Fig. 3. Differential expression of Mitf isoforms during mouse eye development. (A) RT-PCR analysis on RNA isolated from wild-type whole optic vesicles (E9.5-10.5) or separate RPE+choroidal and retinal fractions (E11.5-P0). (B) RT-PCR (left, lanes 1-6) and real-time PCR (right) for RNA isolated from Mitf^mi-bw/mi-bw eyes, which lack neural crest-derived melanocytes. (Lanes 1-3) E15.5 eyes were dissected as in A and 100 individual cells were microscopically selected as described in Materials and methods. Lane 1, mesenchymal cells; lane 2, RPE cells; lane 3, retinal cells. The asterisk in lane 2 indicates that the corresponding cDNA was diluted 1:10 for the reaction with pan-specific primers. (Lanes 4-6) Pooled fractions from dissected E15.5 eyes as indicated. Lane 4, mesenchymal fraction; lane 5, RPE/mesenchymal fraction; lane 6, retinal fraction. (Right) Real-time PCR. RNA was prepared from separately pooled RPE/mesenchymal and retinal fractions from E11.5 and E15.5 eyes. Results are expressed as mean of absolute amounts of the respective cDNAs and are calculated taking into account that RPE cells represent 7% of the cells in the RPE/mesenchymal fraction. Significance of the difference between RPE and retina (P-values, Student's t-test; pools of 20 RPE/mesenchymal and retinal fractions each for E11.5, and of 12 RPE/mesenchymal and retinal fractions each for E15.5; four separate assays in triplicate per pool) for E11.5 A-Mitf, >0.1; J-Mitf, <0.1; H-Mitf, <0.001; D-Mitf, <0.00001; and for E15.5 A-Mitf, <0.01; J-Mitf, <0.01; H-Mitf, <0.0001; D-Mitf, <0.001.