Early events during avian skin appendage regeneration: dependence on epithelial-mesenchymal interaction and order of molecular reappearance

Abstract

Early molecular events during the development and regeneration of skin appendages were studied using cultured chicken skin explants with epithelial-mesenchymal recombination. The explant epithelium was separated from the mesenchyme, rotated 90 degrees or 180 degrees, recombined with the mesenchyme, and cultured. After this procedure, existing feather buds disappeared and new buds were regenerated. The location of the new buds is determined by the original dermal condensations, whereas the orientation is dictated by the original epithelium. The temporal expression of key morphogenetic molecules was examined 3, 6, and 20 h after recombination by whole-mount in situ hybridization and immunostaining. The results showed the following. (i) Placode formation and the expression of wingless-int (Wnt) 7a and Msx-1 in the placode epithelium are mesenchyme dependent. (ii) Hox C6 and neural cell adhesion molecule (NCAM) expression in the anterior mesenchyme is placode epithelium dependent. (iii) Bone morphogenetic protein (BMP)-2, BMP-4, and fibroblast growth factor (FGF)-4 expression in the original dermal condensations was unaffected by recombination. (iv) Old dermal condensations can induce new placodes with new Wnt 7a, sonic hedgehog (Shh), and Msx-1 and -2 expression. (v) The new placode epithelium can then induce new Hox C6 and NCAM microgradients in the feather bud mesenchyme. (vi) The order of appearance can be classified into four groups in the following order: BMP-2, BMP-4, and FGF-4 (peptide growth factors); Wnt 7a and Shh (Drosophila segment polarity gene homologs); Msx-1 and Msx-2 (Msx class homeobox genes); and then Hox C6 (Hox class homeobox genes) and NCAM (adhesion molecules). These results suggest an order for the molecular cascade during the inductive phase of skin appendage development.