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.