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微环境或利基环境的信号如何在正常发育期间和恶性肿瘤中影响造血干细胞。建议的研究集中在原始造血过程中，非规范和规范的Wnt信号在壁龛细胞中的相互作用。规范的和非规范的Wnt通路经常同时被激活，并且相互抑制。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鲜为人知。如下所述，我们已经确定了一种新的非规范Wnt信号激活剂，它是原始造血所必需的，可能有助于在整个发育过程中维持Wnt信号的适当平衡。非洲爪哇胚胎的外胚层需要转录因子GATA2来诱导一种蛋白质的表达，然后该蛋白质向中胚层发出信号，使其能够形成血液。我们的初步数据支持这样的假设，即GATA2诱导一种新的蛋白TRIL的表达，该蛋白激活非规范的Wnt信号，从而使细胞被指定为造血祖细胞。我们将验证这一假说，并确定TRIL信号的机制如下：(1)确定外胚层GATA2是否抑制和激活非典型的Wnt信号，以及这是否是造血所必需的和充分的。我们将在GATA2功能被干扰的非洲爪哇胚胎中检测内源性Wnt通路的激活，并将在整个动物和组织重组中阻断非规范的Wnt信号，以询问Wnt信号在外胚层和/或中胚层是否对于造血是必要的。(2)确定TRIL是否是造血所必需的，以及是否是激活非规范的Wnt信号。我们将分析TRIL表达被下调的非洲爪哇胚胎中的血液形成，并询问非规范Wnt信号的激活是否足以挽救这些胚胎的血液。我们将比较在TRIL表达受到干扰的胚胎中内源性非规范Wnt信号的激活水平。(3)确定TRIL激活非典型Wnt信号的分子机制。我们将在Xenopus中使用功能增益法和功能损失法来测试TRIL功能是否需要上游、下游或与非规范Wnt通路的已知组件并行。此外，我们还将研究TRIL在非洲爪哇胚胎中相对于其他Wnt途径成分的亚细胞定位，并对TRIL进行体内结构和功能分析。最后，我们将在Wnt信号转导的背景下识别与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. )