Fig. 3

Teeth develop only once a periderm-like layer has covered the endoderm. (A) From 48 hpf onwards krt4-positive cells (white arrowheads) cover the (unlabeled) endodermal lining of the pharynx (white arrows) at the posterior margin of pouch 6 (P6). The outline of the endoderm is indicated by a dotted line. (B) Same area as in A, shown in TEM. The originally two layers of endoderm, composed of high cylindrical cells, now become separated from each other by flattened to cuboidal periderm-like cells (white arrowheads, outlined by a dotted line). Between the latter, the first lumina have appeared (red arrow). These will separate dorsal from ventral pharyngeal epithelium, each constituted of a basal endodermal and a superficial, periderm-like layer. (C) The superficial layer (so-called midline cells, outlined by a white dotted line) starts to express sox17, even stronger than the basal endodermal layer (white arrow). (D?F) Control Tg(krt4:gfp) embryos at 72 hpf, treated with DMSO (dimethylsulfoxide) only, showing opening of pouch 2 (P2) into a gill slit (D), yet a still closed pouch 6 (E) and tooth germs clearly present (F, thick arrows). Note the presence of a krt4-positive layer (E and F white arrowheads) covering the unlabeled endoderm (indicated by a dotted outline in E and F). (G?I) Corresponding images of a DAPT-treated 72 hpf Tg(krt4:gfp) embryo at levels corresponding to those shown in D?F. Note opened pouch 2 (G), closed pouch 6 (H), presence of a krt4-positive layer (H and I, white arrowheads) covering the endoderm (outlined by a dotted line in H and I), and clear presence of tooth germs (thick arrows in I), similar to control animal. (J?L) WT embryos at 72 hpf showing an open pouch 2 (J), open pouch 6 (K), and tooth germs clearly present (L, thick arrows). (M?O) mib?/? mutant embryo at levels corresponding to those shown in J?L. Note open pouch 2 (M), closed pouch 6 (N), presence of midline cells covering the endoderm (N, white arrowheads), and clear presence of tooth germs (O, thick arrows). Endoderm is outlined by a dotted line in N and O. 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; 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 in A, C?O, and 10 µm in B.)