Localized mRNAs in vertebrate axis formation

描述（由申请人提供）：人类出生缺陷和癌症是由Wnt信号通路的失调引起的。在早期脊椎动物胚胎中，体轴的发育至关重要，需要激活胚胎一侧的Wnt途径。在蛙爪蟾，轴形成的主要模式生物，这种不对称的信号是通过差异定位的母源性分子储存在鸡蛋。这些蛋白质和mRNA通过受精后卵皮质的旋转运动向未来的背侧重新分布，导致Wnt信号的激活。干扰这一过程会导致胚胎缺乏初级胚层的背侧组织，包括中枢神经系统、脊索和背侧肌肉系统。Wnt在轴形成中激活的确切机制尚不清楚，但来自母体功能丧失研究的数据，包括该提议的初步研究，涉及植物性局部因素。本研究的长期目标是了解这些定位的母体基因产物在胚胎轴形成中的作用。这个建议的初步研究已经确定了新的本地化的mRNA，使用微阵列分析皮质结合的RNA。两个候选基因，trim36和exdl2，这是限制种质，一个亚区的植物皮层，被发现有作用的轴的形成。这些基因分别编码一种三分基序蛋白和一种新的含核酸外切酶结构域的蛋白。在这项建议中的研究将确定这些基因在轴形成中的功能的机制，通过反义介导的耗尽其母体存储和获得的功能测定。此外，这些拟议的研究将通过更有针对性的微阵列筛选来确定其他轴调节器。具体目的是1）通过确定trim 36与Wnt途径相互作用的程度来表征trim 36的作用，2）鉴定trim 36与其在轴形成中的作用相关的生物化学功能，3）进一步表征exdl 2在背侧特化中的作用，以及4）鉴定和表征作为调节轴形成的候选物的另外的胚质定位基因。这些拟议研究的结果与公众健康有关，并将通过加强对细胞和组织分化所涉及的基本信号通路的理解而有益于公众健康，这些信号通路在人类发育中是保守的。对这些机制的新认识将是改善人类出生缺陷和遗传疾病诊断、预防和治疗的重要一步。 公共卫生相关性：这些拟议研究的结果与公众健康相关，并将通过增强对细胞和组织分化中涉及的基本信号通路的理解而受益，这些信号通路在人类发育中是保守的。对这些机制的新认识将是改善人类出生缺陷和遗传疾病诊断、预防和治疗的重要一步。

DESCRIPTION (provided by applicant): Human birth defects and cancers are caused by misregulation of the Wnt signaling pathway. In early vertebrate embryos, development of the body axis critically requires the activation of the Wnt pathway on one side of the embryo. In the frog Xenopus, a predominant model organism for axis formation, this asymmetric signaling is achieved by the differential localization of maternally derived molecules stored in the egg. These proteins and mRNAs are redistributed toward the future dorsal side by rotational movements of the egg cortex following fertilization, resulting in the activation of Wnt signaling. Interference with this process results in embryos lacking the dorsal tissues of the primary germ layers, including the CNS, notochord and dorsal musculature. The exact mechanism of Wnt activation in axis formation is unclear, but data from maternal loss-of-function studies, including Preliminary Studies for this proposal, have implicated vegetally localized factors. The long-term goal of this research is to understand the role of these localized maternal gene products in embryonic axis formation. Preliminary Studies for this proposal have identified novel localized mRNAs, using microarray analysis of cortex-bound RNAs. Two candidate genes, trim36 and exdl2, which were restricted to the germ plasm, a subregion of the vegetal cortex, were found to have roles in axis formation. These genes encode a Tripartite Motif protein and a novel exonuclease domain-containing protein, respectively. The studies in this proposal will determine the mechanisms underlying the function of these genes in axis formation, via antisense-mediated depletion of their maternal stores and gain-of-function assays. Furthermore, these proposed studies will identify additional axis regulators through a more targeted microarray screen. The specific aims are 1) to characterize the role of trim36 by determining the extent of its interaction with the Wnt pathway, 2) to identify biochemical functions of trim36 associated with its role in axis formation, 3) to further characterize the role of exdl2 in dorsal specification and, 4) to identify and characterize additional germ plasm-localized genes as candidates for regulating axis formation. The findings of these proposed studies are relevant to, and will benefit public health by enhancing the understanding of basic signaling pathways involved in cell and tissue differentiation, which are conserved in human development. Novel insights would be gained into these mechanisms will be an essential step toward improving the diagnosis, prevention and treatment of human birth defects and genetic diseases. Public Health Relevance: The findings of these proposed studies are relevant to, and will benefit public health by enhancing the understanding of basic signaling pathways involved in cell and tissue differentiation, which are con- served in human development. Novel insights would be gained into these mechanisms will be an essential step toward improving the diagnosis, prevention and treatment of human birth defects and genetic diseases.