In order to study the function of the nonmuscle myosin II isoforms (NMII-A, II-B, and II-C), homologous recombination has been used to delete each isoform in embryonic stem cells and null mice have been generated. Deletion of nonmuscle myosin heavy chain (NMHC) II-A causes lethality prior to gastrulation (E6.5) and the embryos are disorganized with defects in cell-cell adhesion and a failure to form a columnar visceral endoderm. In order to avoid the early embryonic lethality, a nonmuscle myosin heavy chain (NMHC) II-A floxed mouse has been created. A neomycin-resistance cassette has been inserted into the intron 3 of exon 3 and loxP sites have been inserted flanking both the neomycin cassette and the exon. Matings of these mice to mice bearing Cre recombinase under the control of cell or tissue specific promoters causes the corresponding deletion of NM II-A. The Nkx2.5 homeobox gene is a very early marker of heart development but it is also expressed in the tongue epithelium from at least E13.5 during tongue development. Crosses of the NMHC II-A floxed mice to mice bearing Cre recombinase under control of the Nkx2.5 promoter develop squamous cell carcinoma of the tongue. Nonmuscle myosin II-A is deleted in a mosaic pattern in the post-natal tongue epithelium and this deletion becomes widespread in the epithelial layer in the adult. NM II-B is undetectable but NM II-C is present from the earliest stages of epithelial development in the tongue by immunofluorescence staining with isoform specific antibodies. Mass spectrometry of the wild type tongue epithelium shows a distribution of 62%II-A and 38% II-C with no II-B detected. Earliest signs of an invasive tumor are seen at E17.5 These mice survive up to 3-4 months of age although there are a few mice which live past one year. These latter mice also show evidence for squamous cell carcinoma although it appears to be less extensive than mice which die at 3-4 months. This difference in extent of disease may be due to levels of expression of Cre recombinase. When one of the NMHC II-A flox alleles is replaced by a NMHC II-A null allele the mice develop squamous cell carcinoma with a similar time course, i.e. lethality at 3-4 months.

Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2012
Total Cost
$425,242
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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Type
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