Exploring the role of Wdr68 in craniofacial development in zebrafish

项目总结 发育过程中重要的信号转导通路常常与疾病有关。胰腺 导管腺癌(PDA)是癌症死亡的第四大原因。刺猬(HH)路径是 Ras-Dyrk1b途径下调PDA中的自分泌HH信号。Wdr68 Dyrk1b可以在物理上相互作用，并且对斑马鱼的多种活动都很重要，包括头面部 发展。Wdr68和Dyrk1b对于edn1在颅面发育过程中的表达也是重要的。 有趣的是，Wdr68物理上连接了从MEKK1到Dyrk1b的信号，表明RAS的存在- MAP3K-Wdr68-Dyrk1b信号中继系统。然而，尽管很明显Dyrk1b在 在PDA中调节HH信号，Dyrk1b在正常发育过程中HH信号中的作用尚不清楚。 同样，Wdr68在HH信号中的作用也是未知的。此外，遗传要求和时间选择 Wdr68在edn1介导的颅面发育中的作用仍不清楚。因此，此应用程序的总体目标是 是为了更好地定义Wdr68和Dyrk1b在体内的角色。我们的中心假设是Wdr68是 进化保守的转录共调节复合体，调节多个信号事件 在胚胎和成年时期很重要。核心假设将通过三个具体目标进行检验。 首先，我们将测试wdr68和dyrk1b基因在HH信号转导中的作用。我们假设wdr68 和dyrk1b在颅面发育过程中调节HH信号。实验方法将在现场使用 Wdr68和dyrk1b突变体和变异体的杂交(ISH)分析以确定是否存在 HH信号的缺陷。我们还将用小分子激活剂和HH抑制剂来治疗胚胎。 颌骨发育潜在恢复的信号通路。我们预计至少会看到变化的水平 突变体和变异体中HH信号的某些方面。我们还预计HH拮抗剂将恢复软骨 Wdr68突变体的形成。其次，我们将测试异位表达内皮素-1(EDN1)是否可以 挽救wdr68突变或变异胚胎的下颌骨缺陷。我们假设异位的EDN1 在wdr68突变体中的表达将恢复下颌骨的形成。该实验方法将使用 EDN1表达构建注射的平行方法以及GAL4-UAS系统的使用 在wdr68变异体中过表达edn1。我们预计异位表达edn1将恢复下颌骨。 Wdr68突变体/变异体的形成。第三，我们将确定wdr68的时间需求。 头面部发育中的活动。我们假设wdr68在晚期体细胞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- Wdr68TG(hsp701：GFP-Wdr68)转基因株系。因为几种基因表达缺陷很容易被检测到 到24hpf，我们预计稍早(18hpf)热休克诱导GFP-wdr68拯救转基因wdr68- 莫动物虽然晚期热休克(20hpf或更高)，但未能挽救颅面发育。

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.