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Fig. 6 Loss of RA does not affect pouch endoderm specification. A-E: Ventral views of 36-hpf nkx2.3-labeled control and DEAB-treated embryos. A: In control embryos, nkx2.3 is expressed in pharyngeal pouches 2-5 (arrow) and in the pharyngeal arch ectoderm (black arrowhead). B: In embryos treated with DEAB between 4-10 hpf, the length of the ectodermal domain is normal (white arrowheads) but endodermal pouches 3-6 are missing. The second endodermal pouch is enlarged (white asterisk). Also, nkx2.3 expressing medial cells extend to the posterior edge of the pharyngeal endoderm, indicating that the pharynx as a whole is not shortened (black asterisk). C: In embryos treated with DEAB between 16-36 hpf, the third pouch has formed but pouches 4-5 are absent. D: Treatment with DEAB between 10-36 hpf leads to a similar phenotype as in B. Only an enlarged second pouch is present. E: Shorter treatment with DEAB between 10-14 hpf only causes a slightly less severe phenotype than in D, which demonstrates that DEAB does not get washed out efficiently. F: At 100% epiboly, her5 is expressed in a cell population anterior to the midbrain-hindbrain boundary (mhb), which very likely contains endodermal pouch precursors. No difference in cell number was detected between control (F) and treated embryos (G). H: 30-hpf control embryo hybridized with nkx2.3 and insulin to visualize the pancreas. I: Simultaneous treatment with DEAB and 10-7 M RA between 4-10 hpf leads to almost normal endodermal pouch development by 30 hpf. J: Likewise, treatment of embryos with DEAB between 4-10 hpf, followed by immersion in 10-7 M RA rescues endodermal pouch development normally observed in embryos that are only treated with DEAB. However, RA treatment between 10-30 hpf is unable to restore the formation of the pancreas. This demonstrates that treatment with DEAB during gastrulation does not affect endodermal pouch specification. K-M: Loss of RA signaling between 4-10 hpf causes the loss of the postotic neural crest cells, which are not rescued by subsequent administration of RA. To visualize neural crest cells, treatments were performed in Tg(fli1-EGFP) embryos. fli1 also labels endothelial cells. Embryos were double labeled with nkx2.3 to visualize endodermal pouches. K: 24-hpf control embryo. Neural crest cells are present in pharyngeal arches 1-5 (pa1-pa5). Pharyngeal arches 5-7 have not formed at 24 hpf and the majority of the postotic neural crest cells are present as one population. L: 24-hpf embryo treated with DEAB between 4-10 hpf only possesses neural crest cells in pharyngeal arches 1 and 2 (pa1 and pa2) (L). Subsequent immersion of DEAB-treated embryos in 10-7 M RA rescues the formation of endodermal pouches (ep), as revealed by nkx2.3 but does not rescue the posterior neural crest stream (M). mhb, midbrain-hindbrain boundary; pa, pharyngeal arch; ep, endodermal pouch.