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

这项建议旨在开发脊椎动物模型来阐明已知的分子机制 Myh9基因突变会扰乱发育。有五种疾病是由Myh9基因突变引起的，包括 被归类为Myh9相关疾病的临床综合征障碍：梅-赫格林异常，塞巴斯蒂安， Fetchner和Epstein综合征；以及非综合征性耳聋DFNA17。这些疾病有共同之处 Myh9基因的突变，以包括血小板异常在内的一系列症状为特征， 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)，在细胞分裂、细胞迁移和细胞形状变化中起重要作用。然而， 对于在人类群体中发现的Myh9突变如何影响人类健康，人们的理解存在严重差距 Myh9相关疾病的病原学。我们建议生成斑马鱼(Danio Rerio)的大多数模型 常见Myh9突变用于检查Myh9相关器官的发育 疾病，尤指耳朵、眼睛和肾脏。斑马鱼模型将比目前的模型提供明显的优势 以单细胞分辨率获取和检查早期器官发育能力的哺乳动物模型 使用实时成像。斑马鱼胚胎是光学透明的，发育迅速，基因组编辑 斑马鱼的技术已经很成熟了。我们的长期研究目标是确定分子 Myh9基因突变如何导致Myh9相关疾病的机制。作为迈向我们的 长期研究目标，这项R03提案的总体目标是建立斑马鱼模型和 转基因株系，研究导致Myh9相关疾病的机制。我们的中心假设是 可以产生保守的Myh9突变的斑马鱼模型和NMIIA标记的转基因系，并将 使我们能够阐明导致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来实现假设。目标1是用来产生Myh9突变斑马鱼品系 与Myh9相关的最常见的人类突变相对应的靶向和特定突变 疾病。目标2是建立内源性标记NMIIA蛋白的转基因斑马鱼品系以进行检测 NMIIA在开发过程中的本地化和动力学。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。建议的工作具有创新性， 意义重大，因为这些研究将建立研究所需的发育脊椎动物模型 确定高度保守的Myh9基因突变的作用并确定分子 这些突变如何导致Myh9相关疾病的机制。这项提议的结果是 预计将产生重要的积极影响，因为它们将提供必要的工具来定义 Myh9基因突变会导致发育缺陷。

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.