Multiple neural crest-derived phenotypes in mutant embryos. a Images of embryo morphology at 72 hpf. b to e Melanocyte defects in mutants at 48 hpf. Dorsal view with anterior to top. Scale bars, 1 mm. j to p and r to t) Alcian blue staining of cartilage reveals defects in neurocranium and viscerocranium formation in mutant embryos at 96 hpf. At least 15 embryos for each genotype were analyzed and representative samples are showed. Compared to single brg1 or med14 mutants, embryos bearing an additional copy of med14 or brg1 null allele show a more severe phenotype. Ventral view with anterior to the top. bh, basihyal; cb, ceratobranchial; ch, ceratohyal; ep, ethmoid plate; hs, hyosymplectic; m, Meckel’s cartilage; pc, parachordal plate; pq, palatoquadrate; tr, trabeculae. q Quantification of melanocyte number at 48 hpf on the trunks of control and mutant embryos. Twelve embryos for each genotype were counted. Error bars represent the SD

Neural crest cells are specified normally in mutant embryos. a to l: At 10 somite stage (14 hpf), expression of the neural crest specification markers foxd3, sox9b and snail1b were probed by RNA in situ hybridization. At least 20 embryos for each genotype were analyzed and representative samples are showed. Dorsal views with anterior to the top

Defects in skeletogenic neural crest differentiation in the jaw forming area of med14 and brg1 mutant embryos. a to d Neural crest cells migrated to the oral ectoderm in both control and mutant embryos. a Snapshot of migrating neural crest cells (marked by sox10:EGFP transgene) in control and mutant embryos at 15 somite stage. b and c The migrating neural crest expressed dlx2a and twist1a at 18 somite stage. d At 24 hpf, neural crest cells reached the brachial arches forming region and condensed. a and d: lateral view with dorsal to top and anterior to left. e to g The mutants showed mis-expression of genes involved in mesenchymal condensations and chondrocyte differentiation. b, c, e, f and g RNA in situ hybridization is shown for expression of dlx2a, tiwst1a, sox9a, dlx3b and hand2. At least 15 embryos for each genotype were analyzed and representative samples are shown. Hollow arrowheads indicate pharyngeal arches; red arrowhead in g indicates the heart tube of embryo. Lateral views with anterior to the left. Scale bars, 100 um

No apparent alterations in cell proliferation and cell death in arch-forming regions of mutant embryos. a to l Cell proliferation at 36 hpf was analyzed through BrdU incorporation. Neural crest cells were marked by sox10:EGFP transgene and revealed by immunostaining with anti-GFP antibody. Proliferating cells were marked by BrdU then revealed by immunostaining with anti-BrdU antibody. Lateral views with anterior to left. m to t Cell death at 36 hpf was analyzed through TUNEL assay. Lateral views with anterior to left. u Quantification of BrdU-positive cell in 2nd arch (dotted squares in a, d, g and j) in control and mutant embryos. Bars represent the SD. v Quantification of TUNEL positive cell in one side of pharyngeal arches in control and mutant embryos at 36 hpf. Bars represent the SD. Scale bars, 100 um

Mesoderm of pharyngeal arches and endodermal pouches are not affected in mutants. a to d: Expression of foxa1 at 32 hpf in pharyngeal endoderm. e to h: The expression of shh in the stomodeum epithelium at 48 hpf. i to l: At 36 hpf, expression of the endodermal pouches marker nkx2.3. m to p: At 36 hpf, expression of the pharyngeal arches mesoderm marker tbx1. a to d and i to l, dorsal view with anterior to the top. e to h and m to p, lateral view with anterior to the left

Wild type neural crest can contribute to jaw cartilage in mutant host. a Schematic diagram of the transplantation approach. Wild type sox10:EGFP transgenic donor cells are transplanted to the animal pole of wild type or mutant host embryos at 4 hpf. b to e At 24 hpf, donor-derived neural crest migration to the oral ectoderm is evident regardless of host genotype. Lateral views with anterior to the top. f to i At 72 hpf, donor-derived neural crest persistence and differentiation to cartilage is evident regardless of host genotype. Ventral views with anterior to the top. j to m At 72 hpf, cartilage staining reveals wild type donor-derived cells partially rescuing anterior neurocranium defects in med14 and med14; brg1 double mutant embryos. g and k represent images from the same embryo. Red arrowheads indicate cartilage derived from wild type donor cells; black arrowheads indicate host cell-derived cartilage. Dorsal views with anterior to the top. Scale bars, 100 um

Cell autonomous requirement for med14 and brg1 in neural crest cells for cartilage differentiation. a Schematic diagram of transplantation approach. Donor (wild type or mutant) sox10:EGFP transgenic embryos were injected with rhodamine-dextran lineage tracer and transplanted to the animal pole of wild type host embryos at 4 hpf. b Diagram showed the region was imaged at 80 hpf. c, g, k and o At 24 hpf, donor-derived neural crest migration to the oral ectoderm is evident regardless of donor genotype. Later views with anterior to the top. d, e and f; h, i and j; l, m and n; p, q and r Donor cell contribution to cartilage assayed at 80 hpf. Ventral views with anterior to the top. D’ and H’: higher magnificent views of regions indicated by red squares in b and e. Scale bars, 100 um