Developmental models to determine the molecular mechanisms that cause MYH9-related diseases

该建议旨在开发脊椎动物模型，以阐明已知的 MYH9的突变破坏了发育。有五种疾病是由MYH9突变引起的，包括 分类为MYH9相关疾病的临床综合征疾病：May-Hegglin异常，塞巴斯蒂安， Fetchner和Epstein综合征;和非综合征性耳聋DFNA 17。这些疾病有共同的 MYH9基因突变，并以包括血小板异常在内的许多症状为特征， 肾炎、视力缺陷和听力损失。MYH9编码高度保守的非肌肉肌球蛋白IIA 蛋白质（NMIIA），其在细胞分裂、细胞迁移和细胞形状改变中具有重要作用。然而，在这方面， 在理解人类中发现的MYH9突变如何有助于 MYH9相关疾病的病因。我们建议生成斑马鱼（Danio rerio）模型的最 常见的MYH9突变，以检查MYH9相关的受影响器官的发育 疾病，特别是耳朵，眼睛和肾脏。斑马鱼模型将提供明显的优势， 哺乳动物模型在单细胞分辨率下访问和检查早期器官发育的能力 使用实时成像。斑马鱼胚胎是光学透明的，具有快速发育和基因组编辑 在斑马鱼中的技术已经很成熟。我们的长期研究目标是确定 MYH9基因突变如何导致MYH9相关疾病的机制。作为我们迈向 长期研究目标，本R03提案的总体目标是建立斑马鱼模型， 转基因株系，以研究导致MYH9相关疾病的机制。我们的核心假设是， 可以产生保守MYH9突变的斑马鱼模型和匪IIA标记的转基因系， 让我们能够阐明导致MYH9相关疾病的分子机制。我们将测试我们的中央 通过追求以下具体目标的假设。目的1是产生myh9突变体斑马鱼系， 靶向和特异性突变，对应于MYH9相关的最常见的人类突变， 疾病目的二是建立内源性标记NMIIA蛋白的转基因斑马鱼品系， NMIIA在开发过程中的本地化和动态。我们将通过培育斑马鱼来实现这些目标 使用CRISPR/Cas9基因组编辑的MYH9突变模型。这项工作具有创新性， 重要的是，这些研究将建立研究所需的发育脊椎动物模型， 并确定高度保守的MYH9基因突变的作用， 这些突变如何导致MYH9相关疾病的机制。该提案的结果是 预计将产生重要的积极影响，因为它们将提供必要的工具， MYH9的突变导致发育缺陷。

This proposal aims to develop vertebrate models to elucidate molecular mechanisms by which known mutations in MYH9 disrupt development. There are five diseases that result from mutations in MYH9, including clinical syndromic disorders that are classified as MYH9-related diseases: May-Hegglin anomaly, Sebastian, Fetchner, and Epstein syndrome; and non-syndromic deafness DFNA17. These diseases share common mutations in the MYH9 gene and are characterized by a number of symptoms including platelet abnormalities, nephritis, visual defects, and hearing loss. MYH9 encodes for the highly conserved non-muscle myosin IIA protein (NMIIA), which has essential roles in cell division, cell migration, and cell shape changes. However, there is a critical gap in the understanding of how MYH9 mutations found in the human population contribute to the etiology of MYH9-related diseases. We propose to generate zebrafish (Danio rerio) models of the most common MYH9 mutations to examine the development of the organs that are affected in MYH9-related disease, particularly the ear, eye, and kidney. Zebrafish models will provide distinct advantages over current mammalian models in the ability to access and examine early organ development at single-cell resolution using live imaging. Zebrafish embryos are optically transparent, have rapid development, and genome editing techniques in zebrafish are well established. Our long-term research goal is to determine the molecular mechanisms for how mutations in the MYH9 gene lead to MYH9-related diseases. As the first step towards our long-term research goal, the overall objective of this R03 proposal is to establish zebrafish models and transgenic lines to study the mechanisms that cause MYH9-related diseases. Our central hypothesis is that zebrafish models of conserved MYH9 mutations and NMIIA-labeled transgenic lines can be generated and will allow us to elucidate the molecular mechanisms that cause MYH9-related diseases. We will test our central hypothesis by pursuing the following specific aims. Aim 1 is to generate myh9 mutant zebrafish lines with targeted and specific mutations that correspond to the most common human mutations in MYH9-related diseases. Aim 2 is to create transgenic zebrafish lines with endogenously labelled NMIIA protein to examine NMIIA localization and dynamics during development. We will accomplish these aims by generating zebrafish models of MYH9 mutations using CRISPR/Cas9 genome editing. The proposed work is innovative and significant because these studies will establish developmental vertebrate models that are needed to investigate and identify the role for mutations in the highly conserved MYH9 gene and determine the molecular mechanisms for how these mutations cause MYH9-related diseases. The outcomes from this proposal are expected to have an important positive impact because they will provide the tools necessary to define how mutations in MYH9 lead to developmental defects.