THE ROLE OF TGF-? MODULATORS IN VERTEBRAL DEVELOPMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 脊椎畸形与大量令人痛苦的人类出生缺陷有关。阐明脊椎正常形成的发育和遗传机制，无疑将对人类健康产生重大影响。椎体系统的形成是一个复杂的过程，涉及神经管、脊索、体节的形态发生以及这些组织之间的细胞信号事件。特别是，体节的形成和分化在脊椎发育和脊椎疾病中起着关键作用。每个体节都要建立一个前后(A-P)极，发育过程中这种A-P极的破坏往往会导致椎体和肋骨之间的异常融合，从而导致严重的脊柱和脊髓疾病。脊椎动物尾部的发育为研究体节的形成和分化提供了一个很好的模型系统，因为它的解剖结构与躯干椎体系统相比简单。椎体之间的异常融合通常会导致尾巴弯曲，尾巴扭曲，这是很容易看到的。体节的A-P分化可以通过一系列成熟的分子标记来检测。在本研究中，我们将通过研究两个Smads相互作用因子Zfhx1a和TGIF的功能来研究转化生长因子-b信号通路在体细胞发生中的调节作用。Zfhx1a又称Def1，编码一个锌指蛋白，含有多个DNA结合域和蛋白质结合域，包括同源结构域和Smad结合域。体外研究表明，Zfhx蛋白家族成员可以与Smads蛋白结合，募集转录激活因子p300或抑制因子CtBP。根据招募的辅因子、激活物或抑制物的类型，Zfhx蛋白可以上调或下调转化生长因子-b的活性。生化研究表明，TGIF通过与Smad2结合而拮抗转化生长因子-b信号转导。我们最近对Zfhx1a和TGIF突变小鼠的研究发现，Zfhx1a或TGIF功能的丧失会导致严重的尾部扭曲表型，这有力地表明这些基因在体节的A-P分化中起着重要作用。因此，我们建议研究Zfhx1a和TGIF在小鼠体格发生和脊椎发育过程中的功能需求。此外，我们还将利用斑马鱼系统在体内确定Zfhx1a和TGIF介导的转化生长因子-b抑制的特异性，即脊椎动物体细胞发生中哪些转化生长因子-b成员(S)的活性受到Zfhx1a和TGIF的调节。 拟议的研究结果将具有重要意义，因为它涉及到脊椎动物身体分割这一基本生物学过程中的一个重要途径--转化生长因子-b信号的调节。此外，该项目具有几个优势，适合Cobre应用的目的：1)它是一个适合调查者进入出生缺陷研究的新领域的项目，脊椎疾病；2)额外的模型系统，斑马鱼，将使调查者进行更多的机械性研究，因为斑马鱼更容易获得胚胎和遗传手稿；3)由于斑马鱼系统是发育生物学中流行和强大的模型系统，在实验室拥有斑马鱼模型系统将在很大程度上提高研究实力。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Vertebral malformation associates with a large number of distressful human birth defects. Elucidation of the developmental and genetic mechanisms underlying normal vertebral formation will undoubtedly have great impacts on human health. The formation of vertebral system is a complex process involving the morphogenesis of neural tube, notochord, somites and cell  cell signaling events among these tissues. In particular, the formation and differentiation of somites play a critical role in vertebral development and vertebral disorders. Each somite has to establish an anterior-posterior (A-P) polarity and disruption of this A-P polarity during development often leads to the abnormal fusions between vertebrae bodies and ribs resulting in serious vertebral and spinal cord disorders. The development of vertebrate tail provides an excellent model system to study somite formation and differentiation due to its simple anatomic structure compared with the trunk vertebral system. The abnormal fusion between vertebrae bodies usually cause tail bending, kinky tail, that can be easily visualized. The A-P differentiation of somites can be examined by a set of well established molecular markers. In this study, we will focus on the regulation of TGF-b signaling pathway in somitogenesis by exploring the functions of two Smads interacting factors: Zfhx1a and TGIF. Zfhx1a, also called dEF1, encodes a zinc finger protein that contains multiple DNA and protein binding domains including homeodomain and Smad binding domain. In vitro studies showed that members of Zfhx protein family can bind to Smads proteins and recruit transcription activator p300 or repressor CtBP. Depending on the types of recruited co-factors, activators or repressors, Zfhx proteins can either up- or down- regulate TGF-b activity. Biochemical studies have revealed that TGIF antagonizes TGF-b signaling by binding to Smad2. Our recent studies with Zfhx1a and TGIF mutant mice uncovered that loss of Zfhx1a or TGIF function leads to a severe kinky tail phenotype, strongly indicating that these genes play important roles the A-P differentiation of somites. We therefore propose here to examine the functional requirements of Zfhx1a and TGIF during mouse somitogenesis and vertebral development. In addition, we will employ Zebrafish system to determine the specificity of Zfhx1a and TGIF mediated TGF-b suppression in vivo, namely which TGF-b member(s) activity is regulated by Zfhx1a and TGIF in vertebrate somitogenesis. Results from the proposed research will be significant as it address the regulation of an important pathway, TGF-b signaling, in a fundamental biological process, vertebrate body segmentation. In addition, the project features several advantages that fit the purpose of this COBRE application suitably: 1) It is a suitable project for the investigator to enter a new area of birth defect research, vertebral disorders; 2) An additional model system, Zebrafish, will allow the investigator to conduct more mechanistic studies as Zebrafish is more accessible to embryonic and genetic manuscript; 3) Since Zebrafish system is a popular and powerful model system in developmental biology, having a zebrafish model system in the laboratory will improve the research strength to a great extend.