Fig. 2.

Hedgehog signaling is necessary and sufficient for basement membrane dissolution. (A–C, E–G′) Sagittal sections through primary mouth stained for β-catenin in magenta, fibronectin (FN) and nuclei in green. (Fg) foregut, (BM) basement membrane, (Fb) forebrain, (Cg) cement gland. (A–C) Stage 24. (A) Embryo treated with 10 μM SANT1 from the 2-cell stage. (A′) Magnified view of endoderm–ectoderm interface and basement membrane. FN is observed (white arrowhead) (n=7). (B) Control. (B′) FN immunofluorescence indicates the presence of BM between foregut and ectoderm (white arrowhead, n=7). (C) Embryo treated with 250 μM purmorphamine. (C′) No FN immuofluorescence is observed between foregut and ectoderm (open white arrowhead, n=6/7). (D–D′) Schematic indicating anatomy of sections represented in B–B′. Fibronectin-rich basement membrane separates foregut and ectoderm. (E–G′) Stage 26. (E) Embryo treated with 10 μM SANT1. (E′) shows persistent FN immunofluorescence (white arrowhead, n=8). (F) Stage 26 control. (F′) Almost no BM FN is observed in controls (open arrowhead, n=12). (G) Stage 26 embryo treated with 100 μM purmorphamine. (G′) No FN immuofluorescence is observed (open white arrowhead, n=10). (H–H′) Schematic indicating anatomy of WT sections represented in F–F′. Fibronectin-rich basement membrane is absent or broken, foregut and ectoderm cells mix (red arrow). (J–L) Sagittal sections of stage 39 tadpoles stained for β-catenin (magenta) and DAPI. (J) Tadpole treated with 10 μM SANT1 (n=5). (K) Control tadpole. BPM is observed as a single layer epithelium (n=6). (L) Tadpole treated with 100 μM purmorphamine. No BPM is present (n=4). Scale bars indicate 50 μm. (M) Schematic indicating anatomy of WT sections represented in (K). Buccopharyngeal membrane (BPM) separates foregut from external environment and indicates site of future mouth.