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.
The proposed research is relevant to public health because it will generate novel vertebrate developmental models to identify how human mutations in MYH9 cause MYH9-related diseases. These discoveries will ultimately help decrease the burden of disability caused by these diseases. Thus, the proposed research is particularly relevant to the NICHD mission to advance fundamental and clinical knowledge of complex biological processes and developmental problems.