Pharyngeal teeth develop from the endoderm lining pouch 6 after pouch 6 has made contact with the ectoderm. (A) Development of the placodes of teeth 4V1 (thick arrows) from the endoderm at 48 hpf; on each body side, the contact point of pouch 6 (P6, thin arrows) with the ectoderm is indicated by asterisks. Posterior to this cross-sectional level, there is no longer contact of endoderm with ectoderm. No lumen is present in the pharynx yet. Arrowheads point to flattened midline cells (encircled by yellow dotted line) squeezed between endodermal layers (surrounded by black dotted line). (B) At 96 hpf, more tooth germs have developed. Pouch 6 has opened into a gill slit anterior to this cross-sectional level (visible on Fig. 1J) and the pharynx is now wide open. (C and D) Tooth germs (4V1, thick arrows) at 56 hpf are derived from sox17-positive endoderm (outlined by a black and white dotted line, respectively). Periderm-like cells are outlined by a yellow dotted line. (E) Magnification of the tooth germ shown in D, indicating that folding into the prospective enamel organ occurs by the basal, sox17-positive, endodermal layer only. White and yellow dotted lines surround endoderm and periderm-like cells, respectively. (F–H) Successive stages in the formation of contact of pouch 6 (outlined by a dotted line) with the ectoderm. (F′–H′) Corresponding stages in Tg(sox17:egfp) embryos, immunostained for laminin. Depending on the angle of sectioning, pouch 5 can still be visible in the section along with pouch 6. Note breaking up of the basal lamina at 38 hpf (G′) and basal lamina of pouch 6 continuous with that underlying the ectoderm (H′). Arrowheads point to basal lamina of pouch 6 at its (prospective) contact place with skin ectoderm. (I) Cross-section of 72 hpf embryo at the level of P6 showing ongoing lumen formation (asterisks) (endoderm surrounded by dotted line). (J) At 96 hpf, pouch 6 has opened on either side into a gill slit. Cartoons show representative embryonic and 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: 3V1, 4V1, 5V1: first-generation teeth in positions 3, 4, and 5 of the ventral tooth row; 4V2: second-generation tooth in position 4V (compare ref. 24); b: brain; CB5: ceratobranchial 5; e: eye; nt: notochord; ov: otic vesicle; P6: pouch 6; WT: wild type; y: yolk. (Scale bars in A–D and F–J, 50 µm.), (Scale bar in E, 20 µm.)

Teeth do not form in case of defective pouch 6 contact with ectoderm. (A–F) vgo−/− mutant zebrafish at 5 dpf. (Middle) show midline with (arrow) or without (arrowhead) developing tooth germs; (Left and Right) show corresponding contact of pouch 6 with ectoderm and ensuing gill slit formation (Right) or absence thereof (Left), respectively. (G–I) Edema (thin arrow) causing extremely perturbed pouch formation in a vgo−/− mutant zebrafish at 96 hpf (G), tooth absence (H, arrowheads), and corresponding lack of contact of pouch 6 with ectoderm (I, arrowhead). (J–L) Extreme unilateral edema (J, thin arrow) in a 7 dpf laf−/− mutant zebrafish preventing pouch 6 contact with ectoderm, and corresponding tooth absence on that side (K, arrowhead), while pouch 6 at the contralateral side has opened into a gill slit (L) and a tooth is present (K and L, arrow). (M–O) Unilateral edema in a 96 hpf Tg(krt4:gfp) zebrafish preventing pouch 6 contact with ectoderm, and corresponding tooth absence on that side (M, arrowhead), while pouch 6 at the contralateral side has opened into a gill slit (O) and a tooth is present (N, arrow). Cartoons show the transverse sections from which the different details are taken (boxed areas). Dark gray: cartilage; medium gray: endoderm; light gray: other tissues. Abbreviations: asterisks: pharyngeal lumen; b: brain; ed: edema; nt: notochord; ov: otic vesicle; P6: pouch 6. (Scale bars, 50 µm.)

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.)

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.)