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 gdf6au768 eye (B). (C,D) ccnd1 is highly expressed in proliferating progenitors of wild-type CMZs (C), but reduced in gdf6au768 mutant eyes (D). (E-H) atoh7 and cdkn1c are expressed in committed precursors in wild-type CMZs (E,G) but in the gdf6au768 mutant eyes they are strongly downregulated (F,H). (I,J) Transverse sections of 3dpf sibling and gdf6au900 eyes carrying the vsx2:GFP (green) and atoh7:GAP-RFP (red) transgenes highlighting a reduced CMZ in the gdf6au900 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.).