Pre-oral gut contributes to facial structures in non-teleost fishes

Abstract

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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Figure 1: Micro-CT visualization of pharyngeal development in bichir, sturgeon and gar reveals prominent POGD.
Figure 2: Pharyngeal outpocketing in the pre-oral region, and gene expression patterns in forming POGD in bichir, sturgeon and gar embryos.
Figure 3: Fate-mapping of the primitive gut in bichir, sturgeon and gar embryos reveals endodermal contribution to the orofacial surface.
Figure 4: Oropharyngeal development comprising POGD, and distribution of POGD on chordate phylogenetic tree.

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Acknowledgements

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”.

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Authors

Contributions

R.C. and M.M. conceived the project; M.M. performed most experiments; J.S., P.F. and D.J. performed gene cloning and some experiments; D.J. performed phylogenetic analyses; B.D.M. and M.M. performed micro-CT analyses; M.P. and D.G. provided sturgeon embryonic material; L.A.R. provided gar embryonic material and A.O.P. assisted with gar and sturgeon experiments; R.C., M.M. and I.H. wrote the manuscript with input from all authors.

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Correspondence to Robert Cerny.

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The authors declare no competing financial interests.

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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

Extended Data Figure 1 Micro-CT visualization of bichir embryogenesis.

Stages 19–28 (ref. 34).

Extended Data Figure 2 Micro-CT visualization of sturgeon embryogenesis.

Stages 22–26 (ref. 35).

Extended Data Figure 3 Micro-CT visualization of gar embryogenesis.

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.

ac, 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. df, 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.

Extended Data Figure 6 Micro-CT visualization of sturgeon pharynx at two key developmental stages.

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, dg, 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.

ad, 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. ej, 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

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