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Fig. 4 Defects in signaling and absence of teeth coincide with absence of a periderm-like layer. (A?C) WT embryo at 96 hpf. Pouch 2 is open (A), pouch 6 is opening (B), midline cells cover the endoderm and tooth germs are present (C, thick arrows). (D?F) WT embryo at 96 hpf treated with cyclopamine A to inhibit the Shh pathway. Note open pouch 2 (D), pouch 6 contact (E), presence of midline cells covering the endoderm but clear absence of tooth germs (F, thick arrows). (G?I) WT embryo at 80 hpf, showing an open pouch 2 (G), yet a still closed pouch 6 (H), presence of midline cells (I, black arrowhead) covering the endoderm (outlined by a dotted line in H), and tooth germs clearly present (I, thick arrows). (J?L) WT embryo at 80 hpf, treated with 25 µM SU5402 between 40 and 80 hpf, imaged at levels corresponding to those shown in G?I. Note open pouch 2 (J), closed pouch 6 (K), presence of midline cells (K, black arrowhead) covering the endoderm (outlined by a dotted line), and clear presence of tooth germs (L, thick arrows), similar to control animal. (M?O) WT embryo at 72 hpf, showing an open pouch 2 (M), yet a still closed pouch 6 (N), presence of midline cells (N, black arrowhead) covering the endoderm (outlined by a dotted line in N) and tooth germs clearly present (O, thick arrows). (P?R) WT embryo at 72 hpf, treated with 25 µM SU5402 between 40 and 72 hpf, imaged at levels corresponding to those shown in G?I. Note open pouch 2 (P), closed pouch 6 (Q), presence of midline cells (Q, black arrowhead) covering the endoderm (outlined by a dotted line in Q), and absence of tooth germs (R, thick arrows). Cartoons show representative postembryonic developmental stages and level of transverse sections shown in the different figures (boxed areas). Dark gray: cartilage; medium gray: endoderm; light gray: other tissues. Abbreviations: asterisk: pharyngeal lumen; b: brain; CB5: ceratobranchial 5, e: eye; nt: notochord; ov: otic vesicle; P2: pouch 2; P6: pouch 6; >P6: level posterior to pouch 6; y: yolk. (Scale bars, 50 µm.)