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

Fat3 和 Atropin-2 在斑马鱼颌关节发育中的作用

• 批准号: 8466307
• 负责人: Pierre Le Pabic
• 金额: $ 5.65万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8466307
• 关键词: Adult; Affect; Animals; Award; Binding; Biology; Bone Morphogenetic Proteins; Cadherins; Cartilage; Cell Communication; Cell Polarity; Cell membrane; Cells; Chondrocytes; Cleft Palate; Congenital Abnormality; Defect; Development; Developmental Biology; Diagnosis; Dominant-Negative Mutation; Drosophila genus; Elements; Embryo; Environment; Event; Failure; Fatty acid glycerol esters; Fluorescence; Funding; Head; Heat-Shock Response; Human; Image; Institution; Jaw; Joints; Laboratories; Learning; Life; Mandible; Methods; Micrognathism; Microscope; Modeling; Molecular; Morphogenesis; Neural Crest; Neural Crest Cell; Orthologous Gene; Pathway interactions; Pattern; Physical condensation; Play; Population; Principal Investigator; Reading; Relative (related person); Resources; Role; Shapes; Signal Transduction; Skeletal Development; Skeleton; Specialist; Staging; Supervision; Surface; System; Techniques; Temporomandibular Joint; Testing; Transgenic Organisms; United States National Institutes of Health; Zebrafish; base; bone imaging; career; cartilage cell; craniofacial; cranium; design; fly; gene repression; loss of function; loss of function mutation; mutant; novel; programs; receptor; skeletal

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.

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成人骨骼的大部分建立了蓝图，并且许多相同的发育机制在成人中持续存在。因此，骨骨骼的形态缺陷通常是由软骨骨骼的发育缺陷引起的。因此，骨骼生物学中的一个基本问题是初始软骨元件如何获得其适当的形状，大小和关节。大多数脊椎动物的头部骨骼来源于胚胎中迁移的神经嵴（NC）细胞。很少有人知道的机制，NC细胞间的通讯，必要的组装骨骼元素的适当大小和形状。我们发现了一个新的要求，非典型钙粘蛋白脂肪3在下颌关节形成;斑马鱼脂肪3的损失导致关节融合。在果蝇中，脂肪在细胞间通讯中起着至关重要的作用，作为使细胞群体表现为连贯单位的两个系统的一部分：平面细胞极性（PCP）途径和Hippo途径。脂肪控制的PCP途径的下游因子是转录辅阻遏物Atrophin（Atr）。脂肪和Atr相互结合，并在基因上相互作用，以控制苍蝇体内的五氯苯酚。有趣的是，斑马鱼直系同源基因atr 2a中的功能缺失突变会导致与Fat 3变形子相似的颅面缺陷，包括融合的下颌关节。此外，atr 2a突变体中Fat 3的缺失显著降低了整个咽部骨骼的大小，这表明Fat 3和Atr 2a的相互作用在遗传上与它们的苍蝇亲戚相似。因此，Fat 3和Atr 2a可能是颅面和关节发育中新途径的一部分，这可能在一些人类骨骼出生缺陷中被破坏。我们假设Fat 3和Atr 2a控制关节形成的两个方面：1）在区间软骨细胞分化的转录抑制和2）区间和相对关节面的形态发生。在目标1中，我们将测试这两个要求是否可以区分。为此，我们将：1）使用转基因斑马鱼表征导致野生型胚胎中颌关节形成的形态发生事件，以及其在atr 2a突变体和Fat 3变形体中形成的失败，其中NC衍生的骨骼前体在活胚胎中发荧光，2）确定Atr 2a和/或Fat 3是否非细胞自主地调节关节处的细胞堆积，和3）确定Fat 3和Atr 2a是否物理相互作用。在目标2中，我们将定义Fat 3/Atr 2a和骨形态发生蛋白（BMP）信号传导之间的关系，这是众所周知的控制关节形成。我们将：1）使用bre：eGFP转基因系测试Fat 3和/或Atr 2a是否调节BMP信号传导，和2）使用热休克诱导型显性阴性BMPr 1a转基因系确定BMP是否图案化Fat 3下颌弓表达结构域。该奖项所采用的大多数技术将由候选人在其赞助商的监督下学习，并将为他在发育生物学领域担任首席研究员做好准备。