Figure 3

Gradual depletion of retinal progenitor cell (RPC) population in the Ccnd1^-/- retina is caused by enhanced cell cycle exit. (A-F) Wild-type (wt) and Ccnd1^-/- retinas were labeled with an antibody against PCNA from E12 to P0. Dashed lines in (A, B, D, E) demarcate the differentiated cell layer (DCL) from the neuroblast layer (NBL). Brackets in (F) show the 'apical gap' in the P0 mutant retina. (G) Quantification of PCNA⁺ cells from E12, E14.5 and P0 retinas. (H) Schematic representation of cell cycle exit assay. (I-N) Wild-type and Ccnd1^-/- retina samples, collected at 24 h following a single bromodeoxyuridine (BrdU) injection at E13.5, were co-labeled with antibodies against PCNA and BrdU to measure rate of cell cycle exit, as outlined in (H). Arrowheads in (I-N) indicate cells that had exited the cell cycle in the last 24 h. (O) Quantification of exited cells (BrdU⁺, PCNA^-) as a percentage of BrdU⁺ cells at E14 and P0.5. Abbreviations: DCL, differentiated cell layer; L, lens; NBL; neuroblast layer NR; neural retina. Scale bars: 100 μm; (E) is representative for (B, E); (F) for (A, C, D, F); (N) for (I-N).