Tufan A Cevik, Daumer Kathleen M, Tuan Rocky S
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Dev Dyn. 2002 Mar;223(2):241-53. doi: 10.1002/dvdy.10046.
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.
组织极性基因的卷曲蛋白（Frizzled）家族的产物已被确定为Wnt信号分子家族的假定受体。Wnt信号传导与肢体间充质软骨形成的调节有关，我们最近的研究表明，N-钙黏蛋白及相关活性在功能上参与了Wnt-7a介导的软骨形成抑制作用。通过使用鸡肢体间充质细胞的体外高密度微团培养系统，我们分析了与软骨形成调节有关的两个卷曲蛋白基因Chfz-1和Chfz-7的RCAS逆转录病毒介导的错误表达的时空表达模式及其对软骨形成的影响。在所有检测的时间点，Chfz-1的表达都定位于软骨结节周围区域，而Chfz-7的表达在初始间充质凝聚期间仅限于细胞聚集体，随后在软骨形成分化时从软骨结节的中心向周边下调，类似于N-钙黏蛋白的表达模式。Chfz-7错误表达时，体外软骨形成受到抑制并局限于培养物的较小区域，但不受Chfz-1错误表达的影响。细胞凝聚和软骨形成分化分析表明，Chfz-7的抑制作用不太可能发生在软骨形成分化步骤，而是影响更早的前软骨聚集体形成事件。在24小时时，与对照培养物相比，在Chfz-7错误表达的培养物中，软骨形成细胞凝聚阶段的关键成分N-钙黏蛋白的表达延迟/受到抑制，并且在48小时时局限于整个培养物中明显更小的细胞凝聚区域。在所有时间点，Chfz-1错误表达的培养物看起来与对照培养物相似。然而，Chfz-1和Chfz-7的错误表达均未影响体外间充质细胞增殖。这些结果表明，Chfz-7在肢体间充质软骨形成过程中对N-钙黏蛋白表达的调节中起作用，并且Chfz-1和Chfz-7参与不同的Wnt信号通路。
