Burdsal C A, Damsky C H, Pedersen R A
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143.
Development. 1993 Jul;118(3):829-44. doi: 10.1242/dev.118.3.829.
We have examined the role of cell-cell and cell-extracellular matrix (ECM) interactions during mesoderm differentiation and migration at the primitive streak of the mouse embryo with the use of function-perturbing antibodies. Explants of epiblast or mesoderm tissue dissected from the primitive streak of 7.5- to 7.8-day mouse embryos were cultured on a fibronectin substratum in serum-free, chemically defined medium. After 16-24 hours in culture, cells in explants of epiblast exhibited the typical close-packed morphology of epithelia, and the tissue remained as a coherent patch of cells that were shown to express transcripts of the cytokeratin Endo B by in situ analysis. In contrast, cells in explants of primitive streak mesoderm exhibited a greatly flattened, fibroblastic morphology, did not express Endo B transcripts, and migrated away from the center of the explant. As epiblast cells in vivo undergo the epithelial-mesenchymal transition at the primitive streak, they cease expressing the prominent calcium-sensitive cell adhesion molecule E-cadherin (uvomorulin, Cell-CAM 120/80). We asked whether the loss of E-cadherin expression was a passive result of differentiation or if it might play a more causative role in mesoderm differentiation and migration. Culture with function-perturbing antibodies against E-cadherin caused cells within epiblast explants to lose cell-cell contacts, to flatten, and to assume a mesenchymal morphology; they were also induced to migrate. Anti-E-cadherin antibodies had no effect on explants of primitive streak mesoderm. In immunofluorescence studies, anti-E-cadherin-treated epiblast cells ceased to express SSEA-1, a carbohydrate moiety that is lost as mesoderm differentiates from the epiblast in vivo, and they also ceased to express E-cadherin itself. In contrast, these cells began to express the intermediate filament protein vimentin, a cytoskeletal protein characteristic of the primitive streak mesoderm at this stage of development. As epiblast cells differentiate into mesoderm, their predominant adhesive interactions change from cell-cell to cell-substratum. Therefore, we also investigated the adhesive interactions between primitive streak tissues and extracellular matrix (ECM) components. Epiblast explants adhered well to fibronectin, more poorly to laminin and type IV collagen, and not at all to vitronectin. In contrast, mesoderm explants attached well to all these proteins. Furthermore, epiblast, but not mesoderm, displayed an anchorage-dependent viability in culture. After anti-E-cadherin treatment, epiblast cells that had assumed the mesenchymal morphology did attach to vitronectin, another characteristic shared with primitive streak mesoderm.(ABSTRACT TRUNCATED AT 400 WORDS)
我们利用功能干扰抗体，研究了细胞间以及细胞与细胞外基质（ECM）相互作用在小鼠胚胎原条处中胚层分化和迁移过程中的作用。从7.5至7.8天龄小鼠胚胎的原条处解剖得到的外胚层或中胚层组织外植体，在纤连蛋白基质上于无血清、化学成分明确的培养基中培养。培养16 - 24小时后，外胚层外植体中的细胞呈现出上皮细胞典型的紧密堆积形态，并且该组织仍作为一块连贯的细胞片，通过原位分析显示其表达细胞角蛋白Endo B的转录本。相比之下，原条中胚层外植体中的细胞呈现出极度扁平的成纤维细胞形态，不表达Endo B转录本，并从外植体中心迁移开。由于体内外胚层细胞在原条处经历上皮 - 间充质转变，它们停止表达显著的钙敏感细胞黏附分子E - 钙黏蛋白（卵黄膜蛋白、细胞黏附分子120/80）。我们探究了E - 钙黏蛋白表达的丧失是分化的被动结果，还是它可能在中胚层分化和迁移中发挥更具因果关系的作用。用针对E - 钙黏蛋白的功能干扰抗体进行培养，导致外胚层外植体中的细胞失去细胞间接触、变扁平并呈现间充质形态；它们还被诱导迁移。抗E - 钙黏蛋白抗体对原条中胚层外植体没有影响。在免疫荧光研究中，经抗E - 钙黏蛋白处理的外胚层细胞停止表达阶段特异性胚胎抗原 - 1（SSEA - 1），这是一种随着中胚层在体内从外胚层分化而丢失的碳水化合物部分，并且它们也停止表达E - 钙黏蛋白本身。相比之下，这些细胞开始表达中间丝蛋白波形蛋白，这是发育此阶段原条中胚层特有的一种细胞骨架蛋白。随着外胚层细胞分化为中胚层，它们主要的黏附相互作用从细胞间转变为细胞与基质间。因此，我们还研究了原条组织与细胞外基质（ECM）成分之间的黏附相互作用。外胚层外植体与纤连蛋白黏附良好，与层粘连蛋白和IV型胶原黏附较差，与玻连蛋白完全不黏附。相比之下，中胚层外植体与所有这些蛋白质都黏附良好。此外，外胚层在培养中表现出锚定依赖性活力，而中胚层则不然。经抗E - 钙黏蛋白处理后，呈现间充质形态的外胚层细胞确实与玻连蛋白黏附，这是与原条中胚层共有的另一个特征。（摘要截断于400字）
