Fig. 2.

Ablation of pouch endoderm impairs PAA progenitor specification. (A) Confocal images of live Tg(nkx2.5:ZsYellow;nkx2.3:mCherry) embryos. The formation of mCherry⁺ pharyngeal pouches (red) coincides with the emergence of ZsYellow⁺ PAA clusters (green). Pharyngeal pouches and PAA clusters or sprouts are numbered with yellow or green numbers, respectively. Scale bar: 50 μm. (B) Confocal images of live Tg(nkx2.5:ZsYellow) embryos showed that injection of 8 ng sox32 MO resulted in complete absence of PAA sprouts and PAA tubular structures. The ratios of affected embryos are indicated. Scale bar: 50 μm. (C,D) Embryos were exposed to 10 mM MTZ from bud stage to 38 hpf, and then harvested at the indicated developmental stages for in situ hybridization (C) or in vivo confocal imaging (D). Numbers in C indicate PAA3-PAA6. Scale bar: 50 μm. (E,E′) The scl, etv2 and tie1 transcripts were evaluated by in situ hybridization in Tg(nkx2.3:KalTA4-p2a-mCherry;UAS:NTR-mCherry) embryos treated with DMSO or 10 mM MTZ (E). The average numbers of scl⁺, etv2⁺ and tie1⁺ PAA angioblast clusters were quantified from three independent experiments and the group values are expressed as mean±s.d. (E′). Student's t-test, ***P<0.001. (F) Expression analysis of nkx2.5 in pouch endoderm-depleted embryos. Black arrowheads represent the expression of nkx2.5 in the developing heart.