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

描述(申请人提供)：建议研究项目的目标是了解在哺乳动物发育过程中控制形态发生的发育和分子机制。我们的研究集中在会聚延伸的研究上，这是一种调节组织伸长的过程，涉及高度协调的细胞重排/运动。许多破坏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。对两栖和硬骨脊椎动物的研究表明，非典范的Wnt信号调节汇聚延伸。然而，在小鼠身上的研究表明，在哺乳动物中，收敛延伸可能受到其他分子机制的调节。一项基于表型的正向遗传筛查发现了Chato，这是一种小鼠突变，导致胚胎死亡和收敛的延伸表型。我们对Chato突变体的分析证实，Chato是细胞正确重排所必需的，从而导致最终的内胚层收敛延伸。Chato编码一种新的Kruppel-Associated-Box(KRAB)锌指蛋白ZFP568。对Chato胚胎的分析表明，Chato不执行非规范的Wnt信号，而是通过一个独立的分子机制控制小鼠的伸长。这项提议的目标之一是确定Chato用来控制收敛延伸的分子效应器和信号通路。我们还有兴趣了解Chato利用的发育机制：除了收敛延伸缺陷外，Chato突变体还在胚外组织的形态发生中显示畸形，这可能表明这些支持组织在促进融合延伸方面发挥了作用。我们将进行实验来验证这一假设。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镶嵌胚胎的表型来确定胚外组织是否影响胚胎的收敛延伸。对Chatwo突变体的研究提供了遗传学证据，支持TRIM28作为ZFP568的转录共抑制因子的作用。在特定目标2中，我们确定了影响ZFP568-TRIM28复合体形成的因素，以及介导该复合体专一性控制收敛延伸的机制。最后，在具体目标3中，我们试图识别ZFP 568和ZFP 568-TRIM28复合体的直接和间接靶点，并确定最终控制收敛延伸的ZFP 568效应器。总体而言，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，并为KRAB锌指蛋白利用的分子机制提供见解。 与公共卫生相关：该项目试图了解控制哺乳动物会聚延伸的基因和发育机制是什么，这是一种涉及协调细胞重排的形态发生过程。许多破坏性的先天性畸形是由于细胞运动的异常协调造成的。因此，我们的研究结果将对人类出生缺陷的预防和治疗具有重要意义。

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.