Functional Analysis of Wnt Signaling Modulators Wnt9a, Frzb and Fzd7a in Craniofacial Convergence and Extension Morphogenesis

Background: Wnt signaling is a conserved mechanism regulating convergence and extension movements in vertebrates. Convergence and extension mechanisms mediate movements of facial prominences toward the midline and lengthening of jaw structures. We previously reported requirement of wnt9a in craniofacial morphogenesis. We have identified two frizzled related genes that potentially interact with wnt9a: frzb and fzd7a. This work reports functional as well as biochemical analysis of wnt9a, frzb and fzd7a to gain mechanistic insight into this important signaling pathway. Methods: Gene expression analysis was performed by fluorescent whole mount RNA in situ hybridization. Gene knockdown of wnt9a, frzb and fzd7a were achieved by injecting translation-blocking morpholino knockdown. Lineage tracing analysis of neural crest cells was performed using Tg::sox10: kaede transgenic line. Edu was used for cell proliferation and TUNEL for cell death assays. Endothelin signaling pathway was interrogated using zebrafish edn1 mutants. Results: Spatiotemporal gene expression of wnt9a, frzb and fzd7a were localized to pharyngeal arches and derivatives. Disruption of wnt9a, frzb and fzd7a resulted in absence of lower jaw and shortened palate. Lineage tracing analysis revealed that knockdown of frzb and fzd7a did not preclude migration of NCC to form the facial prominences. However, cell proliferation and convergent extension of the facial prominences failed to occur. We also found a down-regulation of the downstream transcription factor bapx1 in wnt9a, frzb and fzd7a morphants, while its over-expression by mRNA injection can partially rescue formation of the lower jaw. In addition, down-regulation of wnt9a, frzb, fzd7a in edn mutants suggests an interaction between wnt and endothelin signaling pathways, intersecting upon a common downstream transcription factor bapx1. Conclusion: This study underscores the important role of wnt-frizzled signaling in convergence and extension in craniofacial morphogenesis. We also discovered that bapx1 acts downstream of wnt-frizzled to coordinate development of the lower jaw.