Koyama E, Leatherman J L, Shimazu A, Nah H D, Pacifici M
Department of Anatomy and Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003, USA.
Dev Dyn. 1995 Jun;203(2):152-62. doi: 10.1002/aja.1002030204.
The mechanisms by which the early limb cell condensations and interzone mesenchyme give rise to skeletal elements and joints are poorly understood. Previous work from this laboratory has shown that the extracellular matrix protein tenascin-C is associated with articular cartilage and joint tissue development; others have shown that tenascin-C may exert its biological activities via interactions with cell surface receptors, such as syndecans. To further analyze the roles of tenascin-C and its putative receptors in skeletal development, we carried out a detailed in situ hybridization analysis of tenascin-C and syndecan-3 gene expression during development of chick limb skeletal elements and joints. We found that as the early mesenchymal condensations chondrify around day 5 (E5) of development, they become surrounded by a thick syndecan-3 rich perichondrium while tenascin-C transcripts are much fewer and restricted to diaphyseal perichondrium and developing interzones. Similar patterns were observed as distal carpal and digit condensations formed in older embryos. As the cartilaginous long bone models elongated proximo-distally and joint formation proceeded with age, we observed that syndecan-3 transcripts decrease significantly along the diaphysis and remain very abundant along the metaphysis and in the epiphyseal articular cap and interzone. Conversely, tenascin-C RNAs remain abundant along the diaphysis and begin to increase at the epiphysis and in interzone-derived tissues, such as menisci and joint capsule. By E10, the skeletal elements have well-defined morphologies, endochondral ossification has initiated in their diaphysis, and diaphyseal perichondrium has become periosteum. These developmental changes were accompanied by equally marked changes in gene expression; these included a marked increase in tenascin-C gene expression in articular cap, fragmentation of tenascin-C gene expression along the periosteum, reinitiation of syndecan-3 gene expression in periosteum, and differential gene expression in osteoprogenitor cells. The sheer complexity of the gene expression patterns documented in this study attests to the complexity of processes that bring about normal skelatogenesis. Clearly, tenascin-C and syndecan-3 appear to be closely associated with several of these processes, particularly in establishing tissue boundaries (perichondrium and periosteum) between condensations and surrounding mesenchymal cells, in regulating perichondral cell differentiation and incorporation into the growing skeletal elements, and in the genesis of epiphyseal chondrocytes and associated joint tissues.
早期肢体细胞凝聚和中间带间充质形成骨骼元件和关节的机制目前还知之甚少。该实验室之前的研究表明，细胞外基质蛋白腱生蛋白-C与关节软骨和关节组织发育相关；其他人则表明，腱生蛋白-C可能通过与细胞表面受体（如多功能蛋白聚糖）相互作用来发挥其生物学活性。为了进一步分析腱生蛋白-C及其假定受体在骨骼发育中的作用，我们对鸡肢体骨骼元件和关节发育过程中腱生蛋白-C和多功能蛋白聚糖-3基因表达进行了详细的原位杂交分析。我们发现，在发育第5天（E5）左右，随着早期间充质凝聚软骨化，它们被富含多功能蛋白聚糖-3的厚软骨膜包围，而腱生蛋白-C转录本则少得多，局限于骨干软骨膜和正在发育的中间带。在较老胚胎中形成远端腕骨和指骨凝聚时也观察到类似模式。随着软骨性长骨模型向近远侧延伸以及关节随着年龄增长而形成，我们观察到多功能蛋白聚糖-3转录本沿骨干显著减少，而在干骺端、骨骺关节帽和中间带中仍非常丰富。相反，腱生蛋白-C RNA沿骨干仍很丰富，并开始在骨骺和中间带衍生组织（如半月板和关节囊）中增加。到E10时，骨骼元件具有明确的形态，骨干开始软骨内成骨，骨干软骨膜已变成骨膜。这些发育变化伴随着基因表达同样显著的变化；这些变化包括关节帽中腱生蛋白-C基因表达显著增加、腱生蛋白-C基因表达沿骨膜碎片化、骨膜中多功能蛋白聚糖-3基因表达重新启动以及骨祖细胞中的差异基因表达。本研究记录的基因表达模式极其复杂，证明了导致正常骨骼生成过程的复杂性。显然，腱生蛋白-C和多功能蛋白聚糖-3似乎与其中几个过程密切相关，特别是在建立凝聚物与周围间充质细胞之间的组织边界（软骨膜和骨膜）、调节软骨膜细胞分化并使其融入生长中的骨骼元件以及骨骺软骨细胞和相关关节组织的发生方面。
