Fleming T P, McConnell J, Johnson M H, Stevenson B R
Department of Anatomy, University of Cambridge, United Kingdom.
J Cell Biol. 1989 Apr;108(4):1407-18. doi: 10.1083/jcb.108.4.1407.
Tight junction development during trophectoderm biogenesis in the mouse preimplantation embryo has been examined using monoclonal antibodies recognizing the tight junction-specific peripheral membrane protein, ZO-1. In immunoblots, mouse embryo ZO-1 had a molecular mass (225 kD) equivalent to that in mouse liver, was barely detectable in four-cell embryos although later stages exhibited increasing levels. ZO-1 was first detected immunocytochemically at the compacting eight-cell stage, coincident with or just after the expression of basolateral cell adhesion and apical microvillous polarity. Initially, ZO-1 was present as a series of spots along the boundary between free and apposed cell surfaces in intact embryos or cell couplets, but subsequently staining became more linear with blastocyst trophectoderm cells being bordered by a continuous ZO-1 belt. Inhibition of cell adhesion at the 8-cell stage delayed ZO-1 appearance and randomized its surface distribution in a reversible manner. Microfilament disruption, but not microtubule depolymerization, produced major disturbances in ZO-1 distribution. ZO-1 assembly de novo appeared to be independent of proximate DNA and RNA synthesis but was inhibited substantially in the absence of protein synthesis during the eight-cell stage, a treatment that did not prevent intercellular adhesion and polarization. ZO-1 surface assembly, but not adhesion and polarization, was also perturbed when single eight-cells were combined with single four-cells. The results suggest that tight junction development in mouse embryos is a secondary event in epithelial biogenesis, being dependent upon cell adhesion and cytoskeletal activity for normal expression, and can be disrupted without disturbing the generation of a stably polarized phenotype.
利用识别紧密连接特异性外周膜蛋白ZO-1的单克隆抗体，对小鼠植入前胚胎滋养外胚层生物发生过程中的紧密连接发育进行了研究。在免疫印迹中，小鼠胚胎的ZO-1分子量（225 kD）与小鼠肝脏中的相当，在四细胞胚胎中几乎检测不到，尽管后期阶段其水平有所增加。免疫细胞化学检测发现，ZO-1最早在致密化的八细胞阶段被检测到，与基底外侧细胞黏附及顶端微绒毛极性的表达同时或稍晚出现。最初，在完整胚胎或细胞对中，ZO-1沿着游离和相邻细胞表面之间的边界呈一系列斑点状存在，但随后染色变得更呈线性，囊胚滋养外胚层细胞被连续的ZO-1带所环绕。八细胞阶段细胞黏附的抑制延迟了ZO-1的出现，并以可逆的方式使其表面分布随机化。微丝破坏而非微管解聚，对ZO-1的分布产生了重大干扰。从头组装ZO-1似乎独立于近端DNA和RNA合成，但在八细胞阶段缺乏蛋白质合成时会受到显著抑制，这种处理并不妨碍细胞间黏附和极化。当单个八细胞与单个四细胞结合时，ZO-1的表面组装而非黏附和极化也受到了干扰。结果表明，小鼠胚胎中的紧密连接发育是上皮生物发生中的一个次级事件，其正常表达依赖于细胞黏附和细胞骨架活性，并且在不干扰稳定极化表型产生的情况下可能被破坏。
