DEV023663 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Effects of Shh on VT and DN retinal explants outgrowth. Explants from VT or DN E14.5 retinas were cultured in collagen gel in the presence of PBS or Shh (1 µg/ml) directly added to the medium (A) or immobilised on acrylic beads (B). The effect of Shh-beads was antagonised by the addition of cyclopamine (Cyc, 5 µM) directly to the culture medium (B). After 48 hours, explants were fixed and immunostained with anti-tubulin βIII antibody to visualise the extent of outgrowth. (A) Quantification of the area covered by retinal neurites (in pixels) from VT and DN retinal explants in the presence or absence of soluble Shh. Shh significantly increases the outgrowth from VT explants but does not affect that from DN. *P<0.05, Student's unpaired t-test. (B) Quantification of the length (in pixels) of DN or VT retinal neurites grown in the proximity of control or Shh-impregnated beads. Note that Shh-soaked beads significantly reduce neurite length from DN explants, an effect reversed by the Shh signalling inhibitor cyclopamine. No significant effects were observed with VT explants. *P<0.05, **P<0.01, one-way ANOVA. In both charts, bars represent mean ± s.e.m. The number of explants analysed in each condition is indicated in brackets above each bar.

• Supplemental Figure S2 -

  Fig. S2. Neutralisation of Shh causes abnormal turning of C-RGC in the ipsilateral optic tract and growth cone accumulation in the midline region without affecting chiasm specification. (A,B) Confocal images showing DiI-labelled fibres at the chiasm midline of E18.5 control and anti-Shh-treated embryos. An abnormal number of growth cones are present in the path of anti-Shh-treated embryos (inset in B) as compared with controls. (C-F) Frontal sections through the optic chiasm from control (C,E) and anti-Shh-treated (D,F) E18.5 embryos immunostained for Gfap (green; C,D) and SSEA-1 (Fut4) (red; E,F). Sections were counterstained with Hoescht (blue). There is no difference in the chiasm organisation among the embryos. (G,H) Quantification of the area occupied by retrograde DiI-labelled fibres in ipsilateral (G) and contralateral (H) retinas from control (n=4) and anti-Shh-treated (n=7) embryos. The bar charts illustrate the percentage of the total area covered by labelled fibres in the four quadrants of the retinas after retrograde unilateral DiI filling of the optic tract. Control and treated embryos showed no differences in the extent of labelling in the ipsilateral VT quadrant. The ipsilateral DT quadrant of the treated embryo, however, shows a clear increase in labelled fibres. The extent of labelling in contralateral retinas from control or treated embryos appeared fairly similar. cot, contralateral optic tract; oc, optic chiasm. Scale bar: 107 µm in a,B; 36 µm in inset in B; 200 µm in C-f.

• Supplemental Figure S3 -

  Fig. S3. Shh antibodies specifically label floor plate but not DRG cells. (A) Dorsal root ganglia (DRG, a negative control) and (B) floor plate (FP, a positive control) explants were immunostained with antibodies against Shh (green). Cell morphology was highlighted by phalloidin staining (red). As expected, Shh-positive staining was observed in FP cells but not in DRG growth cones. (C) No labelling is present in DN retinal explants incubated only with secondary antibodies. Scale bar: 10 µm.

• Supplemental Figure S4 -

  Fig. S4. Shh blockade causes intraretinal misprojections of C-RGC axons. (A,B) Representative confocal images of flat-mounted retinas from E18.5 anti-Shh-treated embryos electroporated with EGFP to visualize retinal axons. EGFP⁺ fibres do not enter the optic disc but project to the periphery. od, optic disc. Scale bar: 80 µm in A; 165 µm in B.