Genetic and chemical screening in zebrafish to study tendon development

描述（由申请人提供）：肌腱将力量从肌肉传递到骨骼，使其能够运动，而韧带将骨头连接到骨头，提供稳定。运动或年龄相关的肌腱或韧带损伤非常常见，尽管损伤可以缓慢修复，但新组织缺乏原始的生物力学完整性。尽管许多研究都集中在出生后肌腱和韧带组织的结构、组成和组织上，但对它们在胚胎发生期间的发育知之甚少。本项目的目标是发现胚胎中肌腱和韧带形成和分化的调控途径。为了做到这一点，我们将使用斑马鱼，一个强大的遗传脊椎动物模型系统。对于我提案的目标1，我将对斑马鱼鳍和颅骨区域的肌腱进行组织学，超微结构和分子分析。我将开发转基因斑马鱼的实时成像分析，以观察肌腱在真实发育时间内的形成和形态发生，这一技术在其他脊椎动物中是不可行的。在Aim 2中，我将进行正向遗传和高通量化学筛选，以发现和表征调节肌腱发育的新途径。这些研究将直接解决肌腱发育生物学领域的主要问题，并可能最终有助于开发肌腱或韧带损伤的新疗法。由于我的研究生和博士后研究经历，我是开展这个项目的理想人选。在攻读博士学位期间，我在Leonard Zon博士的斑马鱼造血实验室工作，在那里我学习了遗传和化学筛选、定位克隆、转基因、morpholinos和许多其他斑马鱼研究技术。在克里夫·塔宾博士的实验室做博士后期间，我最初研究肢体模式，以深入了解肢体组织最初是如何排列的，最近更关注哺乳动物肌腱随后是如何在胚胎肢体中形成和组织的。哈佛医学院遗传学系实验室丰富的研究环境、设备和设施，以及我的导师Tabin博士和顾问Zon博士、Andrew Lassar博士和Caroline Shamu博士的支持，将使我能够完成本提案指导阶段的目标。此外，该奖项将开发工具和培养在学术教师职位上取得成功所必需的技能。最终，这个项目不仅可以让我从导师那里获得独立的研究计划，而且可以为我作为肌腱生物学领域的独立研究者创造一个独特的利基。

DESCRIPTION (provided by applicant): Tendons transmit force from the muscles to the bone, enabling movement, while ligaments connect bone to bone, providing stabilization. Sports or age-related injuries to tendons or ligaments are extremely common, and although the injury can be slowly repaired, the new tissue lacks the original biomechanical integrity. Although many studies have focused on the structure, composition, and organization of tendon and ligament tissues after birth, very little is known about their development during embryogenesis. The goal of this project is to discover the pathways regulating tendon and ligament formation and differentiation in the embryo. To accomplish this, we will use the zebrafish, a powerful genetic vertebrate model system. For Aim 1 of my proposal, I will perform a histological, ultrastructural, and molecular analysis of zebrafish tendons in the fin and cranial regions. I will develop live imaging assays with transgenic zebrafish to visualize tendon formation and morphogenesis over real developmental time, a technique that is unfeasible in other vertebrates. For Aim 2, I will perform forward genetic and high-throughput chemical screens to discover and characterize new pathways regulating tendon development. These studies will directly address the major questions in the field of tendon developmental biology, and may ultimately, aid in the development of new therapies for tendon or ligament injury. Because of my graduate and post-doctoral research experiences, I am in an ideal position to carry out this project. For my Ph.D., I worked in Dr. Leonard Zon's laboratory on hematopoiesis in the zebrafish where I learned genetic and chemical screening, positional cloning, transgenics, morpholinos, and many other zebrafish research techniques. During my postdoc in Dr. Cliff Tabin's lab, I initially studied limb patterning to gain insight into how limb tissues are first arranged, and have more recently focused on how mammalian tendons subsequently form and organize in the embryonic limb. The rich research environment, equipment and facilities available in the Genetics Department at Harvard Medical School lab along with support from my mentor, Dr. Tabin, and advisors, Dr. Zon, Dr. Andrew Lassar, and Dr. Caroline Shamu will allow me to complete my goals for the mentored phase of this proposal. Furthermore, this award will develop the tools and cultivate the skills necessary for success in an academic faculty position. Ultimately, this project will not only allow me to pursue an independent research plan from that of my mentor, but it also will create a unique niche for me as an independent investigator in the field of tendon biology.