Frizzled-7 and limb mesenchymal chondrogenesis: effect of misexpression and involvement of N-cadherin.
Products of the Frizzled family of tissue polarity genes have been identified as putative receptors for the Wnt family of signaling molecules. Wnt-signaling is implicated in the regulation of limb mesenchymal chondrogenesis, and our recent study indicates that N-cadherin and related activities are functionally involved in Wnt-7a-mediated inhibition of chondrogenesis. By using an in vitro high-density micromass culture system of chick limb mesenchymal cells, we have analyzed the spatiotemporal expression patterns and the effects on chondrogenesis of RCAS retroviral-mediated misexpression of Chfz-1 and Chfz-7, two Frizzled genes implicated in chondrogenic regulation. Chfz-1 expression was localized at areas surrounding the cartilaginous nodules at all time points examined, whereas Chfz-7 expression was limited to cellular aggregates during initial mesenchymal condensation, and subsequently was down-regulated from the centers toward the periphery of cartilage nodules at the time of chondrogenic differentiation, resembling the pattern of N-cadherin expression. Chondrogenesis in vitro was inhibited and limited to a smaller area of the culture upon misexpression of Chfz-7, but not affected by Chfz-1 misexpression. Analyses of cellular condensation and chondrogenic differentiation showed that the inhibitory action of Chfz-7 is unlikely to be at the chondrogenic differentiation step, but instead affects the earlier precartilage aggregate formation event. At 24 hr, expression of N-cadherin, a key component of the cellular condensation phase of chondrogenesis, was delayed/suppressed in Chfz-7 misexpressing cultures, and was limited to a significantly smaller cellular condensation area within the entire culture at 48 hr, when compared with control cultures. Chfz-1 misexpressing cultures appeared similar to control cultures at all time points. However, neither Chfz-1 nor Chfz-7 misexpression affected mesenchymal cell proliferation in vitro. These results suggest that Chfz-7 is active in regulating N-cadherin expression during the process of limb mesenchymal chondrogenesis and that Chfz-1 and Chfz-7 are involved in different Wnt-signaling pathways.
Tufan
A Cevik
AC
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Daumer
Kathleen M
KM
Tuan
Rocky S
RS
D000818:Animals; D030161:Avian Proteins; D015820:Cadherins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured / Q000378:metabolism; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology*; D024043:Collagen Type II / Q000096:biosynthesis; D005121:Extremities / Q000196:embryology*; D051157:Frizzled Receptors; D018507:Gene Expression Regulation, Developmental; D008648:Mesoderm / Q000378:metabolism*; D009924:Organ Culture Techniques; D011506:Proteins / Q000502:physiology; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection; D051153:Wnt Proteins
D030161:Avian Proteins; D015820:Cadherins; D024043:Collagen Type II; C489740:FZD1 protein, human; D051157:Frizzled Receptors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D017981:Receptors, Neurotransmitter; D011993:Recombinant Fusion Proteins; C490509:WNT7A protein, Gallus gallus; D051153:Wnt Proteins; C490507:Wnt7a protein, rat