Fig 7

(A) mCherry fluorescence at the 17-somite stage in Tg(nkx2.3:mCherry) embryos injected with 4 ng cMO, 0.25 ng bmp2b MO, 2 ng bmp4 MO, and 4 ng bmp7a MO, respectively. Scale bar, 50 μm. (B-C) Quantification of the number of the mCherry⁺ pouch progenitors (B) and the pericardial progenitors (C) from embryos of each group shown in (A). Student’s t-test, ****P<0.0001. ns, non-significant. (D-E) Alteration of the nkx2.3 expression pattern in swr^ta72 mutants at the 14-somite stage (D) and embryos injected with 0.25 ng bmp2b MO at the 17-somite stage (E). Black dotted lines indicate the region where the pouch progenitors are located. (F) mCherry fluorescence at the 17-somite stage in Tg(nkx2.3:mCherry) embryos injected with 1 ng cMO or bmp2b MO. Scale bar, 50 μm. (G) The expression of nkx2.3 in wild-type embryos injected with indicated MOs. (H-I) mCherry fluorescence in heated Tg(nkx2.3:mCherry;hsp70l:caBmpr1b-GFP) embryos. Embryos injected with 0.25 ng cMO or bmp2b MOs were heat shocked at 9 hpf for 20 min, and then harvested at the 17-somite stage for confocal imaging (H). Scale bar, 50 μm. The numbers of mCherry⁺ pericardial and pouch progenitors were quantified from heat shocked-embryos with or without GFP expression (I). The group values are expressed as mean±s.d. Student’s t-test, ****P<0.0001. ns, non-significant. (J) p-Smad1/5/8 levels were dramatically reduced in in the pharyngeal endoderm of bmp2b morphants. cMO or bmp2b MO-injected Tg(sox17:GFP) embryos at the 6-somite stage were stained for anti-p-smad1/5/8 and anti-GFP antibodies. Representative dorsal confocal images were shown in the upper panels. Boxed areas are enlarged and presented in the middle panels. The orthogonal sections taken from the boxed areas were shown in the lower panels. Scale bar, 50 μm. (K-L) Knocking down bmp2b resulted in the failure of pharyngeal pouch progenitor specification. Tg(nkx2.3:mCherry;sox17:GFP) embryos were injected with ng bmp2b MO at one-cell stage and harvested at the 10-somite stage for confocal imaging analysis (K). Arrowheads indicate the pouch progenitors expression both mCherry and GFP. Scale bar, 20 μm. The number of mCherry⁺/GFP⁺ cells was statistical analyzed in (L). Student’s t-test, ****P < 0.0001.