Humans with point mutations in MYH9, the gene encoding nonmuscle myosin heavy chain (NMHC) IIA, develop a variety of syndromes including defects in their platelets (macrothrombocytopenia), kidneys (glomerulonephritis) and granulocytes (inclusion bodies). More than 40 different mutations in MYH9 have been reported to date, including both missense and nonsense mutations. The purpose of these studies is to gain insight into the pathological mechanism of the diseases caused by these mutations by creating mouse models for three of the mutations (R702C in the motor domain;D1424N and E1841K in the rod domain) and studying the resultant mouse phenotypes. Previous in vitro work has shown that the R702C mutation, which is in the motor domain of NMHC IIA compromises the MgATPase activity and is responsible for the movement velocity of the myosin;while mutations D1424N and E1841K in the rod domain may affect NMHC IIA filament formation. We have generated three mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with GFP was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5 while homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live cell imaging of megakaryocytes in the bone marrow show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most likely in humans. We also observed premature cataract formation, kidney abnormalities including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domain manifest similar hematological, eye and kidney phenotypes to humans with MYH9-related diseases. These mouse models will be useful in understanding the pathophysiology of human MYH9-related diseases and should also be useful in designing and developing therapies. In addition to using these mutant mice to study the relation between the nonmuscle myosin II-A mutation and disease, we plan to use various cells derived from these mice to study the effects of the mutation on basic properties of the cell. These include cytokinesis, cell-cell and cell matrix adhesion, cell polarity and cell migration. To gain clear insights into the distribution and function of different isoforms of nonmuscle myosin II (NMII) in normal mice, the enhanced GFP or mCherry sequence has been inserted in front of the start codon of the Myh9 gene in the first coding exon. We have obtained both heterozygous and homozygous GFP or mCherry tagged NMIIA mice. The expression level of the tagged NMIIA is similar to that of the endogenously expressed untagged NMIIA in both heterozygous mutants. This tagged NMIIA mouse model will shed light on the function of NM IIA in development. Various cell lines derived from the mice will be used to study the regulation and function of NM IIA in adhesion, cell polarity and cell migration. We also plan to cross mCherry tagged NM IIA mice with GFP tagged NMIIB mouse. The offspring with mCherry tagged NMIIA and GFP tagged NMIIB should be useful to study if NMIIA and NMIIB can form copolymers in vivo. The cell lines (e.g. fibroblasts) derived from this mouse can be used to study the various biological properties of NMIIA and NMIIB in cell polarity and migration with confocal or TIRF microscopy. The purpose of an additional study is to learn whether one isoform of NM II, specifically NM IIC, can functionally replace a second one, NM IIA, in mice. To replace NM IIA with NM IIC, homologous recombination was used to inactivate NM IIA by inserting the cDNA for NM IIC-GFP into the first coding exon of the Myh9 gene. We have obtained heterozygous NM IIC replacing NM IIA mice. However, breeding of heterozygous mutant mice does not produce homozygous NM IIC replacing NM IIA mice. We plan to investigate at which embryonic stage the homozygous mice die and the impact of NM IIC replacing NM IIA on cell polarization and migration.
