlays in A''',B'''. (C) Confocal images of VT (I-RGC) and DN (C-RGC) growth cones exposed for 30 minutes to PBS (control), Shh, SAG, cyclopamine or anti-Shh antibody. (D,E) Percentage of collapsed growth cones in DN and VT explants after activation or inhibition of Shh signalling in the presence or absence of actinomycin D (ActD) or cycloheximide (CHX). The number of growth cones analysed is indicated above each bar. ActD and CHX prevent the decrease in growth cone collapse induced by Shh or SAG. *P<0.05 (contingency table with Fisher's exact test). Scale bar: 25 µm in A,B; 20 µm in C.

 

Figure 5
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  		Fig. 5. Electroporation of Ptc1{Delta}loop2 effectively blocks Shh signal transduction in the central retina. (A-C) Coronal sections of E16.5 retinas electroporated with EGFP and Ptc1{Delta}loop2 (B,C) or EGFP alone (A) and immunostained with anti-Isl2 (C-RGCs) or anti-Zic2 (I-RGCs, arrows). (D) Quantitative RT-PCR analysis of Ptc1, Ptc2 and Hip1 mRNA levels. Values were normalised to 18S rRNA. In Ptc1{Delta}loop2-electroporated retinas, the endogenous levels of Ptc1 (Ptc1 3'UTR), Ptc2 and Hip1 mRNA are significantly reduced. Primers recognising the Ptc1{Delta}loop2 form (Ptc1) confirmed effective plasmid transduction. Values represent the mean ± s.e.m. of two independent experiments performed in triplicate. *P<0.05,**P<0.01 (Student's unpaired t-test). Scale bar: 200 µm.

 

Figure 6
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  		Fig. 6. Repression of Shh signal transduction in C-RGCs causes intraretinal guidance defects. Confocal images of flat-mounted (A-C,H,I) and horizontal sections (D-G) from E16.5 mouse retinas electroporated with EGFP or EGFP-Ptc1{Delta}loop2. Immunolabelling for Pax2 is in red (F). In Ptc1{Delta}loop2-positive retinas, radial organisation is lost (C,F), fibres turn in aberrant directions including towards the periphery (arrows in C,F,G-I). Growth cones (yellow arrows, C) accumulate in the optic disc (od). The boxed regions in A and D indicate the locations of the high-magnification images shown in B and E, respectively. Scale bar: 200 µm in A,D,G; 75 µm in B,C,H,I; 100 µm in E,F.

 

Figure 7
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  		Fig. 7. Additional defects caused by the repression of Shh signal transduction in C-RGCs. Confocal images of the optic nerve (A-C) or chiasm (D-F) from EGFP (C,F) or EGFP/Ptc1{Delta}loop2 (A,B,D,E) transfected mouse embryos at E16.5. In EGFP/Ptc1{Delta}loop2-positive embryos, a few fibres leave the nerve (A,B, arrow), project to the ipsilateral optic pathway (D, arrows), form tangles (E, white arrow) or turn back (E, yellow arrows). The boxed region in A indicates the location of the high-magnification image shown in B. (G,H) Schematic view of the behaviour of visual axons in control (G) and EGFP/Ptc1{Delta}loop2-transduced (H) C-RGCs. C-RGC axons are in green, I-RGC axons in red. Blockade of Shh signal transduction causes aberrant intraretinal growth (1,2), abnormal optic nerve (3) or chiasm/tract (4,5) turnings of C-RGCs axons. oc, optic chiasm; on, optic nerve. Scale bar: 200 µm in A; 155 µm in B-F.

 

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