Fig. 3.

Pouch endoderm is necessary for BMP signal activation in the presumptive PAA progenitors. (A) The bmp2a, bmp2b, bmp4 and bmp5 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. (B) BMP signal was dynamically activated in the forming PAA clusters. Tg(nkx2.5:ZsYellow) embryos were harvested at indicated stages and subjected to immunostaining for p-Smad1/5/8 (red) and ZsYellow (green). The white dotted lines outline the PAA progenitor clusters and the purple dotted lines indicate the PAA sprouts composed of migrating angioblasts. Scale bar: 20 μm. (C) Tg(BRE:EGFP;nkx2.5:ZsYellow) embryos were immunostained for GFP (green) and ZsYellow (red) to visualize BMP-responsive cells and PAA clusters. Scale bar: 50 μm. (D) p-Smad1/5/8 levels were greatly decreased in pouch endoderm-depleted embryos. Tg(nkx2.5:ZsYellow;nkx2.3:KalTA4-p2a-mCherry;UAS:NTR-mCherry) embryos were treated with DMSO or 10 mM MTZ from bud stage to 36 hpf, and then stained for p-Smad1/5/8 (red) and ZsYellow (green). Scale bar: 50 μm. (E) Live confocal images of Tg(BRE:EGFP;nkx2.3:KalTA4-p2a-mCherry;UAS:NTR-mCherry) embryos treated with DMSO or 10 mM MTZ from bud stage to 36 hpf. Scale bar: 50 μm. (F) Tg(sox17:GFP) embryos were injected with 40 pg nkx2.3:noggin3-mCherry plasmids and 200 pg Tol2 transposase mRNA at the one-cell stage, and then harvested for in vivo confocal imaging to visualize pharyngeal pouches (green) and the expression of Noggin3-mCherry fusion proteins (red). Scale bar: 50 μm. (G) Pouch-derived Noggin3 significantly decreased the BMP signal activity in the pharyngeal region. Tg(nkx2.5:ZsYellow;BRE:EGFP) embryos were injected with 40 pg nkx2.3:noggin3-mCherry plasmids and 200 pg Tol2 transposase mRNA at the one-cell stage, and then embryos with abundant mCherry fluorescence in the pouches were selected at 36 hpf for immunostaining. Scale bar: 50 μm.