Fgf signalling is required for gill slit formation in the skate, Leucoraja erinacea.

The gill slits of fishes develop from an iterative series of pharyngeal endodermal pouches that contact and fuse with surface ectoderm on either side of the embryonic head. We find in the skate (Leucoraja erinacea) that all gill slits form via a stereotypical sequence of epithelial interactions: 1) endodermal pouches approach overlying surface ectoderm, with 2) focal degradation of ectodermal basement membranes preceding endoderm-ectoderm contact; 3) endodermal pouches contact and intercalate with overlying surface ectoderm, and finally 4) perforation of a gill slit occurs by epithelial remodelling, without programmed cell death, at the site of endoderm-ectoderm intercalation. Skate embryos express Fgf8 and Fgf3 within developing pharyngeal epithelia during gill slit formation. When we inhibit Fgf signalling by treating skate embryos with the Fgf receptor inhibitor SU5402 we find that endodermal pouch formation, basement membrane degradation and endodermal-ectodermal intercalation are unaffected, but that epithelial remodelling and gill slit perforation fail to occur. These findings point to a role for Fgf signalling in epithelial remodelling during gill slit formation in the skate and, more broadly, to an ancestral role for Fgf signalling during pharyngeal pouch epithelial morphogenesis in vertebrate embryos.

Ectodermal Wnt signaling, cell fate determination, and polarity of the skate gill arch skeleton.

The gill skeleton of cartilaginous fishes (sharks, skates, rays, and holocephalans) exhibits a striking anterior-posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Sonic hedgehog (Shh) signaling from a distal gill arch epithelial ridge (GAER) signaling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here, we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

Distinct proliferative and middle ear skeletal-patterning functions for SHH-expressing epithelia in the chick hyoid arch.

During chick craniofacial development, the second (hyoid) pharyngeal arch expands to close the neck and gives rise to skeletal elements, including the columella of the middle ear (a homologue of the mammalian stapes). Sonic hedgehog (SHH) signalling has been implicated in hyoid arch expansion and columella formation, but spatial and temporal aspects of these signalling interactions within the hyoid arch remain poorly understood. Here, we show that SHH is initially expressed in the posterior endoderm of the hyoid arch, and that this domain subsequently splits into a distal domain at the site of arch expansion (the posterior epithelial margin, PEM), and a proximal domain that lines the foregut (the proximal hyoid epithelium, PHE). Pharmacological manipulations and heterotopic grafting experiments demonstrate that SHH signalling is required for hyoid arch expansion and skeletogenesis, and reveal distinct roles for the PEM and PHE in these processes. The PEM promotes mesenchymal cell proliferation during arch expansion but is not sufficient to repattern the columella. Conversely, the PHE promotes mesenchymal cell survival, and PHE grafts induce partial duplication of the columella. This work demonstrates crucial and distinct roles for endodermal SHH signalling in hyoid arch morphogenesis and patterning of the middle ear skeleton.