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信号通路的错误调节引起的。在早期脊椎动物胚胎中，体轴的发育非常需要激活胚胎一侧的Wnt通路。在爪蟾这种主要的轴形成模式生物中，这种不对称信号是通过储存在卵中的母源分子的不同定位来实现的。这些蛋白和mrna在受精后通过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、脊索和背侧肌肉组织。Wnt在轴形成过程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，包括本提案的初步研究，都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。本提案的初步研究已经确定了新的定位mrna，使用微阵列分析皮质结合rna。两个候选基因，trim36和exdl2，被发现局限于种质，植物皮质的一个亚区，在轴的形成中起作用。这些基因分别编码一个三方基序蛋白和一个新的含有外切酶结构域的蛋白。本提案中的研究将通过反义介导的母体储存耗尽和功能获得测定来确定这些基因在轴形成中的功能机制。此外，这些拟议的研究将通过更有针对性的微阵列筛选确定额外的轴调节因子。具体目标是1)通过确定trim36与Wnt通路相互作用的程度来表征trim36的作用，2)确定trim36在轴形成中的生化功能，3)进一步表征exdl2在背侧规范中的作用，4)鉴定和表征其他胚质定位基因作为调节轴形成的候选基因。这些拟议研究的结果与公共卫生相关，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共卫生，这些信号通路在人类发育中是保守的。对这些机制的新见解将是改善人类出生缺陷和遗传疾病的诊断、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.