DEV02096 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) b-Galactosidase staining in an a-Cre;ROSA26R reporter mouse at P3. The blue signal indicates Cre activity, which in this case is restricted to the peripheral retina. (B-E) BrdU staining in the retina of wild-type and a-Cre;Shh^-/c mice at the indicated ages. White arrows in C and E indicate the region where Gli expression is downregulated. Scale bar: 100 mm.

• Supplemental Figure 2 -

  Fig. S2. RGC development is increased in the absence of Shh. (A) Quantification of RGC generated at E16. RPC were labelled at E16 with BrdU and the number of Brn3b⁺ cells that were heavily BrdU labelled was quantified in the central and peripheral retina of wild-type and a-Cre;Shh^-/c mice at P0. *P<0.005. (B) The distribution of E13 birthdated cells in the outer nuclear layer (ONL), inner nuclear layer (INL) and RGC layer was compared in the central and peripheral retinas of wild-type and a-Cre;Shh^-/c mice at P0. Although the ONL is not fully differentiated at this stage, we counted apically located nuclei for this analysis. The increase in the proportion of birthdated cells in the RGC layer is offset by a corresponding reduction of birthdated cells in the INL in the peripheral retina of a-Cre;Shh^-/c mice.

• Supplemental Figure 3 -

  Fig. S3. RPC proliferation and Müller and bipolar cell development in the perinatal retina is dependent on Hh signalling. Inhibition of Hh signalling reduced the proportion of S-phase cells in reaggregate retinal cultures and the proportion of Muller glia and bipolar cells in retinal explants. Re-aggregate retinal cultures and explants were established from E18 retinas and treated with anti-Hh antibodies for 3 days (re-aggregate) and 10 days (explants), dissociated into single cell suspensions and stained with anti-BrdU, anti-CRALBP and anti-PKC antibodies. *P<0.005, **P<0.05.

• Supplemental Figure 4 -

  Fig. S4. Growth factor signalling on Mycn expression and bipolar cell development in retinal explants. (A) In situ hybridisation for Gli and Mycn mRNA in E18 explants cultured for one day in the presence of recombinant Shh-N protein. (B) In situ hybridisation for cyclin D1 and Chx10 mRNA in retinal explants from P1 wild-type and a-Cre;Shh^-/c(mutant) mice that were cultured under the indicated conditions for 2 days. (C) Retinal explants from P3 wild-type mice were cultured for 6 days under the indicated conditions and stained anti anti-PKC antibodies to detect bipolar cells. Scale bar: 100 mm.