Craniofacial defects are among the most frequent congenital abnormalities, constituting 35% of all birth defects. Studies of animal models offer the opportunity to understand the conserved mechanisms regulating vertebrate development that are responsible for congenital defects. This may help finding new ways to prevent, diagnose and treat craniofacial anomalies. We propose to study the mechanisms controlling craniofacial cartilage morphogenesis by employing a powerful combination of genetic, embryological, and molecular methods uniquely available for zebrafish (Danio rerio). We will test the specific hypothesis that Wnt5 and its putative co-receptor glypican Knypek control the elongation of craniofacial cartilage elements by regulation of chondrocyte polarity. We base this hypothesis on the observations that: 1) wnt5 and knypek mutant embryos exhibit dramatic shortening of the cartilage elements, 2) both mutants have similarly disorganized chondrocytes, 3) Knypek and Wnt5 control cell polarity required for normal convergence and extension movements during early developmental stages (gastrulation), and 4) Knypek cooperates with Wnt11 in the control of gastrulation movements. We will verify our hypothesis by realizing the following Specific Aims: 1. Uncover the cellular basis of wnt5 and knypek mutant cartilage morphogenesis defects, i) We will test if all cartilage elements are similarly affected by the loss of Wnt5 and Knypek. Furthermore, with the aid of in vivo confocal microscopy and a transgenic line of zebrafish expressing membrane localized GFP, we will ii) describe morphogenetic movements and Hi) cellular behaviors controlled by Wnt5 and Knypek activity. 2. Determine if Wnt5 and Knypek functionally interact in the control of pharyngeal cartilage morphogenesis. We will examine whether i) knypek and wnt5 have overlapping expression patterns during pharyngeal cartilage morphogenesis. Next, we will ii) investigate the potential role of Knypek in Wnt5 reception and ligand transport. 3. Verify whether Wnt5 and Knypek control cartilage morphogenesis by signaling on a noncanonical Wnt pathway. We will test the ability of well-established elements of the noncanonical signal transduction pathway, such as Dishevelled and Rho associated kinase 2, to suppress knypek and wnt5 mutant phenotypes. ? ? ?
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