Tavella S, Raffo P, Tacchetti C, Cancedda R, Castagnola P
Laboratorio di Differenziamento Cellulare Istituto Nazionale per La Ricerca sul Cancro, Centro di Biotecnologie Avanzate, Genova, Italy.
Exp Cell Res. 1994 Dec;215(2):354-62. doi: 10.1006/excr.1994.1352.
Mesenchymal cell condensation in chick limb bud occurs at embryonic stage 22 and is the starting event of chondrogenesis. Several mechanisms have been proposed to have an active role in the induction of this process. Among them the establishment of cell-cell contacts represents a key event. Here we have investigated the modulation of N-CAM and N-cadherin gene expression in an in vitro culture system which allows chondrocyte differentiation to proceed from condensation of prechondrogenic cells to hypertrophic chondrocytes and eventually to osteoblast-like cells. Both Northern and Western blots demonstrated that they were developmentally regulated in differentiating chondrocytes. Both cell adhesion proteins were detectable in prechondrogenic cells, increased during cell aggregation, became undetectable in hypertrophic chondrocytes, and resulted in reexpression during their maturation to osteoblast-like cells. The timing of appearance of N-cadherin and N-CAM suggests that N-cadherin initiates the in vitro cell condensation thereafter stabilized by N-CAM. In agreement with the above findings, the immunolocalization of these molecules in the cell aggregates revealed that N-CAM and N-cadherin appear, after 12 h of suspension culture, on the surface of all cells at the membrane regions participating in cell-cell contacts. At 72 h N-CAM became restricted to cells at the aggregate periphery, while N-cadherin was detected both in type II collagen-negative and -positive regions. At this time of culture, electron microscopy shows a number of cell-cell contacts at the perifery of the cell aggregates, while only a few of them were observed in the aggregate interior. The expression of N-CAM and type II collagen by chondrocytes was mutually exclusive and a sorting out between differentiating and nondifferentiating cells occurred.
鸡胚肢芽中的间充质细胞凝聚发生在胚胎第22阶段，是软骨形成的起始事件。已经提出了几种机制在这一过程的诱导中发挥积极作用。其中细胞间接触的建立是一个关键事件。在这里，我们研究了N-CAM和N-钙黏蛋白基因表达在体外培养系统中的调节情况，该系统允许软骨细胞从软骨前体细胞凝聚分化为肥大软骨细胞，并最终分化为成骨样细胞。Northern印迹和Western印迹均表明它们在分化的软骨细胞中受到发育调控。两种细胞黏附蛋白在软骨前体细胞中均可检测到，在细胞聚集过程中增加，在肥大软骨细胞中无法检测到，并在它们成熟为成骨样细胞的过程中重新表达。N-钙黏蛋白和N-CAM出现的时间表明，N-钙黏蛋白启动了体外细胞凝聚，随后由N-CAM使其稳定。与上述发现一致，这些分子在细胞聚集体中的免疫定位显示，悬浮培养12小时后，N-CAM和N-钙黏蛋白出现在参与细胞间接触的膜区域的所有细胞表面。72小时时，N-CAM局限于聚集体周边的细胞，而在II型胶原阴性和阳性区域均检测到N-钙黏蛋白。在这个培养时间点，电子显微镜显示在细胞聚集体周边有许多细胞间接触，而在聚集体内部仅观察到少数细胞间接触。软骨细胞中N-CAM和II型胶原的表达相互排斥，并且在分化细胞和未分化细胞之间发生了分选。
