Regulation of Wnt Signaling in Development

发育过程中 Wnt 信号传导的调控

• 批准号: RGPIN-2016-06207
• 负责人: VanRaay, Terence
• 金额: $ 2.04万
• 依托单位: University of Guelph
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616160
• 关键词: Regulation; Wnt; Signaling; Development

Progressing from a single fertilized cell into a mature organism requires immeasurable coordination of multiple signaling pathways acting in concert. Part of this coordination comes from communication between different signaling pathways and part comes from communication within a signaling pathway, the so-called intrinsic feedback mechanisms. My long-term goals are to understand how these intrinsic feedback mechanisms work to control signaling pathways that, in turn, control development. My short-term goals are to understand the molecular function of how negative feedback regulators control the Wnt pathway. The Wnt pathway is critically important for both early development and in the maintenance of stem cells in the adult. Our investigations will utilize traditional cell culture but also take advantage of the early zebrafish embryo because of its optical transparency, genetics and evolutionary conserved mechanisms. Further, this model provides a native environment in which to investigate the Wnt pathway, which will contribute to our universal understanding of its intrinsic regulation. In this proposal, we will investigate two negative feedback regulators: Nkd1 and Axin2. In Aim 1 we will determine how Nkd1 and Axin2 control the intensity and duration of the Wnt signal during early zebrafish development. We will use state-of-the-art genetic engineering tools to knockout Nkd1 and Axin2 function and observe how these mutants respond to perturbed Wnt signaling. Our current Discovery Grant focuses on the molecular function of Nkd1, where we demystified the function of this protein using the early zebrafish embryo as a cell-signaling model. We found that Nkd1 requires an activation step, which accounts for its unique activity. Here, in collaboration with faculty at the University of Toronto, we will identify the molecular nature of this activation on Nkd1 in Aim 2. This will be accomplished with mass spectrometry on human Nkd1 using traditional cell culture techniques. Putative Nkd1 modifications will be authenticated in our zebrafish model. Critical to this aim is that Nkd1 function is highly conserved from flies to humans. Finally, preliminary results suggest that the activation of Nkd1 is dependent on how the signaling complex is arranged on the cell membrane. Thus, our 3rd Aim will pursue these preliminary results in depth to better understand Nkd1 activation. Here, we will again use the zebrafish embryonic cells where we and others have shown that Wnt signaling becomes compartmentalized on the membrane in these cells. We plan to disrupt these compartments using genetic tools to understand how this affects Nkd1 activation. The experiments described in this proposal will allow us to better understand how signaling pathways communicate within themselves to maintain a balanced signal level that is required for the proper development of the vertebrate embryo.

从单个受精细胞发展成成熟生物体需要多个信号通路的不可估量的协调一致作用。这种协调部分来自不同信号通路之间的通信，部分来自信号通路内的通信，即所谓的内在反馈机制。我的长期目标是了解这些内在反馈机制如何控制信号通路，进而控制发育。我的短期目标是了解负反馈调节器如何控制 Wnt 通路的分子功能。 Wnt 通路对于成人干细胞的早期发育和维持都至关重要。我们的研究将利用传统的细胞培养，但也会利用早期斑马鱼胚胎，因为它的光学透明性、遗传学和进化保守机制。此外，该模型提供了研究 Wnt 通路的原生环境，这将有助于我们对其内在调控的普遍理解。在本提案中，我们将研究两个负反馈调节因子：Nkd1 和 Axin2。在目标 1 中，我们将确定 Nkd1 和 Axin2 在早期斑马鱼发育过程中如何控制 Wnt 信号的强度和持续时间。我们将使用最先进的基因工程工具来敲除 Nkd1 和 Axin2 功能，并观察这些突变体如何响应扰动的 Wnt 信号传导。 我们目前的发现资助项目重点关注 Nkd1 的分子功能，我们使用早期斑马鱼胚胎作为细胞信号传导模型揭开了该蛋白质功能的神秘面纱。我们发现 Nkd1 需要一个激活步骤，这解释了其独特的活性。在这里，我们将与多伦多大学的教师合作，在目标 2 中确定 Nkd1 激活的分子性质。这将通过使用传统细胞培养技术对人类 Nkd1 进行质谱分析来完成。假定的 Nkd1 修饰将在我们的斑马鱼模型中得到验证。这一目标的关键在于 Nkd1 功能从果蝇到人类都高度保守。 最后，初步结果表明 Nkd1 的激活取决于信号复合物在细胞膜上的排列方式。因此，我们的第三个目标将深入研究这些初步结果，以更好地了解 Nkd1 的激活。在这里，我们将再次使用斑马鱼胚胎细胞，我们和其他人已经证明 Wnt 信号在这些细胞的膜上被区室化。我们计划使用遗传工具破坏这些区室，以了解这如何影响 Nkd1 激活。 该提案中描述的实验将使我们能够更好地了解信号通路如何在其内部进行通信，以维持脊椎动物胚胎正常发育所需的平衡信号水平。