Comparative analysis of the expression and regulation of Wnt5a, Fz4, and Frzb1 during digit formation and in micromass cultures

Abstract

Previous studies have shown that three members of the Wnt signaling pathway, the ligand WNT5A, the receptor FZ4, and the Wnt antagonist FRZB1, are implicated in the formation and differentiation of the digits. In this study, we have attempted to establish a functional correlation between them by comparing their expression patterns and their regulation by the signals controlling proliferation and differentiation of the limb mesoderm during formation of the avian digits in vivo and in micromass cultures. In vivo Wnt5a and Fz4 are expressed in the undifferentiated mesoderm of the autopod and in the differentiating digit cartilages. In the undifferentiated mesoderm, the expression of both genes is regulated positively by FGFs and negatively by bone morphogenetic proteins (BMPs). As chondrogenic differentiation starts, Fz4 becomes intensely up-regulated in the aggregate and in the developing perichondrium, whereas transcripts of Wnt5a are excluded from the core of the aggregate but maintained in the surrounding mesenchyme and perichondrium. In addition, at this stage, the expression of both genes become positively regulated by BMPs. These changes in expression and regulation are coincident with the induction of Frzb1 in the chondrogenic aggregate, which is expressed under the positive control of BMPs. Our findings fit with a role of Wnt5a/Fz4 negatively regulating in vivo the initiation and progression of cartilage differentiation. In vitro, only Frzb1 is expressed and regulated in a manner resembling that observed in vivo. Wnt5a and Fz4 are both expressed in the differentiating mesenchyme of micromass cultures, but their expression is not significantly regulated by the addition of FGF-2 or BMP-7 to the culture medium.