Fig 6

(A) bmp2b expression during early somite stages. In situ hybridization of bmp2b in whole mount embryos at the 6-somite stage (left panel). Sagittal section of the same embryo was shown in the right panel. The black dotted line indicates the plane of the section. (B) Whole-mount RNAscope assays showing expression of egfp in lateral pharyngeal endoderm (green) and bmp2b in pharyngeal ectoderm (red) of Tg(nkx2.3:EGFP-CAAX) transgenic embryos at the 10-somite stage. Left panel shows the merged sections in X-Y view (Scale bar, 50 μm); the right panel shows higher magnification orthogonal section of the boxed area in the left panel (Y-Z view; Scale bar, 10 μm). (C) In situ hybridization of nkx2.3 at 36 hpf in wild-type embryos injected with 4 ng cMO, 0.25 ng bmp2b MO, 2 ng bmp4 MO, and 4 ng bmp7a MO, respectively. Note that the expression of the pouch marker nkx2.3 was not notably disrupted in bmp4 MO or bmp7a MO-injected embryos, but was depleted in bmp2b morphants. (D) Malformed lateral pharyngeal endoderm (green) in 0.25 ng bmp2b MO-injected Tg(sox17:GFP) embryos at 36 hpf. Scale bar, 50 μm. (E) Overactivation of BMP signaling rescues the pouch formation in bmp2b-deficient embryos. Tg(hsp70l:caBmpr1b-GFP) embryos injected with 0.25 ng cMO or bmp2b MOs were heat shocked at 9 hpf for 20 min, and then harvested at 36 hpf for in situ hybridizations with nkx2.3 probe. (F-I) The expression of foxa1 (F), nkx2.3 (G), nkx2.7 (H), and pdgfαa (I) at 26 hpf in bmp2b MO-injected embryos. The black dotted lines in (G, H and G) indicate the pharyngeal regions. The black arrowheads in (F) indicate the first pouch.