Lehtonen E, Virtanen I, Saxén L
Dev Biol. 1985 Apr;108(2):481-90. doi: 10.1016/0012-1606(85)90051-x.
The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells.
如果将后肾间充质与诱导组织进行滤膜接触培养，它会转化为上皮小管。在这个用于分化和器官发生的模型系统中，已经研究了用作细胞类型特异性标志物的中间丝（IFs）的表达。在冷冻切片的免疫荧光显微镜检查中，分离的后肾间充质的未分化细胞仅均匀显示波形蛋白类型的IFs。同样，当作为单层培养时，未诱导间充质的细胞仅显示波形蛋白丝。在滤膜外植体的冷冻切片中，培养3天后可见明显对波形蛋白呈阴性的上皮小管，但直到培养第四天，在发育中的小管中才显示细胞角蛋白的表达。进一步培养的外植体切片显示小管细胞具有明显的纤维状细胞角蛋白阳性。使用两种不同的细胞角蛋白抗体进行免疫印迹实验也证实了外植体中细胞角蛋白的出现。在最初暴露于短时间滤膜诱导脉冲的后肾间充质单层培养物中进一步检查了IFs的表达。在这些实验中，培养总共4天后可以证明有细胞角蛋白阳性细胞。双重免疫荧光实验显示细胞角蛋白阳性细胞中波形蛋白的含量各不相同：培养4天后，大多数细胞角蛋白阳性细胞仍显示波形蛋白阳性，尽管通常呈非纤维状形式。在进一步培养过程中，在细胞角蛋白阳性细胞中观察到波形蛋白特异性荧光逐渐消失。结果表明，波形蛋白阳性的后肾间充质细胞在诱导导致肾小管形成时失去其纤维状波形蛋白结构。这种变化对于从未分化的间充质细胞转变为特化的上皮细胞可能至关重要。被视为上皮细胞标志物的细胞角蛋白丝似乎在波形蛋白结构改变的同时或之后不久出现。IF表达的这些变化也发生在最初暴露于短时间滤膜诱导脉冲的间充质细胞单层培养物中。这表明，细胞角蛋白阳性的出现所揭示的上皮分化即使在细胞没有明显形态分化和三维组织的情况下也可能发生。
