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Exploring the role of Wdr68 in craniofacial development in zebrafish

探索 Wdr68 在斑马鱼颅面发育中的作用

基本信息

批准号：
8626186
负责人：
ROBERT M NISSEN
金额：
$ 43.5万
依托单位：
CALIFORNIA STATE UNIVERSITY LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-06 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8626186
关键词：
Adult Agonist Animals Automobile Driving Cancer Etiology Cartilage Cessation of life Complex Data Collection Defect Development Disease Disease Progression Embryo Embryonic Development Endothelin-1 Erinaceidae Event Foundations Future Gene Expression Gene Transfer Techniques Genes Genetic Goals Grant Heat-Shock Response In Situ Hybridization In Vitro Injection of therapeutic agent Jaw Life Light Link MAP3K1 gene Mediating Messenger RNA Methods Modality Modeling Molecular Organism Outcome Pancreatic Ductal Adenocarcinoma Pathology Pathway interactions Pattern Play Publications RNA Reporter Research Research Personnel Resources Role Shapes Signal Transduction Signal Transduction Pathway Somites Staging Structure Study models System Testing Time Transgenic Organisms Vertebrates Work Zebrafish autocrine cancer pain craniofacial graduate student human disease in vivo in vivo Model inhibitor/antagonist innovation insight knowledge base mutant novel novel therapeutics overexpression programs public health relevance research study restoration small molecule smoothened signaling pathway somitogenesis undergraduate student wasting

项目摘要

PROJECT SUMMARY Signal transduction pathways important during development are frequently associated with disease. Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death. The Hedgehog (Hh) pathway is important for development and a Ras-Dyrk1b pathway downregulates autocrine Hh signaling in PDA. Wdr68 and Dyrk1b can physically interact and are important for multiple events in the zebrafish, including craniofacial development. Wdr68 and Dyrk1b are also important for edn1 expression for craniofacial development. Intriguingly, Wdr68 physically bridges signaling from MEKK1 to Dyrk1b suggesting the existence of Ras- MAP3K-Wdr68-Dyrk1b signal relay systems. However, while it is clear that Dyrk1b plays a key role in modulating Hh signaling in PDA, the role of Dyrk1b in Hh signaling during normal development is unknown. Likewise, the role of Wdr68 in Hh signaling is unknown. Furthermore, the genetic requirements and timing for wdr68 in edn1-mediated craniofacial development are still unclear. Therefore, the overall aim of this application is to better define the in vivo roles for Wdr68 and Dyrk1b. Our central hypothesis is that Wdr68 is part of an evolutionarily conserved transcriptional co-regulator complex that modulates multiple signaling events important during embryonic and adult life. The central hypothesis will be tested through three Specific Aims. First, we will test wdr68 and dyrk1b gene activity for roles in Hh signaling. We hypothesize that wdr68 and dyrk1b modulate Hh signaling during craniofacial development. The experimental approach will use in situ hybridization (ISH) analysis of wdr68 and dyrk1b mutants and morphants to determine whether there are defects in Hh signaling. We will also treat embryos with small molecule activators and inhibitors of the Hh signaling pathway for potential restoration of jaw development. We expect to see altered levels of at least some aspects of Hh signaling in mutants and morphants. We also expect Hh antagonists will restore cartilage formation in wdr68 mutants. Second, we will test whether ectopic endothelin-1 (edn1) expression can rescue the lower jaw defects in wdr68 mutant or morphant embryos. We hypothesize that ectopic edn1 expression in wdr68 mutants will restore lower jaw formation. The experimental approach will employ the parallel approaches of edn1 expression construct injection as well as the use of a GAL4-UAS system to overexpress edn1 in wdr68 morphants. We expect that ectopic edn1 expression will restore lower jaw formation in wdr68 mutants/morphants. Third, we will determine the temporal requirement for wdr68 activity in craniofacial development. We hypothesize that wdr68 is required during late-somitogenesis stages for normal craniofacial development. The experimental approach will use a heat shock inducible GFP- Wdr68 Tg(hsp70l:GFP-Wdr68) transgenic line. Because several gene expression defects are readily detected by 24hpf, we expect slightly earlier (by 18hpf) heat shock induction of GFP-Wdr68 to rescue transgenic wdr68- MO animals while late heat shock (20hpf or later) to fail to rescue craniofacial development.

项目摘要在发育过程中重要的信号转导途径经常与疾病相关。胰腺导管腺癌（PDA）是癌症死亡的第四大原因。Hedgehog（Hh）途径 Ras-Dyrk 1b通路下调PDA中自分泌Hh信号。Wdr68 和Dyrk 1b可以物理相互作用，对斑马鱼的多个事件很重要，包括颅面发展Wdr 68和Dyrk 1b对颅面发育的edn 1表达也很重要。有趣的是，Wdr 68在物理上桥接了MEKK 1和Dyrk 1b之间的信号传导，这表明Ras的存在。 MAP 3 K-Wdr 68-Dyrk 1b信号中继系统。然而，尽管很明显Dyrk 1b在虽然Dyrk 1b在PDA中调节Hh信号传导，但在正常发育过程中Dyrk 1b在Hh信号传导中的作用尚不清楚。同样，Wdr 68在Hh信号传导中的作用也是未知的。此外，遗传要求和时间， wdr 68在edn 1介导的颅面发育中的作用尚不清楚。因此，本申请的总体目标是更好地定义Wdr 68和Dyrk 1b的体内作用。我们的中心假设是Wdr 68是一个调节多种信号事件的进化上保守的转录辅助调节因子复合物在胚胎期和成年期都很重要。中心假设将通过三个具体目标进行检验。首先，我们将测试wdr 68和dyrk 1b基因活性在Hh信号传导中的作用。我们假设wdr 68 dyrk 1b在颅面发育过程中调节Hh信号。实验方法将在现场使用 wdr 68和dyrk 1b突变体和morphant的杂交（ISH）分析，以确定是否存在 Hh信号的缺陷。我们还将用Hh的小分子激活剂和抑制剂治疗胚胎，可能恢复颌骨发育的信号通路。我们预计至少会看到 Hh信号在突变体和morphant中的一些方面。我们还希望Hh拮抗剂能够恢复软骨 wdr 68突变体的形成。第二，我们将检测异位内皮素-1（edn 1）表达是否能挽救wdr 68突变体或变形体胚胎中的下颌缺陷。我们假设异位edn 1 在wdr 68突变体中的表达将恢复下颌的形成。实验方法将采用 edn 1表达构建体注射的平行方法以及使用GAL 4-UAS系统，在WDR 68变形体中过表达EDN 1。我们预期异位的edn 1表达会使下颌骨在wdr 68突变体/变形体中的形成。第三，我们将确定wdr 68的时间要求。颅面发育的活动。我们推测，WDR 68在后期体节发生过程中是必需的。正常颅面发育的阶段。实验方法将使用热休克诱导的GFP- Wdr 68 Tg（hsp 70 1：GFP-Wdr 68）转基因株系。因为几种基因表达缺陷很容易被检测到到24 hpf，我们预期GFP-Wdr 68的热休克诱导稍早（到18 hpf）以拯救转基因wdr 68- MO动物，而晚期热休克（20 hpf或更晚）未能挽救颅面发育。