Fig. S3

Fgf3 and Fgf10a are not required during EB placode induction, proliferation, and survival, but they are required for the EB placode and NC interaction. (A, B) Confocal projections of TgBAC(foxi1:d2EGFP) (green) 26 hpf zebrafish embryos immunostained for β-Catenin (magenta). Images show unilateral transverse sections at the level of the glossopharyngeal/vagal placode (arrows). Note columnar morphology of the epithelial cells lateral to the otic vesicle in control (A) and fgf3+10a-MO embryos (B). (C) Average cell height of foxi1:d2EGFP+ cells measured in μm was unchanged in fgf3/10a-MO injected embryos compared to controls, measurement non-placodal cells medial to the foxi1+ cells are significantly shorter (Error bars: standard error of mean. ANOVA multiple comparison with Sidak′s correction; ***P<<0.001; ne25 cells from 5 individual embryos per condition). (D, E) Comparison of TUNEL+ cells or PH3+ cells per unit area of the prospective EB placodes between control and fgf3+10a-MO injected 18 hpf embryos reveals no change in cell death or proliferation at this stage (ne8 embryos per condition). (F, G) Confocal projections of 26hpf embryos derived from crossing Tg(sox10(7.2):mrfp) to TgBAC(foxi1:d2EGFP) parents. Control conditions show properly formed branchial arches (F; arrowheads), and mature placodes assembling within corridor like structures (F′, F′′). In fgf3+10a-MO embryo, a subset of branchial arches is absent (G; arrowheads); however the anterior and posterior most NC derived structures are still present. Foxi1-positive placodal ectoderm is present, albeit not properly organized at this stage (G′, G′′). Scale bars: 25μm (A, A′); 50μm (F).