Zebrafish Wnt9b Patterns the First Pharyngeal Arch into D-I-V Domains and Promotes Anterior-Medial Outgrowth

Disrupted morphogenesis and growth of the embryonic maxillary jaw lead to oral facial clefting in humans (OFC) and result in an incompletely formed secondary mouth and face. A requirement for Wnt signaling and Wnt9b in particular are postulated in the etiology of OFC from association studies in humans and from animal models. Loss of murine Wnt9b leads to reduced upper jaw (maxillary) outgrowth and OFC, though the signaling architecture leading to this phenotype is poorly understood. Previous murine Wnt9b studies largely overlooked cranial neural crest cell (CNCC) patterning events and instead focused on later events during fusion of facial prominences. Using zebrafish and a morpholino-mediated knockdown approach, we demonstrate functional requirements for Wnt9b signaling during two crucial stages of facial development: 1) CNCC patterning into Dorsal-Intermediate-Ventral (D-I-V) domains; and 2) facial outgrowth during the primary to secondary mouth transition (PM to SM). Zebrafish embryos deficient for Wnt9b (Wnt9b morphants) exhibit an open bite with fused jaw joints as well as a flat face. Open bite and jaw joint fusion in Wnt9b morphants phenocopies characteristics of edn1 pathway is mutant zebrafish with disrupted D-I-V patterning of CNCC. Expression studies show Wnt9b morphants exhibit perturbed expression of edn1 signaling targets including dlx2a, jag1b, and msxe, consistent with disrupted CNCC patterning. Wnt9b morphant upper jaws have stunted outgrowth reminiscent of murine Wnt9b mutants and Wnt9b morphant skulls phenocopy the broad class of foreshortened skull zebrafish mutants known as hammerheads. Wnt9b morphants show upregulated expression of pitx2a after the opening of the primary mouth and disrupted expression of Wnt5b which is consistent with disrupted chondrocyte stacking. Strong upregulation of dorsal mesodermal frzb expression in the prechordal plate of Wnt9b morphants suggests a role for Wnt9b in primary mouth induction or maintenance. Collectively these results argue that Wnt9b has a much earlier developmental requirement. This work draws attention to potential vertebrate homologies that pattern CNCC and facial outgrowth and therefore calls for a reexamination of Wnt9b’s role during mammalian craniofacial development.