Fig. 2

gdf6a mutants exhibit decreased expression of stem, progenitor and committed cell genes and reduced proliferation within the CMZ. (A-H) Lateral views of whole-mount eyes from 3dpf embryos (genotype bottom left) showing expression (purple) of various genes (indicated bottom left) in the CMZ. (A,B) col15a1b, a marker of the peripheral putative stem cell compartment, is expressed in the sibling CMZ (A), but reduced in the dorsal and nearly absent in the ventral CMZ of the gdf6a^u768 eye (B). (C,D) ccnd1 is highly expressed in proliferating progenitors of wild-type CMZs (C), but reduced in gdf6a^u768 mutant eyes (D). (E-H) atoh7 and cdkn1c are expressed in committed precursors in wild-type CMZs (E,G) but in the gdf6a^u768 mutant eyes they are strongly downregulated (F,H). (I,J) Transverse sections of 3dpf sibling and gdf6a^u900 eyes carrying the vsx2:GFP (green) and atoh7:GAP-RFP (red) transgenes highlighting a reduced CMZ in the gdf6a^u900 mutant (white arrows). (K-N) Coronal sections immunostained for markers of proliferation (green; BrdU incorporation in K,L or PH3 in M,N) and then counterstained with SYTOX Orange (red nuclei). (O) Graph showing numbers of PH3⁺ mitotic retinal cells in eyes of gdf6a mutant and sibling embryos. All PH3⁺ cells in ten whole-mount 60hpf eyes were counted and graphed with standard error bars (95% confidence limits; Student′s t-test, ***P≤0.0004). (P) Graph showing real-time PCR quantification of gene expression changes of col15a1b, ccnd1, atoh7 and nr2f5 in 3dpf dissected mutant and wild-type retinas normalised to β-actin. Wild-type values for each gene were set to 1 and mutant fold changes were plotted relative to this value (±s.e.).