Fig. 2. Expression of MITF and KIT does not depend on Ednrb, whereas that of tyrosinase does. Neural crest cultures were established from embryos of the indicated genotypes and kept in the absence of EDN3 for 2 days (A-D) or in the presence of EDN3 for 2 weeks (E,F). They were then subjected to double indirect immunofluorescent labeling using MITF- and ß-gal-specific antibodies (A,B), ß-gal-specific antibody alone (C,D), or tyrosinase- and ß-gal-specific antibodies (E,F). (A,B) Merged images show that regardless of the Ednrb genotype, MITF/ß-gal double-positive cells were generated. (C,D) Regardless of the Ednrb genotype, cells double-positive for cytoplasmic ß-gal (marking Ednrb expression) and nuclear ß-gal (marking Kit expression) were generated (arrows). Note that in cultures from Kit^lacZ/+ embryos, ß-gal staining is confined to the nucleus (Hou et al., 2000) (data not shown), and in cultures from Ednrb^lacZ/+ embryos, to the cytoplasm. (E,F) Merged images show Tyr/ß-gal double-positive cells along with ß-gal single-positive cells in Ednrb^lacZ/+ cultures (E) but only ß-gal single-positive cells in Ednrb^lacZ/Ednrb^lacZ cultures (H). Scale bar: 25 µm for A,B,E,F; 20 µm for C,D.