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在正常发育和恶性肿瘤中影响造血干细胞。拟议的研究集中在原始造血过程中的小生境细胞中的非经典和经典Wnt信号之间的相互作用。经典和非经典Wnt通路通常同时激活，并相互抑制。这一点很重要，因为Wnt信号的不平衡会导致人类出生缺陷和癌症;然而，调节这种平衡的机制却知之甚少。如下所述，我们已经鉴定了原始造血所需的非经典Wnt信号传导的新型激活剂，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。非洲爪蟾胚胎的外胚层需要转录因子GATA 2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以下假设：GATA 2诱导新蛋白质TRIL的表达，TRIL激活非经典Wnt信号传导，从而使细胞能够被指定为血液祖细胞。我们将测试这一假设并确定TRIL信号传导的机制，如下：（1）确定外胚层GATA 2是否抑制经典和激活非经典Wnt信号传导，以及这是否是造血所必需和充分的。我们将研究GATA 2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，以询问Wnt信号传导是否是外胚层和/或中胚层血液形成所必需的。(2)确定TRIL是否是造血所需的，以及是否用于激活非经典Wnt信号传导。我们将分析非洲爪蟾胚胎中TRIL表达被敲低的血液形成，并询问非经典Wnt信号的激活是否足以拯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经典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丧失测定来测试TRIL功能是否需要上游、下游或与非经典Wnt途径的已知组分平行。此外，我们将研究TRIL在非洲爪蟾胚胎中相对于其他Wnt通路组分的亚细胞定位，并对TRIL进行体内结构-功能分析。最后，我们将鉴定在Wnt信号转导的背景下与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. )