Novel developmental regulation of non-canonical Wnt signaling

描述（由申请人提供）：我们的长期目标是了解来自微环境或生态位的信号如何影响正常发育和恶性肿瘤期间的造血干细胞。拟议的研究重点是原始造血过程中利基细胞中非经典和经典 Wnt 信号传导之间的相互作用。规范和非规范 Wnt 通路通常同时激活并相互抑制。这一点很重要，因为 Wnt 信号传导失衡会导致人类出生缺陷和癌症；然而，人们对调节这种平衡的机制知之甚少。如下所述，我们已经鉴定出一种新的非经典 Wnt 信号传导激活剂，它是原始造血所需的，并且可能有助于在整个发育过程中维持 Wnt 信号传导的适当平衡。爪蟾胚胎的外胚层需要转录因子 GATA2 来诱导蛋白质的表达，然后向中胚层发出信号，使其能够形成血液。我们的初步数据支持这样的假设：GATA2 诱导一种新蛋白 TRIL 的表达，该蛋白激活非经典 Wnt 信号传导，从而使细胞被指定为血液祖细胞。我们将检验这一假设并确定 TRIL 信号传导的机制，如下： (1) 确定外胚层 GATA2 是否抑制经典 Wnt 信号传导并激活非经典 Wnt 信号传导，以及这对于造血是否是必要和充分的。我们将检查 GATA2 功能受到干扰的非洲爪蟾胚胎中的内源性 Wnt 通路激活，并将阻断整个动物和组织重组体中的非经典 Wnt 信号传导，以探究 Wnt 信号传导在外胚层和/或中胚层中是否是血液形成所必需的。 (2) 确定TRIL是否是造血和非经典Wnt信号激活所必需的。我们将分析 TRIL 表达被敲低的非洲爪蟾胚胎中的血液形成，并询问非经典 Wnt 信号的激活是否足以挽救这些胚胎中的血液。我们将比较 TRIL 表达受到干扰的胚胎中内源性非规范 Wnt 信号传导的激活水平。 (3)确定TRIL激活非经典Wnt信号传导的分子机制。我们将在非洲爪蟾中使用功能获得和功能丧失测定来测试 TRIL 功能是否需要上游、下游或与非经典 Wnt 通路的已知组件并行。此外，我们将检查 TRIL 在非洲爪蟾胚胎中相对于其他 Wnt 通路成分的亚细胞定位，并对 TRIL 进行体内结构功能分析。最后，我们将鉴定在 Wnt 信号转导背景下与 TRIL 相互作用的蛋白质。 ）

DESCRIPTION (provided by applicant): Our long-term goal is to understand how signals from the microenvironment, or niche, affect hematopoietic stem cells during normal development and in malignancies. The proposed studies focus on the interplay between non-canonical and canonical Wnt signaling in niche cells during primitive hematopoiesis. Canonical and non-canonical Wnt pathways are often activated simultaneously and function to inhibit each other. This is important because imbalances in Wnt signaling lead to human birth defects and cancers; however the mechanisms that regulate this equilibrium are poorly understood. As described below, we have identified a novel activator of non-canonical Wnt signaling that is required for primitive hematopoiesis and likely contributes to maintaining the proper balance of Wnt signaling throughout development. The transcription factor GATA2 is required in the ectoderm of Xenopus embryos to induce expression of a protein that then signals to the mesoderm to enable it to form blood. Our preliminary data support the hypothesis that GATA2 induces expression of a novel protein, TRIL, which activates non- canonical Wnt signaling thereby enabling cells to be specified as blood progenitors. We will test this hypothesis and determine the mechanism by which TRIL signals, as follows (1) Determine whether ectodermal GATA2 represses canonical and activates non-canonical Wnt signaling, and whether this is necessary and sufficient for hematopoiesis. We will examine endogenous Wnt pathway activation in Xenopus embryos in which GATA2 function is perturbed, and will block non-canonical Wnt signaling in whole animals and tissue recombinants to ask whether Wnt signaling is necessary in ectoderm and/or mesoderm for blood formation. (2) Determine whether TRIL is required hematopoiesis and for activation of non-canonical Wnt signaling. We will analyze blood formation in Xenopus embryos in which TRIL expression is knocked down and will ask whether activation of non-canonical Wnt signaling is sufficient to rescue blood in these embryos. We will compare the level of activation of endogenous non-canonical Wnt signaling in embryos in which TRIL expression is perturbed. (3) Determine the molecular mechanism by which TRIL activates non-canonical Wnt signaling. We will use gain- and loss-of- function assays in Xenopus to test whether TRIL function is required upstream, downstream or in parallel with known components of non-canonical Wnt pathways. In addition, we will examine the subcellular localization of TRIL in Xenopus embryos relative to other Wnt pathway components and perform an vivo structure-function analysis of TRIL. Lastly, we will identify proteins that interact with TRIL in the context of Wnt signal transduction. )