Functional Analysis of wntless (wls) in Palate Development

Wntless (wls) 在味觉发育中的功能分析

• 批准号: 8747870
• 负责人: Eric Chien-Wei Liao
• 金额: $ 13.05万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8747870
• 关键词: Address; Affect; Area; Behavior; Binding; Cell Proliferation; Cell Transplantation; Cells; Cellular Assay; Cephalic; Chondrocytes; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Defect; Development; Distal; Ectoderm; Elements; Embryonic Development; Endothelin; Erinaceidae; Exhibits; Extracellular Domain; Extracellular Matrix; Future; Gene Expression; Genes; Genetic; Genetic screening method; Golgi Apparatus; Grant; Jaw; Lateral; Ligands; Medial; Mediating; Molecular; Molecular Analysis; Molecular Chaperones; Morphogenesis; Movement; Mutagenesis; Mutation; Neural Crest; Organogenesis; Oropharyngeal; Palate; Pathway interactions; Phase; Phenotype; Principal Investigator; Publishing; Regulation; Reporting; Role; Signal Transduction; Testing; Translations; Work; Zebrafish; base; cell motility; cellular transduction; craniofacial; design; intercalation; loss of function; migration; mutant; orofacial; palatogenesis; programs; public health relevance; receptor; research study

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.

描述（由申请人提供）：Wnt信号传导是胚胎发生和器官发生过程中介导汇聚和延伸（CE）运动的关键途径。影响 Wnt 信号传导各个方面的突变会导致口面部或中线裂痕。我们之前报道过Wnt通路基因wnt9a、frzb和fzd7a调节颅面发育中的CE机制。 Wnt 信号的转导需要细胞内的 wntless (wls) 结合翻译后修饰的 wnt 配体，胞外域中 frzb 结合分泌的 wnt 配体的伴侣，以及 wnt 与邻近细胞的卷曲受体结合。该提案测试了 wls 与 wnt9a 相互作用以调节上颚形态发生过程中控制软骨细胞行为的 CE 机制的假设。目标 1 对 wls 进行分子分析，a) 在 wnt9a、frzb、fzd7a 的背景下描述其在正常腭发育中的基因表达，b) 检查 wls 表达在颅面突变体（edn1、shh、pdgfra）中如何改变，c) wls 突变体中神经嵴和 wnt 基因的表达是如何改变的。目标 2 对 wls 突变体进行机制分析，检查腭软骨细胞如何经历由细胞迁移、增殖、内侧嵌入和腭元件整合介导的 CE。细胞移植实验探讨了上颚发育过程中对wls的需求是否以细胞或非细胞自主方式起作用。目标 3 对 wls 与 wnt9a 的相互作用进行功能分析，利用 CRISPR 介导的诱变检查 wls、wnt9a 和化合物 wls:wnt9a 突变体的表型。 上述目标是合作但独立的，旨在为未来阐明腭和颅面发育中 Wnt 要求的工作提供基础。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，并为未来的遗传学研究提供调节腭和颅面发育的Wnt途径关键基因的突变体。本研究重点关注产生 wnt 信号的细胞内相互作用。未来的工作将检查由细胞外基质中的 frzb 陪伴的 WNT 信号细胞间转导到卷曲受体的遗传调控。 相邻的小区。