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

点击翻译按钮获取中文摘要

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.