Wnt signaling is a key pathway mediating convergence and extension (CE) movements during embryogenesis and organogenesis. Mutations affecting various aspects of Wnt signaling result in orofacial or midline clefts. We have previously reported that Wnt pathway genes wnt9a, frzb and fzd7a regulate CE mechanisms in craniofacial development. Transduction of the Wnt signal requires the binding of post-translational modified wnt ligand by wntless (wls) within the cell, chaperon of secreted wnt ligand by frzb in the extracellular domain, and binding of wnt to frizzled receptor of the neighboring cell. This proposal tests the hypothesis that wls interacts with wnt9a to regulate CE mechanisms that govern chondrocyte behavior during palate morphogenesis.
Aim 1 carries out molecular analysis of wls, a) delineates its gene expression in normal palate development in the context of wnt9a, frzb, fzd7a, b) examines how wls expression is altered in craniofacial mutants (edn1, shh, pdgfra) and c) how expression of neural crest and wnt genes are altered in the wls mutant.
Aim 2 performs mechanistic analysis of wls mutant, examining how the palate chondrocytes undergo CE mediated by cell migration, proliferation, mediolateral intercalation, and integration of palatal elements. Cell transplantation experiments address whether the requirement for wls during palate development acts in a cell or non-cell autonomous manner.
Aim 3 carries forward the functional analysis of wls to its interaction with wnt9a, examining the phenotype of wls, wnt9a and the compound wls:wnt9a mutant, using CRISPR mediated mutagenesis. The above aims are cooperative but independent, designed to provide the basis for future work to elucidate Wnt requirement in palate and craniofacial development. We anticipate these aims will demonstrate that wls is required for chondrocyte intercalation and proliferation, establish the cellular assays we will employ to analyze palate morphogenesis, and provide the mutants in key genes of the Wnt pathway that regulate palate and craniofacial development for future genetic studies. This study focuses on intra-cellular interactions that originate the wnt signal. Future work will examine genetic regulation of the inter-cellular transduction of wnt signal chaperoned by frzb in the extracellular matrix, to frizzled receptors on the neighboring cell.

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Wnt signaling is a key pathway mediating convergence and extension movements during palate development, where wnt9a, frzb, and fzd9a are required for chondrocyte proliferation and extension. Wntless (wls) chaperones wnt ligands, and this proposal tests whether wls interacts with wnt9a to regulate convergence and extension morphogenesis during palate development. This work establishes the basis for future analysis of wnt pathway genes, bridging the gap of understanding from intra-cellular to inter-cellular context.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Small Research Grants (R03)
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NIDR Special Grants Review Committee (DSR)
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Scholnick, Steven
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Massachusetts General Hospital
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Duncan, Kaylia M; Mukherjee, Kusumika; Cornell, Robert A et al. (2017) Zebrafish models of orofacial clefts. Dev Dyn 246:897-914
Ling, Irving Tc; Rochard, Lucie; Liao, Eric C (2017) Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification. Dev Biol 421:219-232
Ciarlo, Christie; Kaufman, Charles K; Kinikoglu, Beste et al. (2017) A chemical screen in zebrafish embryonic cells establishes that Akt activation is required for neural crest development. Elife 6:
Rochard, Lucie; Monica, Stefanie D; Ling, Irving T C et al. (2016) Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis. Development 143:2541-7
Rochard, Lucie J; Ling, Irving T C; Kong, Yawei et al. (2015) Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel's Cartilage. J Vis Exp :e52935