Despite the wide variety of adaptive modifications in the oral and facial regions of vertebrates, their early oropharyngeal development is considered strictly uniform. It involves sequential formation of the mouth and pharyngeal pouches, with ectoderm outlining the outer surface and endoderm the inner surface, as a rule1,2. At the extreme anterior domain of vertebrate embryos, the ectoderm and endoderm directly juxtapose and initial development of this earliest ecto–endoderm interface, the primary mouth3, typically involves ectodermal stomodeal invagination that limits the anterior expansion of the foregut endoderm3,4. Here we present evidence that in embryos of extant non-teleost fishes, oral (stomodeal) formation is preceded by the development of prominent pre-oral gut diverticula (POGD) between the forebrain and roof of the forming mouth. Micro-computed tomography (micro-CT) imaging of bichir, sturgeon and gar embryos revealed that foregut outpocketing at the pre-oral domain begins even before the sequential formation of pharyngeal pouches. The presence of foregut-derived cells in the front of the mouth was further confirmed by in vivo experiments that allowed specific tracing of the early endodermal lining. We show that POGD in sturgeons contribute to the orofacial surface of their larvae, comprising oral teeth, lips, and sensory barbels. To our knowledge, this is the first thorough evidence for endodermal origin of external craniofacial structures in any vertebrate. In bichir and gar embryos, POGD form prominent cranial adhesive organs that are characteristic of the ancient bauplan of free-living chordate larvae. POGD hence seem arguably to be ancestral for all ray-finned fishes, and their topology, pharyngeal-like morphogenesis and gene expression suggest that they are evolutionarily related to the foregut-derived diverticula of early chordate and hemichordate embryos. The formation of POGD might thus represent an ancestral developmental module with deep deuterostome origins.
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Grevellec, A. & Tucker, A. S. The pharyngeal pouches and clefts: development, evolution, structure and derivatives. Semin. Cell Dev. Biol. 21, 325–332 (2010)
Shone, V. & Graham, A. Endodermal/ectodermal interfaces during pharyngeal segmentation in vertebrates. J. Anat. 225, 479–491 (2014)
Dickinson, A. J. G. & Sive, H. Development of the primary mouth in Xenopus laevis. Dev. Biol. 295, 700–713 (2006)
Soukup, V., Horácek, I. & Cerny, R. Development and evolution of the vertebrate primary mouth. J. Anat. 222, 79–99 (2013)
Graham, A. & Richardson, J. Developmental and evolutionary origins of the pharyngeal apparatus. Evodevo 3, 24 (2012)
Choe, C. P. & Crump, J. G. Dynamic epithelia of the developing vertebrate face. Curr. Opin. Genet. Dev. 32, 66–72 (2015)
Couly, G., Creuzet, S., Bennaceur, S., Vincent, C. & Le Douarin, N. M. Interactions between Hox-negative cephalic neural crest cells and the foregut endoderm in patterning the facial skeleton in the vertebrate head. Development 129, 1061–1073 (2002)
Crump, J. G., Swartz, M. E. & Kimmel, C. B. An integrin-dependent role of pouch endoderm in hyoid cartilage development. PLoS Biol. 2, E244 (2004)
Piotrowski, T. & Nüsslein-Volhard, C. The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio). Dev. Biol. 225, 339–356 (2000)
Gillis, J. A., Fritzenwanker, J. H. & Lowe, C. J. A stem-deuterostome origin of the vertebrate pharyngeal transcriptional network. Proc. R. Soc. Lond. B 279, 237–246 (2012)
Lowe, C. J., Clarke, D. N., Medeiros, D. M., Rokhsar, D. S. & Gerhart, J. The deuterostome context of chordate origins. Nature 520, 456–465 (2015)
Kerr, J. G. The Work of John Samuel Budgett (Cambridge Univ. Press, 1907)
Janvier, P. Early Vertebrates (Clarendon, 1996)
Quinlan, R., Martin, P. & Graham, A. The role of actin cables in directing the morphogenesis of the pharyngeal pouches. Development 131, 593–599 (2004)
Alexander, J. & Stainier, D. Y. A molecular pathway leading to endoderm formation in zebrafish. Curr. Biol. 9, 1147–1157 (1999)
Yu, J. K., Holland, L. Z., Jamrich, M., Blitz, I. L. & Holland, N. D. AmphiFoxE4, an amphioxus winged helix/forkhead gene encoding a protein closely related to vertebrate thyroid transcription factor-2: expression during pharyngeal development. Evol. Dev. 4, 9–15 (2002)
Suda, Y. et al. Evolution of Otx paralogue usages in early patterning of the vertebrate head. Dev. Biol. 325, 282–295 (2009)
Rétaux, S. & Pottin, K. A question of homology for chordate adhesive organs. Commun. Integr. Biol. 4, 75–77 (2011)
Yoshida, K., Ueno, M., Niwano, T. & Saiga, H. Transcription regulatory mechanism of Pitx in the papilla-forming region in the ascidian, Halocynthia roretzi, implies conserved involvement of Otx as the upstream gene in the adhesive organ development of chordates. Dev. Growth Differ. 54, 649–659 (2012)
True, J. R. & Haag, E. S. Developmental system drift and flexibility in evolutionary trajectories. Evol. Dev. 3, 109–119 (2001)
Soukup, V., Epperlein, H.-H., Horácek, I. & Cerny, R. Dual epithelial origin of vertebrate oral teeth. Nature 455, 795–798 (2008)
Cooper, M. S. & Virta, V. C. Evolution of gastrulation in the ray-finned (actinopterygian) fishes. J. Exp. Zool. B Mol. Dev. Evol. 308, 591–608 (2007)
Nagasawa, T. et al. Evolutionary changes in the developmental origin of hatching gland cells in basal ray-finned fishes. Zoolog. Sci. 33, 272–281 (2016)
Satoh, N. et al. On a possible evolutionary link of the stomochord of hemichordates to pharyngeal organs of chordates: stomochord and notochord relationship. Genesis 52, 925–934 (2014)
Adachi, N. & Kuratani, S. Development of head and trunk mesoderm in the dogfish, Scyliorhinus torazame: I. Embryology and morphology of the head cavities and related structures. Evol. Dev. 14, 234–256 (2012)
Seifert, R., Jacob, M. & Jacob, H. J. The avian prechordal head region: a morphological study. J. Anat. 183, 75–89 (1993)
Oisi, Y., Ota, K. G., Kuraku, S., Fujimoto, S. & Kuratani, S. Craniofacial development of hagfishes and the evolution of vertebrates. Nature 493, 175–180 (2013)
Sessel, A. in Archiv für Anatomie und Entwickelungsgeschichte (Veit & Comp., 1877)
Godard, B. G. et al. Mechanisms of endoderm formation in a cartilaginous fish reveal ancestral and homoplastic traits in jawed vertebrates. Biol. Open 3, 1098–1107 (2014)
Takeuchi, M., Takahashi, M., Okabe, M. & Aizawa, S. Germ layer patterning in bichir and lamprey; an insight into its evolution in vertebrates. Dev. Biol. 332, 90–102 (2009)
Hatschek, B. & Tuckey, J. The Amphioxus and its Development (Swan, Sonnenschein & Co., 1893)
Brand, M., Granato, M. & Nüsslein-Volhard, C. in Zebrafish (Oxford Univ. Press, 2002)
Metscher, B. D. MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues. BMC Physiol. 9, 11 (2009)
Diedhiou, S. & Bartsch, P. in Development of Non-Teleost Fishes (Science Publishers, 2009)
Dettlaff, T. A ., Ginsburg, A. S . & Schmalhausen, O. I. Sturgeon Fishes—Developmental Biology and Aquaculture (Springer, 1993)
Long, W. L. & Ballard, W. W. Normal embryonic stages of the longnose gar, Lepisosteus osseus. BMC Dev. Biol. 1, 6 (2001)
We thank C. V. H. Baker, M. E. Bronner, M. M. Smith, A. S. Tucker, D. M. Medeiros, P. E. Ahlberg, and S. Kuratani and his laboratory members for their helpful comments; and I. Cˇepicˇka, whose laboratory members initially assisted with the gene cloning procedures. V. Miller and K. Kodejš are acknowledged for their care of bichirs in Prague, and M. Kahanec and M. Rodina for their care of sturgeons in Vodnˇany. D.J. thanks C. V. H. Baker for hosting him in her laboratory and the European Molecular Biology Organization (EMBO) for financial support. The study was supported by GACR project 16-23836S (R.C.); Charles University grant SVV 260 434 / 2017 and Charles University GA UK projects 220213 and 726516 (M.M.), and 1448514 (J.S.). M.M. thanks the OeAD Aktion Österreich-Tschechien scholarship (financed by BMWFW Austria) for financial support during his stay in B.D.M.’s laboratory. M.P. and D.G. thank the Ministry of Education, Youth and Sports of the Czech Republic (projects CENAKVA (CZ.1.05/2.1.00/01.0024), CENAKVA II (LO1205 under the NPU I program)). L.A.R. thanks the SAGARPA-COFUPRO (RGAC-GTOS-2011-027): “Equipamiento para el Fortalecimiento del Núcleo Genético de pejelagarto en el estado de Tabasco”.
The authors declare no competing financial interests.
Reviewer Information Nature thanks A. Gillis, G. Schlosser and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Stages 19–28 (ref. 34).
Stages 22–26 (ref. 35).
Stages 15–26 (ref. 36).
Extended Data Figure 4 Micro-CT visualization of pharyngeal development (yellow) in bichir, sturgeon and gar.
3D reconstructions of key developmental stages.
Extended Data Figure 5 Outpocketing of POGD in bichir and secondary constriction of POGD in sturgeon pharyngulae during development.
a–c, Horizontal vibratome sections of bichir pharyngulae at three succeeding stages, showing formation of POGD and cement glands. Head to the left; white dotted line delineates POGD. Fibronectin (green) marks cell and tissue borders; actin (red) stains contracting actin fibres during POGD formation. Red arrowheads point to the possible role of actin cables in POGD outpocketing. d–f, Plastic parasagittal sections of sturgeon pharyngulae at three succeeding stages. Head to the left; stained with AzureB/eosin. Red dotted line delineates POGD. b, brain; h, heart; pog, pre-oral gut.
3D reconstructions showing the position of pre-oral gut (pog), adenohypophysis (blue), notochord (white), and condensations of head mesenchyme (pink). Endoderm is yellow; roman numerals refer to pharyngeal pouches; red colour shows cranial arteries. aa, aortic arches; h, adenohypophysis; mc, mandibular head cavity.
Extended Data Figure 7 Gene expression patterns (Otx2, Sox17, FoxE4 and Pitx2) in POGD in bichir, sturgeon and gar embryos.
Wholemount views and parasagittal vibratome sections (head to the left); DAPI (blue) stains cell nuclei.
Extended Data Figure 8 CM-DiI fate-mapping of the primitive gut in gars, with endodermal contribution to the orofacial surface.
a, c, Experimental gar embryos, wholemount lateral and ventral view, respectively, with CM-DiI (red) in pharynx and cement glands. Red arrowhead indicates mouth. b, d–g, Horizontal sections, anterior head with CM-DiI (red) in forming cement glands and cranial surface. DAPI (blue) stains cell nuclei; fibronectin (green) marks cell and tissue borders.
Extended Data Figure 9 CM-DiI fate-mapping of the primitive gut in sturgeons, with endodermal contribution to the orofacial surface.
a–d, Experimental sturgeon embryos showing the extent of CM-DiI (red) at stages just before (a), during (b), and after (c, d) mouth opens. Left to right show lateral views, ventral views, SEM images, and schematics. e–j, Sturgeon embryos; bright field images, showing the presence and extent (red arrows) of the yolk-rich cells of the foregut endoderm. These cells appear full of bright yolk granules, which gradually disappear during later development (i, j).
Extended Data Figure 10 SEM images of sturgeon head mapping experimental fate-mapping data with endodermal contribution pseudocoloured yellow.
Antero-ventral views, 10 and 15 days post hatching (d.p.h.); mb, medial barbel; lb, lateral barbel; ao, ampullary organs; rb, rostrum.
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Minarik, M., Stundl, J., Fabian, P. et al. Pre-oral gut contributes to facial structures in non-teleost fishes. Nature 547, 209–212 (2017). https://doi.org/10.1038/nature23008