Developmental & Molecular Mechanisms Employed by Chato, KRAB Zinc Finger Protein

描述（由申请人提供）：拟议研究项目的目标是了解哺乳动物发育过程中控制形态发生的发育和分子机制。我们的研究重点是研究趋同延伸，这是一个介导组织延伸的过程，涉及细胞高度协调的重排/运动。许多毁灭性的先天性畸形是由于细胞运动的异常协调造成的，但这些疾病的遗传基础在大多数情况下是未知的，这阻碍了它们的早期诊断和预防。对两栖动物和硬骨鱼脊椎动物的研究表明，非规范Wnt信号调节趋同扩展。然而，对小鼠的研究表明，在哺乳动物中，趋同延伸可能受到其他分子机制的调控。基于表型的正向遗传筛选鉴定了chato，一种引起胚胎致死和趋同扩展表型的小鼠突变。我们对chato突变体的分析证实，chato是导致最终内胚层收敛延伸的细胞的适当重排所必需的。chato编码ZFP568，一种新的Kruppel-Associated-Box （KRAB）锌指蛋白。对chato胚胎的分析表明，chato不执行非规范Wnt信号，而是通过独立的分子机制控制小鼠伸长。本研究的目标之一是确定chato控制收敛扩展的分子效应物和信号通路。我们也对chato利用的发育机制感兴趣：除了它们的趋同延伸缺陷外，chato突变体在胚胎外组织的形态发生中也表现出畸形，这可能表明这些支持组织在促进趋同延伸中起作用。我们将进行实验来验证这个假设。我们将我们的研究计划分为三个具体目标：在具体目标1中，我们将通过分析四倍体嵌合体和遗传马赛克胚胎的表型来确定胚胎外组织是否影响胚胎的趋同延伸。对chatwo突变体的研究提供了支持TRIM28作为ZFP568转录共抑制因子作用的遗传证据。在Specific Aim 2中，我们确定了影响ZFP568-TRIM28复合物形成的因素，以及介导该复合物特异性控制收敛扩展的机制。最后，在Specific Aim 3中，我们试图确定ZFP568和ZFP568- trim28复合物的直接和间接靶点，并确定控制收敛扩展的最终ZFP568效应物。总的来说，我们的实验结果将增加我们对控制哺乳动物形态发生的分子的理解，并提供有关KRAB锌指蛋白利用的分子机制的见解。

DESCRIPTION (provided by applicant): The goal of the proposed research project is to understand the developmental and molecular mechanisms that control morphogenesis during mammalian development. Our research is focused on the study of convergent extension, a process that mediates elongation of tissues and involves highly coordinated rearrangements/movements of cells. Numerous devastating congenital malformations result from aberrant coordination of cell movements, but the genetic basis of these diseases is in most cases unknown, which impedes their early diagnosis and prevention. Studies in amphibian and teleost vertebrates have revealed that non-canonical Wnt signaling regulates convergent extension. However, studies in mouse have indicated that convergent extension might be regulated by additional molecular mechanisms in mammals. A phenotype-based forward genetic screen identified chato, a mouse mutation causing embryonic lethality and convergent extension phenotypes. Our analysis of chato mutants confirmed that chato is required for the proper rearrangement of cells that lead to definitive endoderm convergent extension. chato encodes ZFP568, a novel Kruppel-Associated-Box (KRAB) Zinc finger protein. Analysis of chato embryos suggests that chato does not implement non-canonical Wnt signaling, but rather controls mouse elongation through an independent molecular mechanism. One of the goals of this proposal is to identify the molecular effectors and signaling pathways employed by chato to control convergent extension. We are also interested in understanding the developmental mechanisms utilized by chato: In addition to their convergent extension defects, chato mutants also display malformations in morphogenesis of extraembryonic tissues, which might indicate a role for these supporting tissues in promoting convergent extension. We will perform experiments to test this hypothesis. We have divided our research plan into three specific aims: In Specific Aim 1, we will determine whether extraembryonic tissues influence convergent extension of the embryo by analyzing the phenotype of tetraploid chimeras and genetic mosaic embryos. The study of chatwo mutants has provided genetic evidence supporting a role for TRIM28 as a transcriptional co-repressor of ZFP568. In Specific Aim 2, we determine the factors that influence formation of ZFP568-TRIM28 complexes and the mechanisms that mediate the specificity of this complex to the control of convergent extension. Finally, in Specific Aim 3, we seek to identify direct and indirect targets of ZFP568 and ZFP568-TRIM28 complexes and determine the ultimate ZFP568 effectors that control convergent extension. Overall, results from our experiments will increase our understanding of the molecules that control mammalian morphogenesis and provide insights about the molecular mechanisms utilized by KRAB Zinc finger proteins. PUBLIC HEALTH RELEVANCE: This project seeks to understand what are the genes and developmental mechanisms that control mammalian convergent extension, a morphogenetic process that involves coordinated cell rearrangements. Numerous devastating congenital malformations result from aberrant coordination of cell movements. Therefore, our results will be important for the prevention and treatment of human birth defects.