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 mouse eye at the indicated time points. Arrowheads at E9.5 indicate expression of exon 1B1b-containing Mitf RNAs throughout the optic vesicle, and the 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 (right-most panel). (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 C-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 the 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.