Functional Analysis of wntless (wls) in Palate Development

描述（由申请人提供）：Wnt信号传导是胚胎发生和器官发生期间介导会聚和伸展（CE）运动的关键途径。影响Wnt信号传导的各个方面的突变导致口面或中线裂。我们以前曾报道过Wnt通路基因wnt9a，frzb和fzd7a调节颅面发育中的CE机制。Wnt信号的转导需要翻译后修饰的wnt配体与细胞内的wnt（wls）结合，分泌的wnt配体与细胞外结构域中的frzb伴侣结合，以及wnt与邻近细胞的卷曲受体结合。该提议验证了wls与wnt9a相互作用以调节在腭形态发生期间支配软骨细胞行为的CE机制的假设。目的1对wls进行分子生物学分析，a）描述wnt 9a，frzb，fzd7a在正常腭发育中的基因表达，b）检测wls在颅面突变体（edn 1，shh，pdgfra）中的表达变化，c）wls突变体中神经嵴和wnt基因的表达变化。目的2对wls突变体进行机制分析，研究腭软骨细胞如何通过细胞迁移、增殖、中外侧嵌入和腭元件整合等途径介导CE。细胞移植实验解决了腭发育过程中对wls的需求是否以细胞或非细胞自主方式起作用。目的3对wls与wnt9a相互作用进行功能分析，利用CRISPR介导的突变技术，检测wls、wnt9a和wls：wnt9a复合突变体的表型。 上述目标是合作的，但独立的，旨在为未来的工作提供基础，以阐明Wnt在腭和颅面发育的需求。我们预期这些目标将证明wls是软骨细胞嵌入和增殖所必需的，建立我们将用于分析腭形态发生的细胞测定，并提供调节腭和颅面发育的Wnt途径关键基因的突变体，用于未来的遗传学研究。这项研究的重点是起源于wnt信号的细胞内相互作用。未来的工作将研究由frzb在细胞外基质中陪伴的wnt信号在细胞间转导的遗传调控， 相邻的细胞。

DESCRIPTION (provided by applicant): 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.