Tbx1 is required in the nkx2.5-positive mesoderm for pouch development. (A-E) Fluorescent in situ hybridization for tbx1 (green) and GFP immunohistochemistry to detect her5:GFP-positive endoderm (red) at 34hpf. In all 58 wild-type siblings, tbx1 expression was observed in her5:GFP-positive endoderm (arrows), adjacent mesoderm (arrowheads) and the ear (asterisk). In all 21 tbx1 mutants, all tbx1 expression was lost. tbx1 expression was selectively restored in the her5:GFP-positive endoderm of all 17 nkx2.3:Tbx1; tbx1-/- embryos and in the mesoderm of all nine nkx2.5:Tbx1; tbx1-/- embryos. Inclusion of both transgenes restored tbx1 expression over a broader area (brackets for presumptive endoderm and arrowheads for presumptive mesoderm) in all four nkx2.3:Tbx1; nkx2.5:Tbx1; tbx1-/- embryos, although for technical reasons her5:GFP was not included. (F-J) In wild-type zebrafish at 34hpf, immunohistochemistry for Alcama (green) labeled five pouches (p1-p5). nkx2.5:Tbx1; tbx1-/- and nkx2.3:Tbx1; nkx2.5:Tbx1; tbx1-/- embryos displayed partial rescue of pouches compared with tbx1 mutants, whereas nkx2.3:Tbx1; tbx1/ embryos did not. Sensory ganglia are indicated with red asterisks. (K-O) Fluorescent in situ hybridization for dlx2a (green) and GFP immunohistochemistry for her5:GFP (red) at 30hpf. In wild-type zebrafish, dlx2a was expressed in the neural-crest-derived mesenchyme of each arch (numbered), with higher expression in posterior arches. In all tbx1-/- (n=9), nkx2.3:Tbx1; tbx1/ (n=6), nkx2.5:Tbx1; tbx1-/- (n=4) and nkx2.3:Tbx1; nkx2.5:Tbx1; tbx1-/- (n=4) embryos, dlx2a expression was reduced in the second arch and lost from the more posterior arches. For technical reasons, her5:GFP was not included in O. (P-T) Ventral whole-mount views of dissected facial cartilages. Wild-type zebrafish (P) invariantly formed five ceratobranchials (CBs) on each side. CBs were missing in all tbx1 mutants (Q, n=41) and not recovered in nkx2.3:Tbx1; tbx1-/- (R, n=23), nkx2.5:Tbx1; tbx1/ (S, n=26) or nkx2.3:Tbx1; nkx2.5:Tbx1; tbx1/ (T, n=24) embryos. (U) Quantification of pouch defects as assessed by Alcama staining in wild-type zebrafish (n=342), tbx1-/- (n=63), nkx2.3:Tbx1; tbx1-/- (n=54), nkx2.5:Tbx1; tbx1-/- (n=22) and nkx2.3:Tbx1; nkx2.5:Tbx1; tbx1-/- (n=23). Data represent mean±s.e.m., P values are shown. n.s., not significant. Scale bars: 40μm (A-O).
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