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Fig. 4. XHes2 is involved in retinal cell fate decisions. (A-F) XHes2 overexpression biases retinal precursors towards gliogenesis. (A) Percentage of retinal cell types observed in stage 41 retinas upon lipofection with GFP alone (control; 1244 cells, 11 retinas) or GFP plus XHes2 (939 cells, 13 retinas). (B-F) Typical sections of retinas transfected with GFP alone (B) or GFP plus XHes2 (C-F) showing the dramatic increase in Müller cell number (white arrows in B and C), as confirmed by anti-CRALBP immunostaining (E,F). White arrows in D-F point to cells with a Müller morphology that are indeed stained by CRALBP. (G-L) Two morpholinos were designed (G) that specifically affect in a dose-dependent manner the expression of a chimaeric XHes2-GFP construct, as assessed by in vitro translation (H) and in vivo GFP fluorescence (I-L) experiments. (M) XHes2 knockdown decreases the percentage of Müller cells (control Mo: 1144 cells, 10 retinas; Mo1: 2028 cells, 12 retinas; Mo2: 1534 cells, 10 retinas; XHes2: 432 cells, four retinas; XHes2+Mo1: 84 cells, three retinas). (N,O) XHes2 affects neuronal cell type specification. (N) Schematic representation of XHes2 variants employed. (O) Percentage of retinal cell types observed in stage 41 retinas co-lipofected with GFP plus XHes2-W (1200 cells, 12 retinas) or GFP plus XHes2-W-VP16 (1072 cells, 11 retinas). Values are given as mean±s.e.m. ^*P<0.05, ^**P<0.01, ^***P<0.001 (Student's t-test). Abbreviations: AM, amacrine cells; BP, bipolar cells; GC, ganglion cells; GCL, ganglion cell layer; HC, horizontal cells; INL, inner nuclear layer; PR, photoreceptor cells; MU, Müller glial cells; ONL, outer nuclear layer.

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