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信号传导是一种关键途径，介导了胚胎发生和器官发生过程中融合和扩展（CE）运动。影响Wnt信号传导各个方面的突变导致口面或中线裂缝。我们以前已经报道过Wnt途径基因Wnt9a，FRZB和FZD7A在颅面发育中调节CE机制。 Wnt信号的转导需要通过细胞内Wntless（WLS）的翻译后修饰的Wnt配体的结合，FRZB在细胞外结构域中的分泌Wnt配体的伴侣，Wnt结合WNT与附近细胞的毛躁受体。该提案检验了WLS与WNT9A相互作用的假设，以调节在pa酸形态发生过程中控制软骨细胞行为的CE机制。 AIM 1对WLS进行了分子分析，a）在WNT9A，FRZB，FZD7A，B）中描述其在正常pa发育中的基因表达，研究了WLS表达如何在颅面突变体（EDN1，SHH，PDGFRA）和C）中如何改变WLS突变体中神经Crest和Wnt基因的表达如何改变。 AIM 2对WLS突变体进行了机械分析，研究了味蕾软骨细胞如何通过细胞迁移，增殖，中外侧插入和pa元元件的整合而介导的CE。细胞移植实验解决了口感发育过程中WLS的需求是否以细胞或非细胞自主方式起作用。 AIM 3将WLS的功能分析与WNT9A相互作用，研究了WLS，Wnt9a和化合物WLS：Wnt9a突变体的表型，使用CRISPR介导的诱变。 上述目的是合作的，但独立的，旨在为未来的工作提供基础，以阐明pa和颅面发展中的Wnt要求。我们预计这些目标将表明，WLS对于软骨细胞插入和增殖需要，建立我们将采用的细胞测定法来分析口服形态发生，并在WNT途径的关键基因中提供突变体，以调节pape pleate和Craniofacial开发未来的遗传研究。这项研究的重点是源自Wnt信号的细胞内相互作用。未来的工作将检查FRZB在细胞外基质中伴随的Wnt信号的细胞间转导的遗传调节，以使毛躁的受体在 相邻的细胞。