The pre-oral gut on the embryonic face reveals novel ectoderm-endoderm interactions during vertebrate head development

The basic vertebrate body plan arises in early development from three embryonic germ layers: ectoderm, endoderm and later mesoderm. In the head, interactions between ecto- and endoderm – without intervening mesoderm – have long been considered to take place only at two strictly defined locations: the primary mouth and pharyngeal gill slits. These key ecto-endodermal interfaces later form the openings allowing communication between outer and inner environments, and give rise to a complex assemblage of organs essential for sensing, feeding, or breathing. In early bichir, sturgeon, and gar embryos, we have previously identified an additional ecto-endodermal interface, whose developmental origin precedes the primary mouth formation as well as pharyngeal slit opening. Here, ectoderm gives way to the endodermal pre-oral gut (POG), which is then integrated into the surface of the embryonic face. However, the ecto-endodermal dynamics during the process of POG externalisation remain largely unknown. Using lineage tracing, high-resolution microscopy and immunohistochemistry, we characterise the complex ecto-endodermal interactions during the process of POG externalisation in bichir, gar, and sturgeon embryos at a cellular level. In bichir and gar, a simple outbulging of the endoderm layer occurs, resulting in exposed endodermal secretory cells constituting their cement glands on the embryonic surface. In sturgeon, however, an intricate intermixing between the POG and the surface periderm cells was revealed, with periderm cells alternating with secretory endodermal cells, expanding the hatching gland cells over the ventral rostrum. Our work further shows how complex changes in the cellular structure of the POG reflect its different fate and function in the studied species. In sturgeon, the POG gives rise to transient hatching gland cells that accumulate and then release hatching enzymes, whereas in bichir and gar, the POG forms cement glands with adhesive function. Our work reveals complex dynamics and architecture of this unique ecto-endoderm interface within the pre-mandibular head domain, which forms ahead of other oropharyngeal openings. We reveal substantial changes in the composition of endodermal secretory cells, compare various architectures of the ectoderm-endoderm interfaces, and discuss possible changes in the germ layer origin of these rostral ephemeral embryonic glands in vertebrates.