Molecular Mechanisms Underlying Xenopus Somitogenesis

描述(由申请人提供)：体细胞发生包括将近轴中胚层分割和分化为成人的骨骼肌、中轴骨骼和真皮。虽然许多参与调节体节形成周期性的基因已经被表征，但这一过程背后的细胞行为仍然知之甚少。这项建议的目的是确定脊椎动物非洲爪哇体节形态发生的分子机制。我们的中心假设是分泌的细胞因子基质衍生因子-1(SDF-1)通过激活特定的肌动蛋白调节分子RhoA、rac1或CDc42来调节非洲爪哇体节的形态发生。对这一假说的具体支持是基于最近对斑马鱼的研究，这些研究表明，与体节旋转相关的细胞行为(Hollway等人，2007年；Stellabte等人，2007年)与我们的实验室和其他实验室在非洲爪哇鱼中记录的类似(Afonine等人，2006年)。此外，分泌的细胞因子基质衍生因子-1(SDF-1)被证明是与斑马鱼体节旋转相关的细胞重排所必需的(Hollway等人，2007年)，这一点意义重大，因为它是第一个被证明调节体节旋转的信号分子。在非洲爪哇，SDF-1被证明在原肠形成期间中胚层迁移中发挥作用(Fukui等人，2006年)，并在整个早期胚胎发育的中轴组织中表达(Braun等人，2002年)。因此，SDF-1出现在合适的时间和地点，在体节的形态发生中发挥作用。 为了验证我们的中心假设，我们将通过使用吗啉来下调SDF-1的表达水平，并通过体内注射mRNA上调细胞中SDF-1及其受体CXCR4的蛋白水平来调节SDF-1信号(目标1)。先前的研究表明，SDF-1信号通过激活特定的Rho GTP酶来影响细胞行为的变化(Tan等人，2006年)。这些肌动蛋白调节分子已被证明在组织重塑中发挥重要作用，因此可能是体细胞发生过程中SDF-1信号通路的下游靶点。为了验证这一假说，我们将确定SDF-1信号是否在非洲爪哇体细胞发生过程中调节特定的Rho GTP酶的活性(目标2)。这项提案的另一个目标是引入新的分子方法，加上现有的成像技术专业知识，将为培训学生提供一个极好的环境，其中许多学生是代表不足的少数群体(目标3)。此外，我们还与三位资深科学家(Amacher博士、Symes博士和Weisblat博士)制定了导师计划，他们的研究专业知识补充了本提案中提出的目标，并将提高我们实验室的研究生产率。与公共卫生的相关性：这项拟议的研究预计将提供有关脊椎动物体节如何形成的关键信息。这些信息将有助于开发诊断或预防脊柱侧弯和其他先天性脊髓畸形等脊椎疾病的工具。

DESCRIPTION (provided by applicant): Somitogenesis consists of the segmentation and differentiation of the paraxial mesoderm into the skeletal muscle, axial skeleton and dermis of the adult. While many genes involved in regulating the periodic nature of somite formation have been characterized, the cell behaviors underlying this process remain poorly understood. The objective of this proposal is to determine the molecular mechanism underlying somite morphogenesis in the vertebrate Xenopus laevis. Our central hypothesis is that the secreted cytokine, stromal-derived factor-1 (SDF-1), regulates Xenopus somite morphogenesis by activating the specific actin regulatory molecules RhoA, Rac1, or Cdc42. Specific support for this hypothesis is based on recent studies in zebrafish that showed that the cell behaviors associated with somite rotation (Hollway et al., 2007; Stellabote et al., 2007) are similar to those documented by our lab and others in Xenopus (Afonin et al., 2006). Furthermore, the secreted cytokine, stromal-derived factor-1 (SDF-1), was shown to be required for the cell rearrangements associated with zebrafish somite rotation (Hollway et al., 2007), which is significant as it is the first signaling molecule shown to regulate somite rotation. In Xenopus, SDF-1 was shown to play a role in mesoderm migration during gastrulation (Fukui et al., 2006) and is expressed in axial tissues throughout early embryonic development (Braun et al., 2002). Thus, SDF-1 is present at the right time and place to play a role in somite morphogenesis. To test our central hypothesis, we will modulate SDF-1 signaling by using morpholinos to knockdown expression levels and in vivo mRNA injections to up-regulate protein levels of SDF-1 and its receptor, CXCR4, in the cell (aim 1). Previous studies have shown that SDF-1 signaling affects changes in cell behaviors by activating specific Rho GTPases (Tan et al., 2006). These actin regulatory molecules have been shown to play an important role in tissue remodeling and thus are likely downstream targets of the SDF-1 signaling pathway during somitogenesis. To test this hypothesis, we will determine whether SDF-1 signaling modulates the activity of specific Rho GTPases during Xenopus somitogenesis (aim 2). Another goal of this proposal is to introduce new molecular approaches that along with the existing expertise in imaging techniques will provide an excellent environment for training students, many of whom are underrepresented minorities (aim 3). Furthermore, we also developed a mentorship plan with three senior scientists (Drs. Amacher, Symes, and Weisblat) whose research expertise complements the objectives presented in this proposal and will increase the research productivity of our lab. Relevance to Public Health: The proposed research is expected to provide critical information on how vertebrate somites are formed. This information will help in developing tools for diagnosing or preventing vertebral disorders such as scoliosis and other congenital spinal cord deformities.