The Roles of Fat3 and Atrophin-2 in Jaw-Joint Development in Zebrafish

描述(申请人提供)：诊断和开发人类骨骼缺陷的治疗方法需要首先确定启动软骨凝结、成熟和重组的分子途径。这是因为这些软骨为成人骨骼的大部分建立了蓝图，而且许多相同的发育机制在成年人身上仍然存在。因此，骨性骨骼的形态缺陷通常是由于软骨骨骼的发育缺陷造成的。因此，骨骼生物学中的一个基本问题是初始软骨元素是如何获得其合适的形状、大小和关节的。大多数脊椎动物的头部骨骼来自胚胎中的迁移神经脊(NC)细胞。人们对NC细胞与细胞之间的通讯机制知之甚少，这是组装适当大小和形状的骨骼元素所必需的。我们发现了在颌骨关节形成中对非典型钙粘附素Fat3的新需求；斑马鱼Fat3的丢失会导致关节融合。在果蝇中，Fat作为两个系统的一部分，在细胞间的交流中起着至关重要的作用，这两个系统使细胞群体表现为一致的单位：平面细胞极性(PCP)途径和河马途径。脂肪控制的PCP途径的下游因子是转录共抑制因子阿托品(ATR)。脂肪和ATR相互结合，并在基因上相互作用，以控制果蝇的五氯苯酚。有趣的是，斑马鱼同源基因atr2a功能突变的丧失会导致类似于Fat3变体的头面部缺陷，包括融合的颌关节。此外，atr2a突变体中Fat3的缺失显著减少了整个咽部骨骼的大小，这表明Fat3和Atr2a在遗传上的相互作用类似于它们的苍蝇亲属。因此，Fat3和Atr2a可能是头面部和关节发育新途径的一部分，这一途径可能在某些人类骨骼出生缺陷中受到干扰。我们假设Fat3和Atr2a控制关节形成的两个方面：1)转录抑制关节间隙软骨细胞的分化；2)关节间隙和相对关节表面的形态发生。在目标1中，我们将测试这两个需求是否可区分。为了实现这一目标，我们将：1)表征导致野生型胚胎中颌关节形成的形态发生事件，以及在使用转基因斑马鱼的atr2a突变体和Fat3变异体中未能形成的形态发生事件；在转基因斑马鱼中，NC衍生的骨骼前体在活胚胎中发出荧光；2)确定Atr2a和/或Fat3是否在调节关节细胞堆积方面发挥非细胞自主作用；以及3)确定Fat3和Fat2a是否存在物理上的相互作用。在目标2中，我们将定义Fat3/Atr2a与骨形态发生蛋白(BMP)信号之间的关系，BMP信号是众所周知的控制关节形成的信号。我们将：1)使用BRE：EGFP转基因系检测Fat3和/或Atr2a是否调节BMP信号转导；2)使用热休克诱导的显性阴性BMPR1A转基因系，确定BMPs是否影响FAT3下颌弓的表达结构域。在该奖项下使用的大部分技术将由候选人在其赞助商的监督下学习，并将为他作为发育生物学领域的首席研究人员的职业生涯做好准备。 与公共卫生相关：头面部缺陷是最常见的出生缺陷之一，例如影响人类颌骨和头骨的缺陷。我们的项目旨在解开发育合适形状和大小的骨骼元素所需的细胞间通讯机制。从长远来看，我们的发现可能有助于开发更好的方法来诊断和治疗骨骼缺陷，如小颌骨畸形和腭裂。

DESCRIPTION (provided by applicant): Diagnosing and developing treatments for skeletal defects in humans requires first identifying the molecular pathways that initiate cartilage condensation, maturation and reorganization. This is because these cartilages establish a blueprint for much of the adult skeleton and many of the same developmental mechanisms persist in adults. Consequently, morphological defects of the bony skeleton often result from developmental defects of the cartilaginous skeleton. Thus, a fundamental question in skeletal biology is how initial cartilage elements acquire their appropriate shapes, sizes and articulations. Most of the vertebrate head skeleton derives from migratory neural crest (NC) cells in the embryo. Little is known about the mechanisms of NC cell-cell communication necessary to assemble skeletal elements of the appropriate size and shape. We found a novel requirement for the atypical cadherin Fat3 in jaw-joint formation; loss of zebrafish Fat3 results in joint fusion. In Drosophila, Fat plays crucial roles in cell-cell communication as part of two systems that make cell populations behave as coherent units: the planar-cell polarity (PCP) pathway and the Hippo pathway. A downstream factor of the Fat-controlled PCP pathway is the transcriptional co-repressor Atrophin (Atr). Fat and Atr bind one another and genetically interact to control PCP in flies. Interestingly, loss of function mutations in the zebrafish ortholog, atr2a, cause craniofacial defects similar to those of Fat3 morphants, including fused jaw-joints. Also depletion of Fat3 in atr2a mutants dramatically reduces the size of the entire pharyngeal skeleton, suggesting that Fat3 and Atr2a interact genetically similar to their fly relatives. Consequently, Fat3 and Atr2a may be part of a novel pathway in craniofacial and joint development, which may be disrupted in some human skeletal birth defects. We hypothesize that Fat3 and Atr2a control 2 aspects of joint formation: 1) transcriptional repression of chondrocyte differentiation at the interzone and 2) morphogenesis of the interzone and opposing articular surfaces. In Aim 1 we will test if these two requirements are distinguishable. To achieve this, we will: 1) characterize the morphogenetic events leading to jaw-joint formation in wild-type embryos, and its failure to form in atr2a mutants and Fat3 morphants using transgenic zebrafish in which NC-derived skeletal precursors fluoresce in the living embryo, 2) determine whether Atr2a and/or Fat3 act non-cell autonomously in regulating cell-stacking at the joint, and 3) determine if Fat3 and Atr2a interact physically. In Aim 2, we will define the relationship between Fat3/Atr2a and Bone Morphogenetic Protein (BMP) signaling, which is well-known to control joint formation. We will: 1) test if Fat3 and/or Atr2a regulate BMP signaling using a bre:eGFP transgenic line, and 2) determine if BMPs pattern the fat3 mandibular arch expression domain, using a heat-shock inducible dominant negative BMPr1a transgenic line. Most of the techniques employed under this award will be learned by the candidate under the supervision of his sponsor, and will prepare him for a career as a Principal Investigator in the field of developmental biology. PUBLIC HEALTH RELEVANCE: Craniofacial defects, such as those that affect the jaw and skull in humans, are among the most common birth defects. Our project is designed to unravel the mechanisms of cell-cell communication required for the development of skeletal elements of the right shape and size. In the long term, our findings may help in developing better methods for diagnosis and treatment of skeletal defects such as micrognathia and cleft palate.