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 30 different mutations in MYH9 have been reported to date, including both mis-sense 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 motor domain;D1424N and E1841K in 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 produced both R702C and D1424N mutant mice by using homologous recombination to replace wild type NMHC IIA with mutant R702C or D1424N in NMHC IIA. The E1841K mutant mice were obtained from Dr. M Kelley at Duke University. Breeding of heterozygous R702C mutant mice has not produced homozygous mutant offspring. The homozygotes die between embryonic day 8.5 and 10.5. On the other hand, breeding of heterozygous D1424N mutant mice produced homozygous mutant offspring at close to normal ratios, and E1841K mutant mice also have homozygous mutant offspring, suggesting that mutations in the motor domain of NMHC IIA may have a more severe effect than mutations in the rod during embryonic development. Interestingly, giant platelets were found in the blood smears from both R702C and D1424N adult heterozygous mice. All three mutants also have significantly higher mean platelet volumes compared to their wild type littermates (6.12 +/- 0.62 fL, wt;10.17 +/- 1.50 fL, heterozygous R702C;10.13 +/- 1.66 fL, heterozygous D1424N;10.92 +/- 1.83 fL, heterozygous E1841K). Kidney function was studied by examining the albumin/creatinine ratio in urine samples. Some but not all adult heterozygotes of both mutant lines have higher albumin/creatinine ratios at 8-9 weeks, indicating that kidney impairment may develop in some heterozygous mutants at an early age. These preliminary results suggest that these mouse models should be useful in understanding the pathophysiology of human MYH9-related diseases. 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 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 mouse, 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 the heterozygous GFP or mCherry tagged NMIIA mice. The expression level of the tagged NMIIA is relatively lower than the endogenously expressed untagged NMIIA in both heterozygous mutants. We have set up breeding cages of the heterozygous tagged NMIIA mice with Cre mice to remove the Neo cassette so the tagged NMIIA protein expression level may increase to the normal level. This tagged NMIIA mouse model will shed light on the function of NM IIA in development. Various cell lines derived from the mouse 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. fibroblast) derived from this mouse can be used to study the distinct kinetic 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 will be 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 several positive embryonic stem cell lines which are heterozygous NMIIC-GFP under the control of NM IIA promoter. These embryonic stem cells will be used to produce chimera mice.
