Regulation of embryonic limb mesenchymal chondrogenesis: Cell adhesion and Wnt signaling
Chondrogenesis is one of the first overt differentiation events to occur in the developing limb bud. An increase in the limb core cell density, which allows for increased cell contact and the formation of gap junctions, is critical to chondrogenic development. N-Cadherin has been shown to play an important functional role in cell adhesion required for the formation of cartilage. Perturbation of N-cadherin function using N-cadherin neutralizing antibodies leads to inhibition of chondrogenesis in vivo and gross skeletal abnormalities in vivo. The goal of this thesis study is to further characterize the localization and function of N-cadherin during chondrogenesis, and to investigate the interactions between cadherin and cell signaling molecules known to be associated with N-cadherin both directly and indirectly. Among those molecules examined in this study are the N-cadherin-associated signaling molecule, β-cadherin, as well as the embyronic patterning molecules, Wnts, which, like N-cadherin, also mediate downstream signaling through β-catenin. To this end, we have utilized the chondrogenesis-stimulating polycation, Poly-L-Lysine (PL) to enhance chondrogenesis in limb mesenchymal cultures in vitro and to analyze the concomitant changes in the level and activity of N-cadherin. PL treatment of chick limb mesenchymal micromass culture increases N-cadherin mRNA expression resulting in a dose-dependent, spatio-temporal increase in N-cadherin protein level associated specifically within cells localized between chondrifying nodules. In addition, functional inhibition of N-cadherin with N-cadherin neutralizing antibodies significantly inhibits the effect of PL. The effect of PL on N-cadherin also extends to N-cadherin-mediated signaling mechanisms. Phosphorylation of β-catenin, a cell signaling molecule required for N-cadherin function, is decreased in PL treated cultures. This effect is consistent with the observation In many cell systems that decreased tyrosine-phosphorylation of β-catenin is linked to increased cell adhesion. Furthermore, these data illustrate the usefulness of PL as a modifying agent in the analysis of the cellular and molecular mechanisms of chondrogenesis. β-Catenin associates with several other proteins in the cell including adenomatous polyposis coli (APC), and the Wnt regulated transcription factor, LEF-1. Examination of the total β-catenin protein profile as well as the subcellular distribution of β-catenin in micromass culture has implicated a role for the Wnt pathway in chondrogenesis. Interestingly, treatment with lithium, a functional mimic of the Wnt signal, inhibits chondrogenesis and abrogates the effect of PL. Indeed, Wnt-3 and Wnt-7a, as well as other members of the Wnt signaling pathway, APC and glycogen synthase kinase-3β are normally expressed in chick limb mesenchymal micromass cultures. Expression of Wnt-3 is developmentally down-regulated over the course of chondrogenesis in chick limb mesenchymal cells, and is not affected by PL treatment. On the other hand, Wnt-7a expression is not altered during chondrogenesis, but is inhibited by PL treatment. These results implicate a chondro-inhibitory role for Writ signaling in chondrogenesis. In addition, these data demonstrate the functional importance of N-cadherin in PL-mediated stimulation of chondrogenesis. We speculate based on the evidence in this study that inhibition of relevant Wnt molecules by PL may play a role in the PL-induced increase in N-cadherin as well as chondrogenesis. (Abstract shortened by UMI.)
Woodward, Wendy Ann, "Regulation of embryonic limb mesenchymal chondrogenesis: Cell adhesion and Wnt signaling" (2000). ETD Collection for Thomas Jefferson University. AAI9965362.