Functional Analysis of wntless (wls) in Palate Development

描述(申请人提供)：WNT信号是在胚胎发生和器官发生过程中调节会聚和延伸(CE)运动的关键途径。影响Wnt信号的各个方面的突变会导致口面部或中线裂隙。我们以前报道过Wnt通路基因wnt9a、frzb和fzd7a调控颅面发育中的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WLS结合，由FRZB在细胞外域结合分泌的WNT配体，以及WNT与相邻细胞的卷曲受体结合。这一建议验证了WLS与wnt9a相互作用以调控CE机制的假说，CE机制控制着软骨细胞在上颌骨形态发生过程中的行为。目的1)利用wnt9a，frzb，fzd7a，b)研究WLS基因在颅面突变体(edn1，shh，PDGFRA)中的表达变化，以及在WLS突变体中神经脊和WNT基因的表达变化。目的对WLS突变体进行机制分析，研究细胞迁移、增殖、内侧嵌插、腭部元素整合等因素对腭部软骨细胞CE的影响。细胞移植实验解决了腭部发育过程中对WLS的需求是以细胞还是非细胞自主方式起作用。目的对wls与wnt9a的相互作用进行功能分析，采用CRISPR介导的突变方法，检测wls、wnt9a及Wls：wnt9a突变体的表型。以上目标是合作的，但又是独立的，旨在为未来阐明腭部和颅面发育中WNT需求的工作提供基础。我们预计这些目标将证明WLS是软骨细胞插入和增殖所必需的，建立我们将用于分析腭部形态发生的细胞分析方法，并为未来的遗传学研究提供调节腭部和颅面发育的Wnt途径关键基因的突变。这项研究的重点是起源WNT信号的细胞内相互作用。未来的工作将研究由细胞外基质中的frzb伴随的wnt信号在细胞间转导到卷曲的受体的遗传调节。 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.