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

描述（由申请人提供）：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许多成人骨骼建立了蓝图，并且许多相同的发育机制在成人中仍然存在。因此，骨性骨骼的形态缺陷往往是由软骨骨骼的发育缺陷引起的。因此，骨骼生物学的一个基本问题是最初的软骨元素如何获得适当的形状、大小和关节。大多数脊椎动物的头部骨骼都是由胚胎时期的迁移神经嵴细胞形成的。对于NC细胞与细胞之间的通讯机制知之甚少，这种通讯是组装合适大小和形状的骨骼元素所必需的。我们发现在下颌关节形成过程中对非典型钙粘蛋白Fat3有新的需求；斑马鱼脂肪3缺失导致关节融合。在果蝇中，脂肪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Atrophin （Atr）。脂肪和Atr相互结合并在基因上相互作用以控制苍蝇体内的PCP。有趣的是，斑马鱼同源基因atr2a的功能突变缺失会导致与Fat3变体相似的颅面缺陷，包括融合的下颌关节。此外，atr2a突变体中Fat3的缺失显著降低了整个咽骨架的大小，这表明Fat3和atr2a在基因上的相互作用与它们的蝇类亲戚相似。因此，Fat3和Atr2a可能是颅面和关节发育新途径的一部分，该途径可能在一些人类骨骼出生缺陷中被破坏。我们假设Fat3和Atr2a控制关节形成的两个方面：1)区间软骨细胞分化的转录抑制和2)区间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)利用nc来源的骨骼前体在活胚胎中发出荧光的转基因斑马鱼，表征野生型胚胎中导致颌骨关节形成的形态发生事件，以及在atr2a突变体和Fat3突变体中无法形成颌骨关节；2)确定atr2a和/或Fat3是否在调节关节处的细胞堆叠中起非细胞自主作用；3)确定Fat3和atr2a是否在物理上相互作用。在Aim 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，2)使用热激诱导显性阴性BMPr1a转基因系确定BMP是否模式化Fat3下颌弓表达域。该奖项中使用的大多数技术将由候选人在其赞助人的监督下学习，并将为他作为发育生物学领域的首席研究员的职业生涯做好准备。

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.