Dynamic expression of the Rb family during mouse retinal development. (A-C) Real-time PCR analysis of p107, Rb and p130 was done at seven stages of mouse retinal development. Data sets of three retinas per stage were analyzed twice, normalized to Gapdh and averaged. The standard deviations were within 5% of the mean. (D) Immunoblot analysis was done at five stages of mouse retinal development. For negative controls, MEF lysates prepared from knockout embryos were used. Positive controls were taken from 293T cells ectopically expressing the full-length cDNAs. Data were normalized to actin. (E-I) Immunofluorescent detection of Rb (green) in E14.5 and E17.5 retinas was overlaid on the nuclear counterstain (red). (J) Radioactive in situ hybridization analysis of Rb (blue silver grains) levels at P0 demonstrated broad expression in the onbl and inbl. This finding was consistent with immunofluorescent detection (green) of the protein (K). (L-O) Immunofluorescent detection of p107 (green) in E14.5 and E17.5 retinas was overlaid on the nuclear counterstain (red). (P) Radioactive in situ hybridization for p107 (blue silver grains) at E17.5 demonstrated broad expression in the onbl and little expression at P3 (Q). (R-T) Immunofluorescence of p130 at P12 (green) was overlaid on the nuclear counterstain (red). (U) Expression of p130 mRNA at P12 was consistent with the protein expression data. (V) Summary of the dynamic expression of the Rb family during retinal development. Abbreviations: GCL, ganglion cell layer; inbl, inner neuroblastic layer; INL, inner nuclear layer; MEF, mouse embryonic fibroblast; onbl, outer neuroblastic layer; ONL, outer nuclear layer. Scale bars: E, G, I, N, O and T, 10 μm; J, K, L, M, P, R and S, 25 μm.