A dissection of the mechanisms generating and stabilizing polarity in mouse 8- and 16-cell blastomeres: the role of cytoskeletal elements

Abstract

Pairs of 8-cell or 16-cell blastomeres were cultured for up to 9 h after their formation from isolated 1/4 or 1/8 blastomeres respectively. Blastomeres were examined for the incidence and orientation of their surface polarity, as assessed by binding of FITC-Con A and by distribution of microvilli, and of their cytoplasmic polarity, as assessed by distribution of cytoplasmic actin, clathrin and a 100 kD antigen associated with the lysosomal/acid vesicle fraction of membranous organelles. The effect on polarity of incubating the pairs of cells in taxol, nocodazole, cytochalasin D or in a combination of nocodazole plus cytochalasin D for different parts of the incubation period was examined. Neither the development nor the stability of the surface polarity in 8-cell blastomeres was blocked by any treatment and only the use of CCD in combination with nocodazole affected the incidence of surface polarity appreciably. However, with some treatments, the form and position of the surface poles were modified. In the presence of microtubule inhibitors surface poles extended over a larger area of the cell surface, while exposure to CCD led to poles that were not opposite to the contact point between cells. In contrast to surface polarity, the development of cytoplasmic polarity was suppressed by both microtubule- and microfilament-inhibiting drugs, which also reversed it rapidly. In polar 16-cell blastomeres surface polarity was influenced in a similar manner to that of 8-cell blastomeres, only the combined use of cytochalasin D and nocodazole having any major effect. Polarization of clathrin in polar 16-cell blastomeres was inhibited almost completely by all drug treatments applied including cytochalasin D. The focal concentration of lysosomal antigen that occurs during the 16-cell stage was reduced only in the continuous presence of nocodazole plus cytochalasin D, but once established was not reversed appreciably by any drug. However, the localization of the lysosomal antigen to the basal region of polarized cells did not seem to occur in the presence of any drug. The dissociation of surface and cytoplasmic polarity revealed in these experiments leads us to conclude that either surface polarity is a prerequisite for the organization of cytoplasmic polarity, and mediates the latter via the cytoskeleton, or surface and cytoplasmic polarity develop by parallel but separate mechanisms.