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
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Micro-CT visualization of bichir embryogenesis. (384 KB)
Stages 19–28 (ref. 34).
- Extended Data Figure 2: Micro-CT visualization of sturgeon embryogenesis. (552 KB)
Stages 22–26 (ref. 35).
- Extended Data Figure 3: Micro-CT visualization of gar embryogenesis. (455 KB)
Stages 15–26 (ref. 36).
- Extended Data Figure 4: Micro-CT visualization of pharyngeal development (yellow) in bichir, sturgeon and gar. (690 KB)
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. (803 KB)
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.
- 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.
- 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.
- 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, 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. (933 KB)
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. (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 (2.3 MB)
This file contains Supplementary Table 1.