Spatiotemporal profile of N-cadherin expression in the developing limb mesenchyme.
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 Ca(2+)-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 Ca(2+)-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.
Oberlender
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SA
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Tuan
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RS
D000818:Animals; D000911:Antibodies, Monoclonal; D015820:Cadherins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002449:Cell Aggregation / Q000187:drug effects; D002642:Chick Embryo; D046508:Culture Techniques; D005121:Extremities / Q000196:embryology* / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry; D008648:Mesoderm / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D000911:Antibodies, Monoclonal; D015820:Cadherins; D012333:RNA, Messenger; D002118:Calcium