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