SPATIOTEMPORAL PROFILE OF N-CADHERIN EXPRESSION IN THE DEVELOPING LIMB MESENCHYME
SPATIOTEMPORAL PROFILE OF N-CADHERIN EXPRESSION IN THE DEVELOPING LIMB MESENCHYME
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DOI：
10.3109/15419069409014216
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发表时间：
1994-01-01
期刊：
影响因子：
--
通讯作者：
TUAN, RS
中科院分区：
文献类型：
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作者：
OBERLENDER, SA;TUAN, RS
在胚胎肢体发育过程中，间充质的软骨形成之前是一个关键的细胞凝聚阶段。由于冷凝是可能导致从特定的细胞-细胞相互作用，我们已经研究了可能参与的N-钙粘蛋白，钙依赖性细胞粘附分子，在这个冷凝事件。之前我们已经报道过N-钙粘蛋白在鸡胚胎肢芽中表达，并且N-钙粘蛋白介导的细胞粘附的干扰显着抑制体内和体外肢体间充质细胞聚集和软骨形成。为了进一步研究N-cadherin的表达和软骨形成之间的关系，我们已经量化和表征的时空表达的N-cadherin mRNA和蛋白质在发育中的鸡胚肢体，并分析了机制，外源性钙离子刺激软骨形成在体外。免疫组化结果显示，在肢芽成熟过程中，N-cadherin以发育特异性的方式在间充质细胞表面表达。N-Cadherin在早期肢芽中央核心的间充质中有表达，在24 ~ 26期表达最强，与体内细胞凝聚期相对应。成熟软骨不表达N-cadherin。在鸡肢间充质微团培养物在体外，N-钙粘蛋白的蛋白表达被认为与不同的细胞聚集的凝聚间充质，但不是在成熟的软骨结节或间充质之间的凝聚聚集。原位杂交定位N-cadherin mRNA表达在25/26期肢芽的肢间充质致密区;在后期阶段，N-cadherin mRNA表达见于软骨膜和致密间充质中，但不见于成熟软骨中。因此，mRNA和蛋白质数据之间的协议表明，N-钙粘蛋白的表达在发育中的肢芽在转录水平上进行调节。Western免疫印迹分析进一步证实，从第19期到第36期，肢芽中的N-钙粘蛋白水平增加了5倍，直到第25/26期，然后在软骨形成和骨形成的过程中下降。在体外微团肢间充质培养物中也观察到平行的轮廓。通过保留或去除N-钙粘蛋白的间充质细胞的选择性蛋白水解解离来检查N-钙粘蛋白对Ca 2+介导的间充质细胞聚集和软骨形成的重要性。显示具有N-钙粘蛋白的细胞表现出Ca 2+依赖性聚集，并且与没有N-钙粘蛋白的细胞相比始终更具有软骨形成性。这些结果为N-钙粘蛋白在软骨形成中的功能作用提供了强有力的支持。
During embryonic limb development, chondrogenesis of the mesenchyme is preceded by a crucial cellular condensation phase. Because condensation is likely to result from specific cell-cell interactions, we have examined the possible involvement of N-cadherin, a Ca2+-dependent cell adhesion molecule, in this condensation event. previously we have reported that N-cadherin is expressed in chick embryonic limb bud, and that perturbation of N-cadherin-mediated cell adhesion significantly inhibits limb mesenchymal cellular aggregation and chondrogenesis both in vivo and in vitro. To further examine the relationship between N-cadherin expression and chondrogenesis, we have quantified and characterized the spatiotemporal expression of N-cadherin mRNA and protein in the developing chick embryonic limb, and analyzed the mechanism whereby exogenous Ca2+ stimulates chondrogenesis in vitro. Immunohistochemistry revealed that N-cadherin is expressed on mesenchymal cell surface in a developmentally specific manner during limb bud maturation. N-Cadherin was detected in the mesenchyme of the central core of the early limb bud, and was maximally expressed between stages 24 to 26, Corresponding to the period of cellular condensation in vivo. Mature cartilage did not express N-cadherin. In chick limb mesenchyme micromass cultures in vitro, N-cadherin protein expression was seen associated with distinct cellular aggregates of condensing mesenchyme, but not in mature cartilaginous nodules or the mesenchyme situated between the condensing aggregates. In situ hybridization localized N-cadherin mRNA expression in condensing regions of limb mesenchyme in a stage 25/26 limb bud; at later stages, N-cadherin mRNA expression was seen in the perichondrium and the dense mesenchyme, but not in mature cartilage. The agreement between the mRNA and protein data thus suggests that N-cadherin expression in the developing limb bud is regulated at the transcriptional level. Western immunoblot analysis further confirmed that, from stages 19 to 36, the level of N-cadherin in the limb bud increased 5-fold through stage 25/26, then decreased during the progression of chondrogenesis and osteogenesis. A parallel profile was also seen in micromass limb mesenchyme cultures in vitro. The importance of N-cadherin for Ca2+ mediated mesenchymal cell aggregation and chondrogenesis was examined by selective, proteolytic dissociation of the mesenchymal cells with retention or removal of N-cadherin. Cells with N-cadherin were shown to exhibit Ca2+-dependent aggregation and were consistently more chondrogenic than those without N-cadherin. These results provide strong support for the functional role of N-cadherin in chondrogenesis.