Fig. 4. Hh signalling inhibits RGC development in vitro. (A-C) Brn3b staining in E12 retinal explants cultured for 48 hours under control conditions (A) or in the presence of recombinant Shh-N (B) or anti-Hh (C). The most dramatic change in Brn3b staining is observed in the peripheral region (closer to the lens) of the retinal explants. (D) Co-localization of silver grains marking [³H]thymidine labelled cells and Brn3b staining. RPC were labelled with [³H]thymidine for 4 hours, washed and cultured for an additional 48 hours. Arrows indicate double-labelled cells, arrowheads indicate Brn3b⁺ [³H]thymidine^– cells. (E) Dissociated cell scoring to quantify the proportion of RGCs among the [³H]thymidine⁺ cohort in E12 retinal explants cultured for 48 hours under the indicated treatment conditions. Treatment with isotype-matched anti-LFA antibodies (1E6) was no different than control and is not shown. n3 for each condition. (F) Proportion of S-phase, Brn3b⁺ and the non-RGC component of the Tuj1⁺ population (determined by subtracting the number of Brn3b⁺ from the number of Tuj1⁺ cells) in E12 explants cultured for 48 hours under the indicated conditions and analyzed by dissociated cell scoring. *P<0.005, ** P<0.005. Scale bars: 100 µm in A-C; 25 µm in D.