Expression of genes of type I and type II collagen in the formation and development of the blastema of regenerating newt limb

We cloned cDNAs of alpha 1(I) and alpha 1(II) collagen, and studied their e xpression profiles in regenerating limbs of newts, Cynops pyrrhogaster. The expression of the al(I) gene was markedly up-regulated at the early bud st age of the blastema. In situ hybridization experiments revealed that the al pha 1(I) gene was expressed in not only mesenchymal cells of the blastema, but also the basal cells of the wound epidermis at the wound healing stage when the epidermal basement membrane was absent. This unique expression con tinued until 21 days (late bud stage), while the basement membrane began to form at 14 days. These results indicate biochemical differences between th e wound and normal epidermis, and suggest the direct involvement of the for mer in the synthesis of blastemal matrices of type I collagen. Actually, im munohistochemistry revealed that type I collagen began to be deposited bene ath the wound epidermis at 8 days, and accumulated there and around blastem al mesenchymal cells at 14 to 21 days. Undifferentiated mesenchymal cells a ssociated with the amputated muscle fibers actively expressed the alpha 1(I ) gene. Mesenchymal cells in the central region of blastemas deposited type I collagen fibers around them. Concomitantly with the appearance of precho ndrocytes, the alpha l(II) collagen gene became activated. The present stud y clearly shows that the expression of the genes of both type I and type II collagen in blastemal cells is temporally and regionally well-regulated in a cooperative manner. Dev Dyn 1999;216:59-71. (C) 1999 Wiley-Liss, Inc.