Fig. 2.

Expression of Mitf RNAs and proteins containing exon 1B1b. (A) In situ hybridization with an exon 1B1b-specific probe on frontal sections of the developing eye at the indicated time points. Arrowheads at E9.5 indicate expression throughout the optic vesicle, and arrowhead at E10.5 indicates reduced expression in the distal optic vesicle. In a control section of an Mitf^mi-rw/mi-rw embryo, which lacks exon 1B1b, there is no labeling. (B) Antibody characterization and Mitf expression in RPE/choroid and retina. A-, H-, D-, and M-Mitf cDNAs were expressed in NIH3T3 cells and extracts blotted with polyclonal anti-1B1b MITF antibodies (top panel) or monoclonal anti-MITF carboxyl terminal-domain antibodies (anti-CTD, middle panel). The bottom left panel shows a combined immunoprecipitation (IP)/immunoblot (IB) of NIH-3T3 cells transfected with A-Mitf. The bottom right panel shows a similar IP/IB of extracts from RPE/choroidal and retinal fractions from 100 wild type eye primordia (E12.5). Arrowheads point to MITF isoforms with distinct electrophoretic mobilities. (C) Immunohistochemistry of frontal cryosections of embryos of the indicated genotypes and ages using anti-1B1b MITF and anti-pan MITF antibodies. Arrows in panel labeled wt-E15.5 point to M-MITF expressing neural crest-derived melanocytes that are only seen with pan-MITF-specific antibodies. Note that “wt” refers to Tyr^c (albino) embryos carrying a wild type Mitf allele. RPE, retinal pigment epithelium; ret: retina; CMZ: ciliary margin zone.