Multiple Epithelia Are Required to Develop Teeth Deep Inside the Pharynx

Multiple epithelia are required to develop teeth deep inside the pharynx

Veronika Oralováa,1, Joana Teixeira Rosaa,2, Daria Larionovaa, P. Eckhard Wittena, and Ann Huysseunea,3

aResearch Group Evolutionary Developmental Biology, Biology Department, Ghent University, B-9000 Ghent, Belgium

Edited by Irma Thesleff, Institute of Biotechnology, University of Helsinki, Helsinki, Finland, and approved April 1, 2020 (received for review January 7, 2020) To explain the evolutionary origin of vertebrate teeth from closure of the gill slits (15). Consequently, previous studies have odontodes, it has been proposed that competent epithelium spread stressed the importance of gill slits for pharyngeal tooth formation into the oropharyngeal cavity via the mouth and other possible (12, 13). channels such as the gill slits [Huysseune et al., 2009, J. Anat. 214, Gill slits arise in areas where ectoderm meets endoderm. In 465–476]. Whether tooth formation deep inside the pharynx in ex- vertebrates, the endodermal epithelium of the developing pharynx tant vertebrates continues to require external epithelia has not produces a series of bilateral outpocketings, called pharyngeal been addressed so far. Using zebrafish we have previously demon- pouches, that eventually contact the skin ectoderm at corre- strated that cells derived from the periderm penetrate the oropha- sponding clefts (16). In primary aquatic osteichthyans, most ryngeal cavity via the mouth and via the endodermal pouches and pouch–cleft contacts eventually break through to create openings, connect to periderm-like cells that subsequently cover the entire or gill slits (17–19). In teleost fishes, such as the zebrafish, six endoderm-derived pharyngeal epithelium [Rosa et al., 2019, Sci. pharyngeal pouches form from anterior to posterior, separating Rep. 9, 10082]. We now provide conclusive evidence that the epi- the prospective pharyngeal arches (19–21). The first pouch (P1) is thelial component of pharyngeal teeth in zebrafish (the enamel homologous to the spiraculum of chondrichthyans. It separates organ) is derived from medial endoderm, as hitherto assumed based mandibular from the hyoid arch but does not usually open any on position deep in the pharynx. Yet, dental morphogenesis starts longer in teleosts. The second pouch (P2) separates the hyoid arch only after the corresponding endodermal pouch (pouch 6) has made from the third pharyngeal arch (also called first branchial or gill contact with the skin ectoderm, and only after periderm-like cells arch), pouch 3 (P3) separates pharyngeal arch 3 from 4, and so on. have covered the prospective tooth-forming endodermal epithe- The pouches in vertebrates give rise to different organs crucial for DEVELOPMENTAL BIOLOGY lium. Manipulation of signaling pathways shown to adversely affect immune responses and calcium homeostasis (16, 22). In zebrafish, tooth development indicates they act downstream of these events. teeth develop on the seventh (last) pharyngeal arch, i.e., posterior – We demonstrate that pouch ectoderm contact and the presence of to pouch 6. Using various approaches, we have recently shown a periderm-like layer are both required, but not sufficient, for tooth that periderm (the initial epithelial covering of the embryo) par- initiation in the pharynx. We conclude that the earliest interactions tially invades the pouches and that endogenous cells that resemble to generate pharyngeal teeth encompass those between different the periderm cells phenotypically, spread over the endoderm epithelial populations (skin ectoderm, endoderm, and periderm-like – along the midline (23). Thus, at the time tooth formation is cells in zebrafish), in addition to the epithelial mesenchymal inter- initiated the pharynx epithelium is composed of a double layer: actions that govern the formation of all vertebrate teeth.

pharyngeal teeth | tooth evolution | germ layers | zebrafish Significance

Many vertebrates possess teeth deep in the pharynx. While n chondrichthyans, basal sarcopterygians, amphibians, and teeth are known to derive from odontodes (skin denticles), it is actinopterygians not only the jaw margins but also the roof and I unknown if an external epithelium is still required to produce a floor of the pharynx can be tooth bearing, constituting a pharyngeal— pharyngeal tooth, such as for odontode formation. Here, we next to an oral—dentition. Teeth—whether oral or pharyngeal— show that the epithelial enamel organ of pharyngeal teeth in are evolutionarily derived from odontodes, also called skin den- zebrafish is formed from endoderm, i.e. the internal germ layer. ticles, dermal skeletal elements of ancient jawless vertebrates. The However, teeth develop 1) only when this endoderm becomes homology between odontodes and teeth is now well documented covered by a layer of cells with features of a periderm, i.e., the (1–6). Apart from being elements of the dermal skeleton, teeth outer epithelial covering of the embryo; and 2) only when the and odontodes belong to the complex of ectodermal appendages endoderm has physically contacted the skin at the prospective whose development depends on reciprocal interactions between gill slits. Thus, multiple epithelia are engaged in tooth formation, the surface epithelium (ectoderm) and the underlying (neural whether oral (mammals) or pharyngeal (teleosts). crest-derived) mesenchyme (7, 8). Accordingly, mutations of the

ectodysplasin gene (EDA) or its receptor (EDAR) cause deficient Author contributions: P.E.W. and A.H. designed research; V.O., J.T.R., D.L., and A.H. per- development of hair, sweat glands, and teeth in humans, but also formed research; V.O., J.T.R., D.L., and A.H. analyzed data; and V.O., J.T.R., P.E.W., and of pharyngeal teeth, scales, and dermal fin rays in zebrafish (9). As A.H. wrote the paper. already noted by Charles Darwin, “Hairless dogs have imperfect The authors declare no competing interest. teeth” (ref. 10, p. 30). However, different from the mammalian This article is a PNAS Direct Submission. dentition that develops in an ectoderm-covered oral cavity (11), Published under the PNAS license. pharyngeal teeth in extant vertebrates develop in an endoderm- See online for related content such as Commentaries. covered pharynx. How it was possible for dermal skeletal elements 1Present address: Institute of Animal Physiology and Genetics, v.v.i., Czech Academy of to develop deep inside the pharynx remains to be elucidated. It Sciences, Laboratory of Odontogenesis and Osteogenesis, 602 00 Brno, Czech Republic. has been proposed that competent epithelium may have invaded 2Present address: Comparative, Adaptive and Functional Skeletal Biology, Centre of Ma- the pharyngeal cavity via any channel that provides communica- rine Sciences, University of Algarve, 8005-139 Faro, Portugal. tion between the skin and the pharynx (12–14). In gnathostomes 3To whom correspondence may be addressed. Email: [email protected]. with open gill slits the pharynx can be covered with teeth, whereas This article contains supporting information online at https://www.pnas.org/lookup/suppl/ in sarcopterygians (the lineage of tetrapods), pharyngeal teeth doi:10.1073/pnas.2000279117/-/DCSupplemental. eventually disappear in the course of evolution together with the First published May 12, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.2000279117 PNAS | May 26, 2020 | vol. 117 | no. 21 | 11503–11512 Downloaded by guest on October 2, 2021 a basal endoderm, that is overlain by a layer with periderm-like ectodermal layer covered by a periderm layer and sharply characteristics. delimited from the underlying mesenchyme by a distinct basal Using mutant and transgenic (Tg) zebrafish, as well as ex- lamina (Fig. 1 F and F′). At 38 hpf, the pouch endoderm has perimental manipulations (pharmaceutical inhibition experi- merged with the skin ectoderm, and the basal lamina between ments as well as mechanical perturbation of pouch development), pouch 6 endoderm and ectoderm is now fragmented (Fig. 1 G this study now tests whether pharyngeal tooth initiation depends and G′). At 40 hpf the pouch has thinned into a bilayer, covered on the presence of pouch–cleft contacts and/or the presence of the by a basal lamina, which continues imperceptibly with the basal periderm-like layer. We demonstrate that the enamel organ de- lamina underlying the ectoderm (Fig. 1 H and H′). These shape velops from the endodermal epithelium, as hitherto assumed changes match those observed on whole mounts (SI Appendix, basedonthelocalizationoftheteeth deep in the pharynx. However, Fig. S3 A–C). At the cross-sectional level of pouch 6, several dental morphogenesis starts only after pouch 6 has made contact lumina first appear along the midline at around 56 hpf and soon with the ectoderm, and only after a layer of periderm-like cells has become confluent (Fig. 1I). The pouch itself persists as a bilay- covered the prospective tooth-forming endodermal epithelium. We ered sox17-positive endodermal epithelium until the lumina conclude that the earliest interactions required to make a tooth merge into a single pharyngeal lumen and extend outwards, thus deep in the pharynx encompass those between different epithelial finally producing the open gill slit between the sixth and seventh populations, in addition to the epithelial–mesenchymal interac- arch at 72 hpf or beyond (Fig. 1J). tions that govern the formation of all vertebrate teeth. Taken together, tooth buds start to form from the endodermal layer ∼10 h after pouch 6 has contacted the ectoderm, yet well Results before the gill slit opens. Given this consistent developmental Pharyngeal Teeth Develop from the Endoderm Lining Pouch 6. The 1 sequence of contact of pouch 6 with the ectoderm (below referred first pair of teeth (designated as 4V , ref. 24) appears at around to as “pouch 6 contact”) and start of tooth formation, we next 48 h postfertilization (hpf) as a placodal thickening of the pha- tested whether this contact is a prerequisite for teeth to form, ryngeal epithelium on both sides of the midline at the level of using mutant zebrafish with anomalous pouches, and embryos pouch 6 (P6). More precisely, these teeth develop where the having experienced mechanical perturbation of pouch formation contact zone of pouch 6 with the ectoderm ends posteriorly 1 (SI Appendix, Table S1). (Fig. 1A). The development of tooth 4V is soon followed by Earlier, it was reported that van gogh (tbx1) mutants display that of its successor, 4V2, and the germs of teeth 3V1 and 5V1, 1 defective pouch formation (28, 29), thus providing an excellent medially and laterally of 4V , respectively (Fig. 1B). Using way to test for a link between pouch 6 contact and tooth for- Tg(sox17:egfp) zebrafish, we could establish that the enamel or- mation. We examined 30 specimens ranging from 48 to 144 hpf, gans are derived from sox17-positive epithelium (Fig. 1 C and D, – sectioned serially (not to miss any contact or tooth germ), and and see SI Appendix, Fig. S1 A C), clearly indicating that the assessed each body side separately (n = 56; not all sides could be epithelial component of the teeth is of endodermal origin. In scored unequivocally for the presence of pouch 6 contact). We particular, only the single layer adjoining the basal lamina folds determined whether tooth germs were present, and whether into the prospective enamel organ (compare, e.g., figure 2J in ref. pouch 6 endoderm contacted the ectoderm anterior to the po- 25 with Fig. 1E). The requirement for endoderm in pharyngeal sition of the tooth germs (Fig. 2 A–F and SI Appendix, Table tooth formation was confirmed by the study of casanova mutants. S1A). Teeth were present if pouch 6 had made contact with the Cas encodes a protein of the SoxF family that also includes ectoderm, possibly even opened into a gill slit (n = 20/56) (Fig. 2 Sox17 but it works upstream of Sox17 (26). casanova mutants do B and C, arrow). Teeth were absent if there was no such contact not develop endoderm (26). Serial sections of casanova mutants (n = 18/56) (Fig. 2 A and B, arrowheads). Frequently, teeth were revealed the absence of pharyngeal pouches and of teeth (SI absent despite the presence of pouch 6 contact (n = 16/56). In Appendix, Fig. S2), in line with the observation that enamel or- only 2 cases out of 56, teeth were present in the apparent ab- gans derive from sox17-positive endodermal epithelium. Thus, the endoderm layer is essential to produce the enamel sence of pouch 6 contact (Fig. 2 D and E; compare to contra- organ of the pharyngeal teeth. lateral side, Fig. 2 E and F). However, at least in the case pictured, the epithelial buds are questionable as tooth germs Teeth Develop Only When Pouch 6 Has Made Contact with the (Fig. 2E). Interestingly, four embryos had teeth unilaterally. In Ectoderm. Earlier, we suggested that an ectodermal cellular two of those, absence of teeth corresponded with absence of contribution or signal may be essential for tooth formation (12, pouch 6 contact at that body side, while the contralateral side – 13). Given that teeth always develop from midline endoderm presented with pouch 6 contact and teeth (Fig. 2 A C). In the immediately caudal to the contact area between the endodermal two others, one side presented with pouch 6 contact and teeth, pouch 6 and the skin ectoderm, we investigated the possible the contralateral side pouch 6 contact but no teeth. contribution of ectoderm via this pouch. To do so, we first ex- To further test the relationship between tooth formation and amined the timing of contact of pouch 6 with the ectoderm. pouch 6 contact with ectoderm, we examined zebrafish in which Observations on whole mount Tg(sox17:egfp) zebrafish of pouch development and contact with ectoderm was perturbed closely spaced developmental stages revealed that pouches 1 to 5 mechanically. Pericardial or yolk sac edema provokes a severe (P1–P5) are fully formed by 32 hpf, confirming earlier observa- disruption of contact between ectoderm and pouch endoderm or tions by Kopinke et al. (21) (i.e., P5 formed around 30 hpf) and impedes this contact altogether. We examined serial sections of Choe et al. (27) (i.e., P5 formed around 32 hpf). At 36 hpf, a 14 embryos between 96 and 168 hpf that had developed peri- bud-like thickening projects from the posterior end of pouch 5 cardial edema, either naturally, or as the result of mutation or and is recognizable as pouch 6 by 38 hpf. By 40 hpf, this pouch induction by ethanol. Given the (infrequent) asymmetric presence has thinned, while the pharynx continues to extend posteriorly to of teeth in the vgo mutants, and the occasional unilateral occur- meet the alimentary canal (SI Appendix, Fig. S3 A–C and A′–C′). rence of edemas, we scored each body side separately (n = 28) (SI − − Sections of WT, as well as Tg(sox17:egfp) zebrafish stained for Appendix, Table S1B). In all four vgo / mutants that had de- laminin, reveal details of the contact between pouch and ecto- veloped an edema, teeth were absent on both sides (n = 8) and no derm. At 36 hpf, the lateral part of the pouch has taken on a club pouch 6 contact was observed (Fig. 2 G–I). In nine other speci- shape and the lateralmost pouch cells extend filopodia toward mens with edema, teeth were present on both sides (n = 18) along − − the ectoderm. At this time point, the skin is thickened at the with pouch 6 contact. Finally, in one (168 hpf old) laf / mu- prospective contact point and is bilayered, with a single basal tant (Fig. 2 J–L) and in one (96 hpf old) Tg(krt4:gfp) specimen

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Fig. 1. 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 barsinA–D and F–J,50μm.), (Scale bar in E,20μm.)

(Fig. 2 M–O), teeth were absent unilaterally, coinciding with ex- these krt4-positive, periderm-like cells back to a source of cells tremely disturbed pouches on the edentulous side. more anteriorly, at the level of pouch 2, expanding posteriorly in In vertebrates, pouch–cleft contacts have been reported to act the pharynx along the midline. These cells express sox17 from as signaling areas as judged by the localized expression of certain early onwards (SI Appendix, Fig. S1D). They reach the level of genes, such as Eya1 (30) or wnt4a (27). These genes are im- pouch 6 at around 48 hpf (Fig. 1A, and see ref. 23), followed by portant for normal pouch development, respectively in mammals the appearance of small lumina in between them (Fig. 3B and SI (31) and zebrafish (27). Rather surprisingly, the six dog-eared tm90b Appendix, Fig. S5). The separate lumina coalesce into a larger (eya1, dog ) mutant specimens (12 sides) did not display a lumen that eventually separates the future floor from the roof tooth phenotype (SI Appendix, Fig. S4 A and B and Table S1C). of the pharynx (Fig. 3C). The future pharynx floor is then wnt4a Mutants for also displayed a normal dentition (27). constituted of a deep layer of endodermal cells resting on the Taken together, our data provide a strong indication that basal lamina, covered by a superficial layer of more flattened pouch 6 needs to contact the overlying ectoderm before tooth periderm-like cells (further referred to as “midline cells”), facing formation can occur, yet the molecular player(s) remains elusive. the lumen (Fig. 3C). The same two tiers, in mirror image, form Teeth Develop Only once a Periderm-Like Layer Has Covered the the future roof of the pharyngeal cavity. While the endoderm Endoderm. Using Tg(krt4:gfp) zebrafish, we observed that the loses its sox17 expression, the midline cells continue to strongly placode of the first tooth forms only after a layer of krt4-positive express sox17 (Fig. 3C), while maintaining krt4 expression cells has appeared along the midline, squeezed between the two (Fig. 3A and SI Appendix, Fig. S1E). Pouch 6 itself persists as a layers of endodermal epithelium, and with cells more flattened sox17-positive endodermal bilayer until at least 56 hpf (Figs. 1D than in the endoderm (Fig. 3A). Earlier, we traced the origin of and 3C).

Oralová et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11505 Downloaded by guest on October 2, 2021 Fig. 2. 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.)

Because of the striking temporal coincidence between the Signaling Pathways Known so Far to Affect Tooth Development Do appearance of these krt4-positive midline cells and the start of Not Interfere with Pouch 6 Contact or Presence of Periderm-Like tooth formation, we scored all 50 embryos—previously examined Cells. The above findings hint at the potential importance of for pouch 6 contact—for the presence of these cells in the midline endoderm–ectoderm contacts and presence of periderm-like, mid- at the level of pouch 6. Their presence was established either line cells in tooth formation. Thus, we next tested whether ma- through a GFP signal, or by microscopic observation at high nipulating signaling pathways, some of which are known to perturb magnification (SI Appendix, Table S1 and Fig. S6). In all but the zebrafish tooth development, also affect either pouch 6 contact, two youngest (48 hpf) embryos such cells were present, either as the presence of midline cells, or any combination. Because we previously established that these midline cells blend with periderm individual cells, or up to a complete additional layer. Tooth germs invading via pouch 2, we included observations on pouch 2 in our were present in only half of the embryos (n = 50/100 sides), but analysis, again using serial sections and scoring each body side present, without exception, only when midline cells were present separately not to miss potential asymmetries. We assigned pouch as well. number only as far as it could be identified in case of highly de- Because we previously established that the midline cells, ob- formed pouches. For this analysis, we used mutants targeting served to cover the enamel organ-forming endoderm, do not particular signaling pathways, and/or pharmaceutically inhibi- derive from skin periderm (23), we expected that mutants with ted the pathway (SI Appendix,TableS2). perturbed skin periderm would not yield a tooth phenotype. This Because the close cellular contact between midline cells and was confirmed by studying goosepimple mutants, which carry a tooth-forming endodermal epithelium suggested potential juxtacrine mutation in myosin Vb, involved in peridermal plasma membrane signaling, we started by investigating the role of Delta-Notch homeostasis (32) (SI Appendix, Fig. S4 C and D). signaling. DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S- In conclusion, tooth germs form from the endodermal phenylglycine t-butyl ester), a known and carefully characterized layer but only after periderm-like cells have covered this γ-secretase inhibitor (33), severely interferes with Notch signaling endoderm. in zebrafish embryos. DAPT was applied at 40 or 44 hpf, i.e., when

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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 μminB.)

midline cells have not yet expanded far enough posteriorly as to hedgehog signaling through cyclopamine A (CyA) from 40 hpf cover the endoderm of the future tooth-forming region. Controls onwards until killing at 96 hpf did not prevent spreading of midline and treated specimens were killed at 72 hpf. In controls of this age, cells until the posterior pharynx. Yet, despite the presence of mid- pouch 2 is open (Fig. 3D), pouch 6 makes contact with the ecto- line cells in the tooth-forming region, and despite pouch 6 contact, derm (Fig. 3E), but is not open yet, and a layer of periderm-like, teeth did not develop, consistent with findings reported in ref. 35 krt4-positive, midline cells is present, as are tooth germs (Fig. 3F). (Fig. 4 A–F)(SI Appendix,TableS2B). Continuous application of DAPT until 72 hpf did not prevent Next to Shh signaling, Fgf signaling has been shown to be expansion of the periderm-like cells, pouch 6 contact, or tooth essential for tooth formation (25). We performed different ex- formation (n = 6/6) (Fig. 3 G–I). mind bomb (mib) is a ubiquitin periments whereby SU5402 was applied starting either from ligase that is essential for efficient activation of Notch signaling by 30 or from 40 hpf onwards (i.e., before, respectively after fully Delta (34). In mib mutants, like in controls, pouch 2 was observed established contact between pouch 6 and ectoderm), until killing to be open, pouch 6 made contact with the ectoderm, teeth were at 56, 72, or 80 hpf. Again, we scored each side separately. present, as was a layer of midline cells (n = 6/6) (Fig. 3 J–O)(SI Whereas controls displayed normal tooth development associ- Appendix,TableS2A). ated with the presence of at least two epithelial tiers, pouch 6 Hedgehog signaling is important at multiple stages of tooth contact and an open pouch 2 (25 embryos, n = 50/50) (Fig. 4 development in zebrafish (35). Thus, we investigated the effect of G–I), the treated 30 embryos showed a variable, yet symmetric impeding hedgehog signaling on spreading of periderm-like cells outcome. In about half of the embryos treated from 30 hpf on- over the midline endoderm, and on tooth formation. Inhibiting wards (n = 16/30), and about half of the embryos treated from

Oralová et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11507 Downloaded by guest on October 2, 2021 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.)

40 hpf onwards (n = 14/30), tooth germs were present (albeit each side separately (n = 14), tooth germs were present, along delayed), as was a layer of midline cells (Fig. 4 J–L). In the other with pouch 6 contact and presence of midline cells. Teeth were half of the embryos treated from 30 or 40 hpf, tooth germs were not necessarily in full numbers, their presence indicating never- = missing, with the layer of midline cells either absent (n 10/30), theless that tooth initiation is not fully impeded (SI Appendix, = – or frequently also present (n 20/30) (Fig. 4 M R)(SI Appendix, Fig. S4 E and F and Table S2E). Table S2C). In all 30 embryos, pouch 2 was normally developed Finally, we reexamined masterblind (mbl) mutants for the and pouch 6 contact was present on both sides. status of the pouches and the presence or absence of midline Recently, FoxF genes have been shown to have an essential role in − − − − − − cells. mbl encodes for axin1, part of the beta-catenin destruction tooth formation, as triple FoxF mutants (foxf1 / ;foxf2a / ;foxf2b / ) are edentulous (36). Close examination nevertheless revealed an complex, and thus these mutants mimic overactivation of Wnt open pouch 2, pouch 6 contact, and presence of midline cells (SI signaling. These mutants were previously reported to have a Appendix,Fig.S4G and H and Table S2D). normal tooth phenotype (37) (SI Appendix, Table S2F). In all 11 Zebrafish defective in eda/edar signaling display perturbed specimens studied and scored for each side (n = 22), pouches 2 tooth development (9). We examined serial sections of 10 finless and 6 presented a normal contact with ectoderm and midline (edar) mutants; in seven embryos older than 48 hpf, scored for cells were present.

11508 | www.pnas.org/cgi/doi/10.1073/pnas.2000279117 Oralová et al. Downloaded by guest on October 2, 2021 Together, these data indicate that signaling pathways known Malformation of endodermal pouches in zebrafish prdm1a mu- so far to affect tooth development do not interfere with pouch 6 tants is accompanied by the loss of pharyngeal teeth (43). To- contact or presence of periderm-like midline cells. gether these observations suggest that some sort of signaling may occur at the pouch–cleft contact to allow tooth initiation from Pouch 6 Contact with Ectoderm and the Presence of Periderm-Like midline endoderm. Signaling between endoderm and ectoderm is Midline Cells Are Required but Not Sufficient for Tooth Formation. To required for the development of several organs. For example, assess the role of pouch contact with ectoderm and the presence defective development of the thymus in nude mice is because third of periderm-like cells in tooth initiation, we pooled all of the data pouch endoderm no longer contacts the ectoderm, thus depriving on mutant, transgenic, and experimental animals (SI Appendix, it from its normal inducing agent (44). Both in the chick (45) and Fig. S7 and Table S3). We did not consider embryos younger than in zebrafish (46), neurogenesis in epibranchial placodes requires a 48 hpf because tooth formation in WT becomes morphologically signal from pouch endoderm. In zebrafish vgo mutants studied discernible only from 48 hpf onwards. Taking all data together, it here, teeth were frequently absent despite the presence of an can be inferred that teeth are sometimes present in the absence of endodermal–ectodermal contact (n = 16/56), but in those cases, pouch 2, while its contact with ectoderm, or even opening, is not the contact zone could be established as being narrower than sufficient to allow tooth formation, suggesting that pouch 2 has no 16 μm, i.e., less than two cell diameters (i.e., the width of a pouch). causal role in tooth initiation (SI Appendix, Fig. S7 and Table Interestingly, Holzschuh et al. (46) likewise found a close corre- S3C). There are two questionable exceptions (2/30) (discussed lation between enlarged or reduced contacts made by pouches further) to the observation that pouch 6 must contact the ecto- with the ectoderm and epibranchial defects in vgo mutants. derm in order for teeth to be present. Yet, as for pouch 2, the One could raise the argument that the contact between pouch contact is not sufficient for tooth initiation, as around 30% of 6 and the ectoderm on the one hand, and the start of tooth the body sides examined display pouch 6 contact without teeth. formation on the other hand, is purely coincidental, and merely a The most stringent requirement appears to be the presence of reflection of overall sufficient maturation of endoderm. How- − − periderm-like cells; in not one single case were tooth germs ob- ever, observations on the vgo / mutants argue against this in- served in the absence of such cells. Finally, we scored the absence terpretation, as some specimens have teeth on just one body or presence of teeth if both pouch 6 contact and the presence of side, i.e., the side where pouch 6 makes contact with ectoderm. midline cells are considered. With the same two exceptions, it can Asymmetric pouch development is also observed in ace (fgf8) be inferred that both periderm-like cells and pouch 6 contact must mutants, yet teeth develop symmetrically, albeit that they are be present if teeth are to be found, but that they are not sufficient malformed, smaller, and less mature (20, 25, 47). However, it is for tooth initiation. not clear from these studies whether defective pouches actually DEVELOPMENTAL BIOLOGY fail to contact the ectoderm. Discussion The nature of the putative inductive signal mediating endo- Our findings show that 1) the epithelial component of pharyn- derm–ectoderm interactions remains elusive. Genes important in geal teeth in zebrafish (the enamel organ) is derived from medial zebrafish pouch outgrowth and cellular rearrangement into a endoderm just posterior to pouch 6, yet, 2) dental morphogen- bilayer are tbx1, fgf8a, and wnt4a, yet none of the mutants for esis starts only after pouch 6 has made contact with skin ecto- these genes lacks teeth (this study and ref. 25, 27). Different derm, and 3) only after a layer of periderm-like cells has covered bmps (Bmp7 in the chick, bmp2b and bmp5 in zebrafish), as well the prospective odontogenic epithelium (SI Appendix, Fig. S8), as fgf3, have been identified as crucial components of the en- and finally, 4) interfering with signaling pathways known to affect dodermal signals that induce epibranchial neurogenesis (45, 46, tooth development supports the conclusion that pouch 6 contact 48). Knockdown in zebrafish of bmp2b and bmp4 does not cause and the presence of midline cells are required, but not alone tooth absence (49). Likewise, mutants for other genes expressed sufficient, for tooth initiation. at pouch–cleft contacts and candidates for mediating ectodermal–endodermal signaling, such as eya1 (50) or pax9 (51), have The Enamel Organ of Pharyngeal Teeth Derives from Endoderm. That not yielded a tooth phenotype so far. The absence in zebrafish of a the epithelial component of pharyngeal teeth in teleosts derives tooth phenotype both in eya1 mutants and pax9 morphants is re- from endoderm has long been assumed based on circumstantial markable also because of the high conservation of the Pax-Six-Eya evidence, such as their position on the posteriormost pharyngeal regulatory network in vertebrate pharyngeal pouches (16). arches, but conclusive evidence was missing so far. A reconsider- One, largely unexplored, possibility is that tissue mechanics, ation of the outside in theory for the evolutionary origin of teeth together with molecular effectors, coordinate morphogenesis (52). had furthermore fueled the idea that ectoderm may invade the Thus, mechanical cues issuing from pouch–cleft contact, rather pouches and occupy the tooth-forming region (12, 13). This had than a molecular prepattern, may promote morphogenesis of the cast doubt on the endodermal nature of the enamel organ. Here basal endodermal layer to form the enamel organ. Our data on we show that only the layer adjoining the basal membrane, i.e., the failure of tooth initiation in the case of mechanically perturbed sox17-positive endodermal layer, undergoes morphogenesis to pouch–cleft contacts support this idea. Thus, while Fgf and Shh produce the early enamel organ of pharyngeal teeth. The absence, signaling pathways are crucial for tooth initiation, they could act in zebrafish, of teeth on the mandibular arch precludes any con- downstream of pouch 6 contact (as well as of appearance of clusion on the germ layer contributing to oral teeth in teleosts. In midline cells, see below). urodele amphibians, the enamel organ can be formed in endoderm, ectoderm, or both (reviewed in refs. 38, 39). In contrast, in Pharyngeal Tooth Initiation Requires a Periderm-Like Cell Layer mammals it has now been convincingly shown that all teeth derive Covering the Presumptive Enamel Organ. At the time tooth mor- from ectodermal epithelium (11). phogenesis starts (48 hpf), pouch 6 contact is still closed. The tight bilayer nature of the pouch prevents any precocious invasion Pouch–Ectoderm Contact Is Required for Pharyngeal Tooth Formation. of ectoderm or periderm into the pouch, similar to conclusions for Teeth start to develop morphologically only after pouch 6 has medaka (42). However, recently we have demonstrated that a made contact with the skin ectoderm (two questionable exceptions group of krt4-positive, periderm-like cells appears at the level of out of 216 body sides examined), yet before the pouch opens into a pouch 2 at around 26 hpf, and that this population expands gill slit. Likewise, in medaka (Oryzias latipes), pharyngeal teeth throughout the pharynx along the midline, thereby covering the start to form first on pharyngobranchial 4 at stage 29 (40, 41), endoderm (23). These cells, here called midline cells, connect to which is after pouch 5–ectoderm contact is established (42). the superficial skin layer, the periderm, first via pouch 2, later via

Oralová et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11509 Downloaded by guest on October 2, 2021 more posterior pouches. The remarkable correlation between the nonteleost actinopterygian lineages, instead of the telolecithal presence of midline cells and the presence of teeth (no exceptions eggs of zebrafish. out of 216 sides), strongly suggests that these midline cells are Since the periderm in zebrafish partially invades the mouth required for initiation of tooth formation. Again, one could argue cavity (23), the absence of oral teeth cannot be attributed to a that the presence of a periderm-like layer and the start of tooth lack of such cells. This is in line with conclusions reached in ref. formation is coincidental but not causally linked. While we have 66 that changes in transacting regulators of Dlx genes were re- no other argument than correlative evidence, it is useful to point sponsible for cypriniform tooth loss. On the other hand, the out that we know of no instances where vertebrate teeth are ini- periderm-like layer expands into the esophagus, but not further tiated from a nonstratified epithelium. It may well be that enamel caudally (23). Based on our current findings, we predict that organs simply cannot form from a monolayer and that the teeth can be present in the digestive system but not beyond the periderm-like cells may play a mechanical role similar to that of esophagus. Interestingly, several fish species indeed possess the suprabasal canopy in mammalian tooth development (53). In esophageal teeth (67), while we know of no teleosts with teeth in mice, Fgf8-expressing cells migrate toward the molar placode’s the stomach or beyond. Shh-expressing cells to initiate tooth development (54). It is interesting to note that in zebrafish, two Fgf receptors, fgfr1a and Conclusion fgfr2, are expressed in pharyngeal endoderm (55). Knockout of Summarizing the data from mutant and transgenic zebrafish and three Fgf receptors, fgfr1a, fgfr1b, and fgfr2, apparently leads to from our experimental approaches, we conclude that there is a tooth absence (figure 6C in ref. 56), consistent with results double requirement before pharyngeal teeth are initiated. These reported in ref. 25 and here on the use of SU5402. That endoderm are contact of the endodermal pouch with skin ectoderm and the morphogenesis is essential for normal craniofacial development presence of a periderm-like layer covering the endoderm. Both has been firmly established in numerous papers (57); yet, our data are necessary, but not sufficient, for tooth formation. We find – warrant an investigation into whether the midline cells, in addition that pouch cleft contacts are necessary, although not to allow an – to the endoderm, play a crucial role. The intimate contact between influx of ectodermal (or peridermal) cells. Pouch cleft contacts the apical periderm-like layer and the basal endodermal layer, as may act as signaling centers, as demonstrated for other verte- well as the long cell processes issuing from the former and con- brate pharyngeal derivatives. Alternatively, they may deliver tacting the basal lamina (23), provide a further incentive to do so. mechanical cues to coordinate endoderm morphogenesis. In the mammalian oral cavity, a periderm is present, superficial to Likewise, the need for a layer of cells covering the endoderm, the tooth germs (58). Different from zebrafish, this periderm is with periderm-like features, is strongly underscored by observa- derived from ectoderm. Its role in mammalian tooth initiation has tions on more than a hundred embryos, deriving from various not properly been investigated, although its reported timing of mutant and transgenic lines and experiments. The nature of the appearance (59) clearly warrants an investigation into this signal issuing from the midline cells and allowing initiation of question. tooth placode formation in the endoderm remains elusive. Our results are nevertheless in line with the findings for mouse molar Pharyngeal Teeth from an Evolutionary Developmental Biology teeth, where two spatially distinct cell populations, one expressing Perspective. The primary incentive for the present study was to Shh, the other Fgf8, are involved in tooth initiation (54). Whether test whether a contribution from the ectoderm (cellular or via the spatial expansion of periderm-like cells can be likened to the signaling) is still required for pharyngeal teeth to develop, as intraepithelial migration found in the mouse, still needs to be proposed in the modified outside-in hypothesis (12–14). Atu- assessed. Nevertheless, it is clear that both in zebrafish and in the korala et al. (42) tested this hypothesis on medaka. They saw no mouse, the earliest interactions necessary to produce a tooth in- clude those between different epithelial populations, in addition to evidence of any ectodermal contribution to pharyngeal tooth – formation and concluded for an intrinsic odontogenic compe- the known epithelial mesenchymal interactions that govern all tence of the rostral endoderm. However, we have demonstrated, vertebrate teeth (8). This suggests a high level of conservation of both in ref. 23 and in the present paper, that cells with periderm- tooth development, whether oral or pharyngeal. like characteristics, different from endoderm, come to overlie Materials and Methods the endoderm and appear to be required to start the process of Transgenic Zebrafish Lines. Tg(sox17:egfp) zebrafish (68), in which the en- tooth formation. Importantly, in medaka the endoderm is cov- doderm is labeled, were obtained from the laboratory of R. Opitz, Vrije Universiteit ered by hatching gland cells, itself overlain by flattened cells Brussel (VUB), Brussels, Belgium. Tg(krt4:gfp)(69)andTg(krt4:tomatoCAAX) reminiscent of the midline cells observed in zebrafish (60). Other zebrafish, in which the outer skin layer, the periderm, is GFP- respectively teleost species also possess a layer of flattened periderm-like tomato-positive, were a gift from M. Hammerschmidt, University of Köln, cells lining the pharynx (23). The decisive experiment to dem- Köln, Germany. Tg(fli1:gfp) were obtained from A. Willaert, Ghent University onstrate a key role for the periderm-like layer will be a selective Hospital, Ghent, Belgium. Adult fish were maintained and spawned according ablation of these cells. to ref. 70. Embryos were raised in egg water at 28.5 °C and staged according The developmental origin of the midline cells still needs to be to ref. 18. elucidated but they have been termed periderm-like because of Mutant Zebrafish Embryos. We selected a slate of mutant zebrafish, chosen to shared morphology and markers, and compatible adhesion with cover a broad range of defects in the pharyngeal epithelium (SI Appendix, periderm (23). The periderm itself is the first epithelial covering Supplementary Material and Methods). All mutants, along with their wild of the embryo. In teleosts the periderm derives from the envel- type (WT) siblings, were obtained as embryos, fixed either in a mixture of oping layer (EVL) that surrounds the embryo during gastrulation. 1.5% paraformaldehyde (PFA) and 1.5% glutaraldehyde in 0.1 M cacodylate The EVL segregates from the blastoderm and becomes restricted buffer (a mixture abbreviated as PG), or in 4% PFA, and processed for em- to a peridermal fate by 4 h, at late blastula stage (61). Still, ho- bedding in epon or glycol methacrylate (GMA), as described below. mology of the EVL across taxa is not clearly established (62). However, periderm (upper layer) and ectoderm (lower layer) to- Pharmaceutical Inhibition. Inhibition experiments were conducted for three gether constitute the bilayered epidermis of the skin of teleost different signaling pathways, as described in SI Appendix, Supplementary Material and Methods. The role of Notch signaling was investigated using embryos and posthatching stages, and the periderm persists well DAPT (33). The role of Shh signaling was examined through the use of CyA, into juvenile life (63, 64). Given that early embryonic development an established hedgehog pathway antagonist, known to affect pharyngeal is to a large extent determined by the amount of yolk (65), it will tooth development (35). Fgf dependency was tested by inhibition with the be interesting to study the periderm in representatives of basal, generalized Fgf inhibitor SU5402 (25).

11510 | www.pnas.org/cgi/doi/10.1073/pnas.2000279117 Oralová et al. Downloaded by guest on October 2, 2021 Mechanical Perturbation of Pharynx Development. Zebrafish embryos that had Ethical Statement. Animal care, experimentation, and killing complied with − − developed pericardial or yolk sac edema, either provoked by mutation (laf / ; European Directive 2010/63/EU of September 22, 2010. All animal procedures − − vgo / ), or as an occasional phenotype in transgenic lines [Tg(fli1:gfp) and used in this study were carried out under the laboratory permit number Tg(krt4:gfp)], were considered separate from nonaffected siblings. Edemas LA 1400452. were also artificially induced in Tg(sox17: egfp) embryos by ethanol treat- ment (for details, see SI Appendix, Supplementary Material and Methods). Data Availability. All sections used for this study are kept in the slide collection of the Research Group “Evolutionary Developmental Biology” at the Biology Immunohistochemistry. Immunohistochemistry for laminin on whole mount Department of Ghent University, and are available for inspection upon embryos was performed as described in ref. 71 using a polyclonal rabbit anti- request. laminin primary antibody (Sigma-Aldrich, L9393) and a goat anti-rabbit sec- ondary antibody (DyLight 488 nm, Abcam). After immunohistochemistry, ACKNOWLEDGMENTS. We gratefully acknowledge the following persons or embryos were processed for embedding in GMA. institutions for generous gifts of transgenic or mutant lines: Dr. R. Opitz (VUB) for Tg(sox17:egfp) zebrafish; Dr. M. Hammerschmidt (University Köln) Histology and Transmission Electron Microscopy. High-resolution histology for Tg(krt4:gfp) and Tg(krt4:tomatoCAAX) zebrafish; Dr. A. Willaert (Ghent (light and transmission electron microscopy [TEM]) was achieved by fixation University) for Tg(fli1:gfp) zebrafish; T. F. Schilling (University of California, Irvine) for cas mutants; T. Piotrowski (then University of Utah) for vgo mu- in PG and embedding in epon as described previously (72) (for details, see SI tants; M. Sonaware (Tata Institute of Fundamental Research, Mumbai, India) Appendix, Supplementary Material and Methods). To visualize GFP on sec- for gsp/myoVb mutants; G. C. Crump (University of Southern California) for tions, embryos were fixed in 4% PFA. The GMA embedding protocol fol- foxF triple mutants; T. Whitfield (University of Sheffield) for eya1 (dogtm90b) lowed (73), a technique that preserves GFP signals and requires no extra mutants; P. C. Yelick (then at the Forsyth Institute) for laf (alk8) mutants; staining (SI Appendix, Supplementary Material and Methods). M. Fürthauer (Université de Nice) for mib mutants; M. Harris (then MPI Tübingen) for edar mutants; and the Hubrecht Laboratory, Utrecht, the Observations and Microphotography. Observations of epon or GMA sections Netherlands, for mbl mutants. TEM was carried out at the Ghent University were done on a Zeiss Axio Imager Z1 (https://www.zeiss.com/corporate/int/ TEM-Expertise Center (Life Sciences), facility Nematology Research Unit. M. Soenens provided valuable technical help. We also thank G. T. Eisenhoffer home.html). Photomicrographs were taken with a Zeiss Axiocam 503 camera (Department of Genetics, The University of Texas MD Anderson Cancer and processed using ZEN software (Zeiss, https://www.zeiss.com/corporate/ Center) for fruitful discussions, and two anonymous reviewers for their int/home.html). All photomicrographs were manually aligned so as to be constructive comments. V.O., J.T.R., and A.H. acknowledge a grant of the able to count and identify individual pouches. Computer-generated images Ghent University Research Fund (BOF24J2015001401). A.H. acknowledges a were processed for color balance, contrast, and brightness only, and applied grant for sabbatical leave from the FWO (Research Foundation Flanders to all parts of the figures equally. FWOSAB2019000601).

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