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

Journal name:
Nature
Volume:
547,
Pages:
209–212
Date published:
DOI:
doi:10.1038/nature23008
Received
Accepted
Published online

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.

At a glance

Figures

  1. Micro-CT visualization of pharyngeal development in bichir, sturgeon and gar reveals prominent POGD.
    Figure 1: Micro-CT visualization of pharyngeal development in bichir, sturgeon and gar reveals prominent POGD.

    a, Micro-CT technique and 3D rendered visualization of pharyngeal endoderm (yellow) within an embryo. b, g, l, Wholemount view of early pharyngulae of bichir (b), sturgeon (g) and gar (l), head to the left, showing the anterior extent of the foregut endoderm (green arrowhead). ce, hj, mo, 3D models of pharyngeal development in bichir (ce), sturgeon (h–j) and gar (m–o); endoderm yellow, POGD marked by green arrowhead, mouth marked by red arrowhead, notochord white, adenohypophysis blue, roman numerals refer to pharyngeal pouches. f, k, p, SEM images of bichir (f), sturgeon (k) and gar (p) at stages just after hatching; anteroventral views mapping micro-CT data (pseudocoloured yellow). cg, cement gland; hg, hatching gland; nt, neural tube; st, embryonic stage; y, yolk.

  2. Pharyngeal outpocketing in the pre-oral region, and gene expression patterns in forming POGD in bichir, sturgeon and gar embryos.
    Figure 2: Pharyngeal outpocketing in the pre-oral region, and gene expression patterns in forming POGD in bichir, sturgeon and gar embryos.

    ag, Early pharyngulae of bichir (a), sturgeon (b, d, e) and gar (c, f, g); parasagittal sections, head to the left. ac, Foregut outpocketing with POGD (yellow arrowheads). DAPI (blue) stains cell nuclei; fibronectin (green) marks cell and tissue borders; actin staining (red) indicates that apical constriction is involved in the process of POGD formation. dg, Sox17, FoxE4 and Otx2 expression in forming POGD (yellow arrowheads; see Methods for details of probes). Insets show wholemounts; main images show parasagittal sections, head to the left; DAPI (blue) stains cell nuclei. ac, archenteron cavity; br, brain.

  3. Fate-mapping of the primitive gut in bichir, sturgeon and gar embryos reveals endodermal contribution to the orofacial surface.
    Figure 3: Fate-mapping of the primitive gut in bichir, sturgeon and gar embryos reveals endodermal contribution to the orofacial surface.

    a, CM-DiI fate-mapping. b, Median section of sturgeon neurula. Asterisk shows injection site and enlarged inset shows incorporation of CM-DiI (red) into the endoderm internal to the basal lamina (fibronectin, green) at the anteriormost pole. DAPI (blue) stains cell nuclei. c, Experimental sturgeon and gar larvae with CM-DiI-filled gut cavities. d, e, Bichir larvae with cement glands visualized by anti-tubulin staining (d) (lateral view), and by mucus secretion (e) (ventral view; pink, PAS staining). f, Experimental bichir embryo with CM-DiI in pharynx and cement glands. g, h, Horizontal sections showing CM-DiI in the secretory layer of cement glands. DAPI (blue) stains cell nuclei; fibronectin (green) marks cell and tissue borders. i, j, Gar larvae with cement glands visualized by anti-tubulin staining (i) (lateral view), and by mucus secretion (j) (ventral view; pink, PAS staining). k, Experimental gar embryos. Wholemount lateral (left) and ventral (right) views with CM-DiI in pharynx and cement glands. Red arrowhead indicates mouth. l, Horizontal section showing anterior head with CM-DiI in cement glands. DAPI (blue) stains cell nuclei; fibronectin (green) marks cell and tissue borders. m, n, Experimental sturgeon embryos showing the extent of CM-DiI at stages just before (m) and after (n) mouth (red arrowhead) opens. Left to right show lateral views, ventral views, SEM images and schematics. os, Parasagittal sections, head to the left, showing contribution of the foregut endoderm (CM-DiI) in rostrum (r), barbels (b), upper lip (ul), and teeth (white arrowhead) on the upper (r) and lower (s) jaws. DAPI (blue) stains cell nuclei; fibronectin (green) marks cell and tissue borders. EC, ectoderm; nEC, neural ectoderm; EN, endoderm; e, eye; ph, pharynx; uj, upper jaw; lj, lower jaw; ll, lower lip; m, mouth; n, nostril; llo, lateral line organs.

  4. Oropharyngeal development comprising POGD, and distribution of POGD on chordate phylogenetic tree.
    Figure 4: Oropharyngeal development comprising POGD, and distribution of POGD on chordate phylogenetic tree.

    a, A common scheme of vertebrate pharynx (left) with typical pharyngeal pouches in post-oral position; the ectoderm is outside (blue), and endoderm inside (yellow). In non-teleost fishes, outpocketing of the primitive gut cavity forms prominent POGD (either single as in sturgeon, or paired as in bichir and gar) that penetrate the epidermis, and develop by morphogenesis similar to pharyngeal pouch formation. Endodermal epithelium is yellow, cavity of the primitive gut is brown, brain structures are grey. b, Simplified phylogenetic scheme of chordates, with the presence of POGD in hemichordates (stomochord in acorn worms24), cephalochordates (Hatschek’s diverticula of amphioxus31), and in non-teleost fishes (framed). POGD seem to be reduced in teleosts24 and can be observed as rudimentary in hagfishes27, sharks25 and amniotes7, 26 in the form of Seessel’s pouch28. The top right (framed) scheme has been redrawn from ref. 26.

  5. Micro-CT visualization of bichir embryogenesis.
    Extended Data Fig. 1: Micro-CT visualization of bichir embryogenesis.

    Stages 19–28 (ref. 34).

  6. Micro-CT visualization of sturgeon embryogenesis.
    Extended Data Fig. 2: Micro-CT visualization of sturgeon embryogenesis.

    Stages 22–26 (ref. 35).

  7. Micro-CT visualization of gar embryogenesis.
    Extended Data Fig. 3: Micro-CT visualization of gar embryogenesis.

    Stages 15–26 (ref. 36).

  8. Micro-CT visualization of pharyngeal development (yellow) in bichir, sturgeon and gar.
    Extended Data Fig. 4: Micro-CT visualization of pharyngeal development (yellow) in bichir, sturgeon and gar.

    3D reconstructions of key developmental stages.

  9. Outpocketing of POGD in bichir and secondary constriction of POGD in sturgeon pharyngulae during development.
    Extended Data Fig. 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.

  10. Micro-CT visualization of sturgeon pharynx at two key developmental stages.
    Extended Data Fig. 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.

  11. Gene expression patterns (Otx2, Sox17, FoxE4 and Pitx2) in POGD in bichir, sturgeon and gar embryos.
    Extended Data Fig. 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.

  12. CM-DiI fate-mapping of the primitive gut in gars, with endodermal contribution to the orofacial surface.
    Extended Data Fig. 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.

  13. CM-DiI fate-mapping of the primitive gut in sturgeons, with endodermal contribution to the orofacial surface.
    Extended Data Fig. 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).

  14. SEM images of sturgeon head mapping experimental fate-mapping data with endodermal contribution pseudocoloured yellow.
    Extended Data Fig. 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.

References

  1. Grevellec, A. & Tucker, A. S. The pharyngeal pouches and clefts: development, evolution, structure and derivatives. Semin. Cell Dev. Biol. 21, 325332 (2010)
  2. Shone, V. & Graham, A. Endodermal/ectodermal interfaces during pharyngeal segmentation in vertebrates. J. Anat. 225, 479491 (2014)
  3. Dickinson, A. J. G. & Sive, H. Development of the primary mouth in Xenopus laevis. Dev. Biol. 295, 700713 (2006)
  4. Soukup, V., Horácek, I. & Cerny, R. Development and evolution of the vertebrate primary mouth. J. Anat. 222, 7999 (2013)
  5. Graham, A. & Richardson, J. Developmental and evolutionary origins of the pharyngeal apparatus. Evodevo 3, 24 (2012)
  6. Choe, C. P. & Crump, J. G. Dynamic epithelia of the developing vertebrate face. Curr. Opin. Genet. Dev. 32, 6672 (2015)
  7. 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, 10611073 (2002)
  8. 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)
  9. Piotrowski, T. & Nüsslein-Volhard, C. The endoderm plays an important role in patterning the segmented pharyngeal region in zebrafish (Danio rerio). Dev. Biol. 225, 339356 (2000)
  10. 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, 237246 (2012)
  11. Lowe, C. J., Clarke, D. N., Medeiros, D. M., Rokhsar, D. S. & Gerhart, J. The deuterostome context of chordate origins. Nature 520, 456465 (2015)
  12. Kerr, J. G. The Work of John Samuel Budgett (Cambridge Univ. Press, 1907)
  13. Janvier, P. Early Vertebrates (Clarendon, 1996)
  14. Quinlan, R., Martin, P. & Graham, A. The role of actin cables in directing the morphogenesis of the pharyngeal pouches. Development 131, 593599 (2004)
  15. Alexander, J. & Stainier, D. Y. A molecular pathway leading to endoderm formation in zebrafish. Curr. Biol. 9, 11471157 (1999)
  16. 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, 915 (2002)
  17. Suda, Y. et al. Evolution of Otx paralogue usages in early patterning of the vertebrate head. Dev. Biol. 325, 282295 (2009)
  18. Rétaux, S. & Pottin, K. A question of homology for chordate adhesive organs. Commun. Integr. Biol. 4, 7577 (2011)
  19. 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, 649659 (2012)
  20. True, J. R. & Haag, E. S. Developmental system drift and flexibility in evolutionary trajectories. Evol. Dev. 3, 109119 (2001)
  21. Soukup, V., Epperlein, H.-H., Horácek, I. & Cerny, R. Dual epithelial origin of vertebrate oral teeth. Nature 455, 795798 (2008)
  22. Cooper, M. S. & Virta, V. C. Evolution of gastrulation in the ray-finned (actinopterygian) fishes. J. Exp. Zool. B Mol. Dev. Evol. 308, 591608 (2007)
  23. Nagasawa, T. et al. Evolutionary changes in the developmental origin of hatching gland cells in basal ray-finned fishes. Zoolog. Sci. 33, 272281 (2016)
  24. 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, 925934 (2014)
  25. 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, 234256 (2012)
  26. Seifert, R., Jacob, M. & Jacob, H. J. The avian prechordal head region: a morphological study. J. Anat. 183, 7589 (1993)
  27. Oisi, Y., Ota, K. G., Kuraku, S., Fujimoto, S. & Kuratani, S. Craniofacial development of hagfishes and the evolution of vertebrates. Nature 493, 175180 (2013)
  28. Sessel, A. in Archiv für Anatomie und Entwickelungsgeschichte (Veit & Comp., 1877)
  29. Godard, B. G. et al. Mechanisms of endoderm formation in a cartilaginous fish reveal ancestral and homoplastic traits in jawed vertebrates. Biol. Open 3, 10981107 (2014)
  30. 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, 90102 (2009)
  31. Hatschek, B. & Tuckey, J. The Amphioxus and its Development (Swan, Sonnenschein & Co., 1893)
  32. Brand, M., Granato, M. & Nüsslein-Volhard, C. in Zebrafish (Oxford Univ. Press, 2002)
  33. 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)
  34. Diedhiou, S. & Bartsch, P. in Development of Non-Teleost Fishes (Science Publishers, 2009)
  35. Dettlaff, T. A., Ginsburg, A. S. & Schmalhausen, O. I. Sturgeon Fishes—Developmental Biology and Aquaculture (Springer, 1993)
  36. Long, W. L. & Ballard, W. W. Normal embryonic stages of the longnose gar, Lepisosteus osseus. BMC Dev. Biol. 1, 6 (2001)

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Author information

Affiliations

  1. Department of Zoology, Charles University, Prague, Czech Republic

    • Martin Minarik,
    • Jan Stundl,
    • Peter Fabian,
    • David Jandzik,
    • Ivan Horácek &
    • Robert Cerny
  2. Department of Zoology, National Museum, Prague, Czech Republic

    • Jan Stundl
  3. Department of Zoology, Comenius University, Bratislava, Slovakia

    • David Jandzik
  4. Department of Theoretical Biology, University of Vienna, Vienna, Austria

    • Brian D. Metscher
  5. University of South Bohemia in České Bud ějovice, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodňany, Czech Republic

    • Martin Psenicka &
    • David Gela
  6. Universidad Juárez Autónoma de Tabasco, División Académica de Ciencias Biológicas, Villahermosa, Mexico

    • Adriana Osorio-Pérez &
    • Lenin Arias-Rodriguez

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

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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 Figures

  1. Extended Data Figure 1: Micro-CT visualization of bichir embryogenesis. (384 KB)

    Stages 19–28 (ref. 34).

  2. Extended Data Figure 2: Micro-CT visualization of sturgeon embryogenesis. (552 KB)

    Stages 22–26 (ref. 35).

  3. Extended Data Figure 3: Micro-CT visualization of gar embryogenesis. (455 KB)

    Stages 15–26 (ref. 36).

  4. Extended Data Figure 4: Micro-CT visualization of pharyngeal development (yellow) in bichir, sturgeon and gar. (690 KB)

    3D reconstructions of key developmental stages.

  5. Extended Data Figure 5: Outpocketing of POGD in bichir and secondary constriction of POGD in sturgeon pharyngulae during development. (803 KB)

    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.

  6. Extended Data Figure 6: Micro-CT visualization of sturgeon pharynx at two key developmental stages. (408 KB)

    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.

  7. Extended Data Figure 7: Gene expression patterns (Otx2, Sox17, FoxE4 and Pitx2) in POGD in bichir, sturgeon and gar embryos. (862 KB)

    Wholemount views and parasagittal vibratome sections (head to the left); DAPI (blue) stains cell nuclei.

  8. Extended Data Figure 8: CM-DiI fate-mapping of the primitive gut in gars, with endodermal contribution to the orofacial surface. (635 KB)

    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.

  9. Extended Data Figure 9: CM-DiI fate-mapping of the primitive gut in sturgeons, with endodermal contribution to the orofacial surface. (933 KB)

    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).

  10. Extended Data Figure 10: SEM images of sturgeon head mapping experimental fate-mapping data with endodermal contribution pseudocoloured yellow. (337 KB)

    Antero-ventral views, 10 and 15 days post hatching (d.p.h.); mb, medial barbel; lb, lateral barbel; ao, ampullary organs; rb, rostrum.

Supplementary information

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Additional data