A Biochemical, Proteomic and Functional Delineation of Dcbld1 and 2 Signaling during Zebrafish Neural Retina Development

尽管正常的视力对所有类型的动物都很重要，但眼睛中与大脑(视网膜)沟通的部分是如何发育的，人们只了解了一部分。目前的研究项目是在斑马鱼身上进行的，因为有有利的遗传和实验工具可用于研究早期眼睛发育。该研究小组之前进行的研究发现，有两种蛋白质需要正常运作才能使视网膜发育(称为DCBLD1和DCBLD2)；如果不制造这些蛋白质，视网膜就不能正常形成。用人的DCBLD2蛋白取代有缺陷的鱼DBCLD2蛋白，使鱼的视网膜能够正常发育，这表明DCBLD蛋白在所有有脊椎的动物中的功能相似。本项目将结合解剖学、生化、细胞生物学、功能遗传学和蛋白质组学技术研究DCBLD蛋白调控适当的视网膜形成的机制，并将提供对视网膜形成的更完整的理解。这项研究的更广泛影响包括对高中生、大学本科生、研究生和工作人员科学家进行许多现代实验方法的实验室培训。其中几名学生将专门从科学界代表性不足的人群中招募。这项研究和培训将由一个跨学科的团队在佛蒙特州(佛蒙特州大学和诺里奇大学)、马萨诸塞州(波士顿学院)和波多黎各(波多黎各大学-里约热内卢大学和大都会大学)进行。主要研究人员之前的工作确定，假定的神经元引导受体DCBLD1和2作为CRK适配器家族的支架，已知该家族在神经元发育过程中发挥重要作用。对转基因斑马鱼的组织学研究发现，Dcbld1和Dcbld2对于视网膜神经节细胞的正常发育和视网膜细胞类型的正确定位和/或分化都是必不可少的。人DCBLD2被证明可以挽救dcbld2表型。本研究旨在阐明Dcbld1和Dcbld2在斑马鱼眼睛发育中的作用：Aim1将利用免疫组织化学方法确定Dcbld1和Dcbld2的缺失如何影响细胞增殖和斑马鱼视网膜细胞类型的分化。Aim 2将使用功能发育方法来确定Crk/CrkL结合所需的DCBLD1和2个酪氨酸磷酸化位点是否对斑马鱼视网膜的发育至关重要。AIM 3将使用生化、蛋白质组学、细胞生物学和功能遗传学的方法来表征DCBLD蛋白、DCBLD结合伙伴的磷酸化以及它们在斑马鱼神经视网膜发育中所起的作用。

Even though proper vision is important to all types of animals, how the parts of the eyes that communicate with the brain (the retinas) develop is only partly understood. The present research project is conducted in zebrafish because of advantageous genetic and experimental tools available for studying early eye development. Previous studies carried out by this research team found two proteins that need to function properly for retinas to develop (called DCBLD1 and DCBLD2); if these proteins aren't made, retinas don't form properly. Replacing a defective fish DBCLD2 protein with a human DCBLD2 protein enables the fish retina to develop normally, suggesting that DCBLD proteins function in a similar manner in all animals with backbones. The present project will examine the mechanisms by which DCBLD proteins govern proper retina formation using a combination of anatomical, biochemical, cell biological, functional genetic and proteomic techniques, and will provide a more complete understanding of retina formation. Broader impacts of this research include laboratory training for high school students, university undergraduates, graduate students, and staff scientists in many modern experimental approaches. Several of the students will be specifically recruited from populations that are underrepresented in science. The research and training will be conducted by an inter-disciplinary team in Vermont (the University of Vermont and Norwich University), Massachusetts (Boston College) and Puerto Rico (the University of Puerto Rico-Rio Piedras and the Metropolitan University).Previous work by the principal investigators determined that the putative neuronal guidance receptors DCBLD1 and 2 act as scaffolds for the Crk family of adaptors, which are known to play important roles in the positioning of neurons during development. Histological studies in transgenic zebrafish identified Dcbld1 and 2 as essential for both proper retinal ganglion cell development and proper positioning and/or differentiation of retinal cell types. Human DCBLD2 was shown to rescue dcbld2 morphant phenotypes. The following research aims will be conducted to delineate the roles of Dcbld1 and 2 in zebrafish eye development: Aim1 will use immunohistochemistry to determine how loss of Dcbld1 and 2 affects cell proliferation and the differentiation of zebrafish retinal cell types. Aim 2 will use functional developmental approaches to determine if the DCBLD1 and 2 tyrosine phosphorylation sites required for Crk/CrkL binding are essential for the development of the zebrafish retina. Aim 3 will use biochemical, proteomic, cell biological and functional genetic approaches to characterize the phosphorylation of DCBLD proteins, DCBLD binding partners and the roles these play in zebrafish neural retina development.