Fig. 4.

Mitf^mi-rw/mi-rw mutant mice carry a genomic deletion in Mitf encompassing the exons 1H, 1D and 1B. (A) Phenotype of an Mitf^mi-rw/mi-rw mutant mouse. Generally, coat pigment patches and eye size are not correlated, as eye size is determined by the development of the RPE and coat patches by neural crest-derived melanocytes. The sequence on the right marks the base pairs flanking the genomic deletion, with the numbers of the first and last base of the depicted sequence referring to the positions on chromosome 6 according to assembly NCBIM36. The schematic diagram below shows the extent of the deletion and highlights the novel splice junctions that are generated between the upstream exons 1A, 1J, 1C, 1MC and 1E and the downstream exon 2A. For details, see text. (B) RT-PCR using RNA from E12.5 wild type and Mitf^mi-rw (rw) mutant embryos and the corresponding P0 newborns. Note increased band intensity in rw with exons 1A, 1J and 1E but decreased band intensity at E12.5 with pan-specific primers (exon 9). (C) Real time PCR from RNA of whole eyes harvested at the indicated ages, using primers as in (B). The results are expressed as RNA levels in rw mutants relative to those in corresponding wild type embryos (groups of 14 eyes each, three measurements each in triplicates). The increases in rw over wild type in 1A and 1J as well as the decrease in exon 9 at E12.5 are statistically significant (p<0.01, Student’s t test). P values for the increase in 1E are <0.02 at E12.5 and =0.13 at P0. No significant difference was found for exon 9 at P0 (p=0.27). (D) RT-PCR using primers spanning four exons. Note different-size products in wt and rw for isoform 1A. For isoform 1J, the expected larger product in wt is not seen because its relative level is low (see Fig. 2), but the smaller-size product in rw is visible. In 1E-4, white arrowhead points to the correct E-Mitf band.