Organization of non-muscle myosin during early murine embryonic differentiation

Abstract

Abstract A monoclonal antibody (3D10) recognizing myosin heavy chain was isolated following immunization with a synthetic peptide sequence of eight amino acids. The antibody reacted with purified rabbit skeletal myosin and light mero-myosin in enzyme-linked immunosorbent assays and Western immunoblotting. A band of approximately 200 kDa was detected in cell extracts of an embryonal carcinoma (EC) cell line (P19EC) and one of its cloned differentiated derivatives, suggesting reactivity against non-muscle myosin. By indirect immunofluorescence, typical myosin banding patterns were observed in cryostat sections of human skeletal and cardiac muscle tissue. In undifferentiated P19EC cells, speckled immunofluorescent staining was observed in the cytoplasm that became organized in cortical rings where the cells made direct contact with each other. These rings consisted of circular bundles of F-actin decorated by myosin. Undifferentiated embryonic stem (ES) cells derived directly from mouse embryos shared the same features, although the pattern was less pronounced. Human testicular primary germ cell tumours showed cortical staining in the embryonal carcinoma component reminiscent of the staining of EC cells in vitro while cytoplasmic staining was observed in tumour cells with a differentiated morphology. In preimplantation embryos, the immunofluorescent staining was observed at cell apices of blastomeres of morula stage embryos. In blastocysts, staining of inner cell mass cells was not detectable. By contrast, various differentiated derivatives of P19EC contained extensive F-actin microfilament bundles throughout the cytoplasm decorated with myosin. Thick stress fibers in filopodious extensions of cells were particularly highly decorated by myosin. Over the nucleus, linear arrays of myosin containing speckled patterns of immunofluorescence were observed that were not associated with F-actin. The same pattern of staining could be observed in trophectoderm cells of the blastocyst. We conclude that embryonic non-muscle myosin is organized in specific patterns depending on the state of differentiation. As the myosin is primarily associated with F-actin we suspect that it forms part of a contractile apparatus that may have significance during embryonic development.