Alterations in the spatiotemporal expression pattern and function of N‐Cadherin inhibit cellular condensation and chondrogenesis of limb mesenchymal cells in vitro

Abstract

Cartilage formation in the embryonic limb is presaged by a cellular condensation phase that is mediated by both cell-cell and cell-matrix interactions. N-Cadherin, a Ca(2+)-dependent cell-cell adhesion molecule, is expressed at higher levels in the condensing mesenchyme, followed by down-regulation upon chondrogenic differentiation, strongly suggesting a functional role in the cellular condensation process. To further examine the role of N-cadherin, we have generated expression constructs of wild type and two deletion mutants (extracellular and intracellular) of N-cadherin in the avian replication-competent, RCAS retrovirus, and transfected primary chick limb mesenchymal cell cultures with these constructs. The effects of altered, sustained expression of N-cadherin and its mutant forms on cellular condensation, on the basis of peanut agglutinin (DNA) staining, and chondrogenesis, based on expression of chondrocyte phenotypic markers, were characterized. Cellular condensation was relatively unchanged in cultures overexpressing wild type N-cadherin, compared to controls on all days in culture. However, expression of either of the deletion mutant forms of N-cadherin resulted in decreased condensation, with the extracellular deletion mutant demonstrating the most severe inhibition, suggesting a requirement for N-cadherin mediated cell-cell adhesion and signaling in cellular condensation. Subsequent chondrogenic differentiation was also affected in all cultures overexpressing the N-cadherin constructs, on the basis of metabolic sulfate incorporation, the presence of the cartilage matrix proteins collagen type II and cartilage proteoglycan link protein, and alcian blue staining of the matrix. The characteristics of the cultures suggest that the N-cadherin mutants disrupt proper cellular condensation and subsequent chondrogenesis, while the cultures overexpressing wild type N-cadherin appear to condense normally, but are unable to proceed toward differentiation, possibly due to the prolonged maintenance of increased cell-cell adhesiveness. Thus, spatiotemporally regulated N-cadherin expression and function, at the level of both homotypic binding and linkage to the cytoskeleton, is required for chondrogenesis of limb mesenchymal cells.

Hydrogels that mimic developmentally relevant matrix and N-cadherin interactions enhance MSC chondrogenesis

“…Therefore, the diminishing effect of the coupled cadherin peptides was potentially a concomitant consequence of decreasing N-cadherin density on hMSCs with chondrogenesis. In fact, persistent expression of N-cadherin via viral transfection in limb mesenchymal cell culture had an inhibitory effect on chondrogenesis, possibly due to prolonged maintenance of increased cellcell adhesiveness, stressing the importance of precise temporal presentation of N-cadherin (21). Moreover, the encapsulated hMSCs elaborate and deposit increasing amounts of cartilage matrix with culture, which could block interactions with the peptide with time.…”

“…Studies have shown that the expression of the deletion mutant form of N-cadherin, which lacks either the extracellular homotypic interaction domains or the intracellular β-catenin binding site, results in decreased cellular condensation and impaired chondrogenesis (21,22). These findings suggest that both extracellular homotypic interaction and intracellular interaction with the catenin complex are essential for proper N-cadherin signaling (23).…”

Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap

“…A region of interest (ROI) was selected around the thresholded aggregate using the wand tool, and ROI area plotted against aggregate age. The development and strength of aggregate physical integrity (through cell-cell adhesion) was determined by sedimentation analysis (Coakley et al, 2004;Edwards et al, 2007). Ultrasound was terminated following aggregate formation which led to sedimentation onto the USWT surface.…”

“…Chondrogenesis is a multi-step developmental process leading to the formation of skeletal tissues such as cartilage (reviewed in Delise et al, 2000 andShimizu et al, 2007). Mesenchymal cells in the developing limb bud deposit extracellular matrix, form tightly-packed condensations, then differentiate into chondrocytes, proliferate and mature.…”

“…hydrophobicity or van der Waals) result in close molecular interactions between adjacent cells (Bazou et al, 2005b). Stable 2-D aggregates of interacting mammalian cells in physiological media can be rapidly assembled that form adhesive complexes and show cytoskeletal remodelling (Bazou et al, 2005a(Bazou et al, , 2006Edwards et al, 2007). The current study was designed to determine how initial intercellular interactions could initiate chondrogenesis in limb buds in vitro, independent from contact with an artificial substratum, by levitation in an USWT.…”