NULL MUTATION OF A NEURON-SPECIFIC EXON OF NONMUSCLE MYOSIN II HEAVY CHAIN-B

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Vertebrate nonmuscle myosin II exists as at least two isoforms encoded by two different genes. Nonmuscle myosin B1 (NMM-B1) contains a 10 amino acid insert near the ATP binding region of the NMM-B, generated by alternative splicing of a 30 nucleotide exon. In avian and mammalian tissues, NMM-B1 is confined to neuronal cells where the expression begins early in development and is correlated with neuronal differentiation. In an effort to understand the function of the NMM-B1 in vertebrates, we deleted the 30 nucleotide exon from NMM-B gene in mice. This was done by replacement of the exon by neomycin resistance gene through homologous recombination in embryonic stem cells. Analysis of the mRNA from various tissues of homozygous mice showed the absence of the NMM-B1 and an unexpected decrease in NMM-B RNA and protein levels. Analysis of the heart from adult homozygous mice showed hypertrophy determined by macroscopic examination and an increase in heart to body weight ratio. Microscopic examination showed hypertrophy of the cardiac myocytes. In cerebellum, the Purkinje cells were found to lack NMM-B, by immunofluorescence staining. However, this did not cause any dysfunction in cerebellar motor coordination judged by rotarod and footprint tests. Microscopic analysis of the eye showed displaced retinal ganglionic cells in the inner nuclear layer. Initial experiments using cultured superior ganglionic cells showed a reduction in outgrowth of neurites and growth cone area of approximately 30%. Previously, we had generated NMM-B ablated mice in our laboratory. By crossing the NMM-B heterozygous mice with NMM-B1 homozygous mice, we generated mice that carry the NMM-B gene without the 30 nucleotide exon, on only one allele. These mice developed hydrocephalus varying between 1-3 weeks after birth to 10 months. Preliminary analysis of the heart in these mice showed ventricular septal defects and, in one case, transposition of the great vessels. The retinal defect seen in NMM-B1 (-/-) mice was also apparent in these mice, with an increase in the number of misplaced ganglionic cells. Comparison of the protein and RNA levels in these mice with the other genotypic mice should help us to evaluate the phenotypic variability we are observing in the NMM-B mutated mice.