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In a previous study, we showed that the proline-rich divergent homeobox gene Hex/Prh is expressed in dorsal skin of the chick embryo before and during feather bud development and that the pattern of Hex mRNA expression in the epidermis is similar to that of Wnt7a mRNA. In order to study the function of Hex and the relationship between Hex and Wnt7a in feather bud development, sense and/or antisense sequences of Hex or Wnt7a were ectopically and transiently expressed in the dorsal skin with the epidermal side toward the cathode by electroporation at the placode stage and then the skin was cultured. Increased expression of Wnt7a and beta-catenin mRNA was observed in the same region where Hex-EGFP fusion protein was expressed 2 days after culture, which was followed by extra bud formation a few days later as a result of the stimulation of cell proliferation. Concomitantly, expression of Notch1 mRNA, which is expressed in normal bud development, increased in Hex-overexpressing skin. However, ectopic Wnt7a expression induced neither Hex expression nor extra bud formation in normal skin. Antisense Wnt7a specifically inhibited bud initiation in Hex-overexpressing skin but did not in normal skin. Taken together, these results suggest that Hex is upstream of Wnt7a and beta-catenin and regulates the Wnt signaling pathway in feather bud initiation and that some other Wnt signals in addition to Wnt7a may be required for bud initiation.

Involvement of Hex in the initiation of feather morphogenesis