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. Rep. pharyngeal pouches form from anterior to posterior, separating 9, 10082]. We now provide conclusive evidence that the epi- – thelial component of pharyngeal teeth in zebrafish (the enamel the prospective pharyngeal arches (19 21). The first pouch (P1) is organ) is derived from medial endoderm, as hitherto assumed based homologous to the spiraculum of chondrichthyans. It separates on position deep in the pharynx. Yet, dental morphogenesis starts mandibular from the hyoid arch but does not usually open any only after the corresponding endodermal pouch (pouch 6) has made longer in teleosts. The second pouch (P2) separates the hyoid arch contact with the skin ectoderm, and only after periderm-like cells from the third pharyngeal arch (also called first branchial or gill have covered the prospective tooth-forming endodermal epithe- arch), pouch 3 (P3) separates pharyngeal arch 3 from 4, and so on. DEVELOPMENTAL BIOLOGY lium. Manipulation of signaling pathways shown to adversely affect The pouches in vertebrates give rise to different organs crucial for tooth development indicates they act downstream of these events. immune responses and calcium homeostasis (16, 22). In zebrafish, We demonstrate that pouch–ectoderm contact and the presence of teeth develop on the seventh (last) pharyngeal arch, i.e., posterior a periderm-like layer are both required, but not sufficient, for tooth to pouch 6. Using various approaches, we have recently shown initiation in the pharynx. We conclude that the earliest interactions that periderm (the initial epithelial covering of the embryo) par- to generate pharyngeal teeth encompass those between different tially invades the pouches and that endogenous cells that resemble epithelial populations (skin ectoderm, endoderm, and periderm-like the periderm cells phenotypically, spread over the endoderm cells in zebrafish), in addition to the epithelial–mesenchymal inter- along the midline (23). Thus, at the time tooth formation is actions that govern the formation of all vertebrate teeth. initiated the pharynx epithelium is composed of a double layer: pharyngeal teeth | tooth evolution | germ layers | zebrafish Significance n chondrichthyans, basal sarcopterygians, amphibians, and Iactinopterygians not only the jaw margins but also the roof and Many vertebrates possess teeth deep in the pharynx. While floor of the pharynx can be tooth bearing, constituting a pharyngeal— teeth are known to derive from odontodes (skin denticles), it is next to an oral—dentition. Teeth—whether oral or pharyngeal— unknown if an external epithelium is still required to produce a are evolutionarily derived from odontodes, also called skin den- pharyngeal tooth, such as for odontode formation. Here, we ticles, dermal skeletal elements of ancient jawless vertebrates. The show that the epithelial enamel organ of pharyngeal teeth in homology between odontodes and teeth is now well documented zebrafish is formed from endoderm, i.e. the internal germ layer. (1–6). Apart from being elements of the dermal skeleton, teeth However, teeth develop 1) only when this endoderm becomes and odontodes belong to the complex of ectodermal appendages covered by a layer of cells with features of a periderm, i.e., the outer epithelial covering of the embryo; and 2) only when the whose development depends on reciprocal interactions between endoderm has physically contacted the skin at the prospective the surface epithelium (ectoderm) and the underlying (neural gill slits. Thus, multiple epithelia are engaged in tooth formation, crest-derived) mesenchyme (7, 8). Accordingly, mutations of the whether oral (mammals) or pharyngeal (teleosts). ectodysplasin gene (EDA) or its receptor (EDAR) cause deficient development of hair, sweat glands, and teeth in humans, but also Author contributions: P.E.W. and A.H. designed research; V.O., J.T.R., D.L., and A.H. per- of pharyngeal teeth, scales, and dermal fin rays in zebrafish (9). As formed research; V.O., J.T.R., D.L., and A.H. analyzed data; and V.O., J.T.R., P.E.W., and already noted by Charles Darwin, “Hairless dogs have imperfect A.H. wrote the paper. teeth” (ref. 10, p. 30). However, different from the mammalian The authors declare no competing interest. dentition that develops in an ectoderm-covered oral cavity (11), This article is a PNAS Direct Submission. pharyngeal teeth in extant vertebrates develop in an endoderm- Published under the PNAS license. 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 www.pnas.org/cgi/doi/10.1073/pnas.2000279117 PNAS Latest Articles | 1of10 Downloaded by guest on October 1, 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).