Fig. 5. Fgf3 and Fgf8 are necessary for EB placode induction. (A) fgf3+/-;fgf8+/- embryos were crossed to generate various genotypic combinations, including fgf3;fgf8 mutants. Resulting embryos were processed for in-situ hybridization with eya1, foxi1, pax2a, ngn1 and phox2b riboprobes, photographed and genotyped (genotypes are shown in the bottom left of each panels). All panels show lateral views, except eya1-expression panels, which show dorsal views. PPE is not affected in fgf3 or fgf8 mutants or fgf3;fgf8 double mutant. Consistent with our previous observations (Nechiporuk et al., 2005), fgf3 mutants lacked ngn1 and phox2b expression in glossopharyngeal and small vagal ganglia, whereas foxi1 and pax2a expression was normal. All markers were expressed normally in fgf8-/- embryos. However, foxi1 expression (brackets) was strongly reduced and pax2a, ngn1, and phox2b expression was either absent or strongly reduced in fgf3;fgf8 double mutants. Vagal neural crest is not affected in fgf3;fgf8 double mutants (arrowheads). fgf3+/-;fgf8-/-embryos displayed intermediate phenotypes, where expression of all markers were reduced but not absent. (B,C) Transverse sections of fgf3/8 double mutants and wild-type siblings were obtained from the whole-mounts processed for foxi1 (B) and pax2a (C) in situ (dashed lines in A indicate level of cross section). In contrast to wild type, the epithelium appears disorganized and foxi1 expression is not restricted to a single cell layer in fgf3;fgf8 mutants. pax2a expression is absent from the ectoderm in fgf3;fgf8 mutants. Scale bars: 50 µm. Abbreviations are as in Fig. 1; a, acoustic ganglion; al, anterior lateral line ganglion; op, otic placode.