Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface

Abstract

The influence of the wound epithelium on the cellular events preceding blastema formation was examined by comparing dedifferentiation, DNA labeling indices, and mitotic indices of the distal mesodermal tissues in control regenerating newt forelimbs and in amputated forelimbs covered with a flap of full thickness skin. Three kinds of results were seen following the skin-flap graft operations. Epidermal migration across the amputation surface was completely inhibited in 22% (8) of the cases and these limbs repaired the amputation wound but did not form regeneration blastemas. In 11% (4) of the experimental limbs, essentially normal wound epithelia displaced the skin flaps and the limb stumps formed blastemas and regenerated. The majority of the skin grafts (67%) exhibited epidermal migration restricted to the free edges of the flaps. These limbs formed eccentric blastemas on the ventral side of the limb next to the dermis-free epidermis and regenerated laterally in that direction.

Mechanisms of urodele limb regeneration

“…Interestingly, between the twin blastemas a small area of basement membrane was reconstituted, but not under the blastema AEC, suggesting differences in MMP expression between these two regions. RA‐treated double posterior limbs did not form a blastema, similar to the results of grafting full‐thickness skin over limb stumps (Chew & Cameron, 1983; Mescher, 1976; Tassava & Garling, 1979) or providing only skin from one quadrant of the limb to provide blastema cells (Lheureux, 1975). A basement membrane and thick mat of connective tissue was quickly formed under the wound epidermis (Kim & Stocum, 1986b) (Fig.…”

“…Transplanting whole skin over the amputation surface (Chew & Cameron, 1983; Mescher, 1976), or inserting the ends of amputated limbs or regenerates into a pocket made under flank skin (Butler, 1955; Polezhaev & Faworina, 1935) or into the coelom (Deck, 1955; Goss, 1956a, b) results in lack of blastema formation. Thornton (1954) noted that the AEC is always present during blastema growth and patterning and fails to form in anuran late tadpole limb buds that have lost the ability to regenerate (Thornton, 1956).…”

“…Labeling of amputated limbs deprived of nerves or wound epidermis with [3H]‐thymidine showed that DNA synthesis of nascent blastema cells is independent of both these tissues, but that in the absence of either one the labeled cells were arrested in G 2 of the cell cycle (Kelly & Tassava, 1973; Mescher, 1976; Tassava et al., 1974). The cells can be rescued by re‐innervation (Olsen, Barger, & Tassava, 1984), but otherwise undergo apoptosis and are removed by macrophages (Mescher et al., 2000; Yun et al., 2015).…”

Looking proximally and distally: 100 years of limb regeneration and beyond

“…An important function of the MMPs encoded by the basal layer of the wound epidermis is thought to be the prevention of basement membrane reassembly beneath it, thus maintaining communication between the wound epidermis and subjacent blastema cells. Loss of such communication, either by removing the wound epidermis (Stocum and Dearlove, 1972;Mescher, 1976) or conditions under which a pad of connective tissue becomes prematurely interposed between wound epidermis and blastema cells (Stocum and Crawford, 1987), inhibits regeneration. These MMPs might also diffuse into the underlying tissues to participate in the degradation of other ECM components.…”

“…Blastema formation and growth are also inhibited in the absence of the AEC (Goss 1956a, b;Mescher, 1976;Tassava and Garling, 1979;Loyd and Tassava, 1980). Well-established blastemas of larval A. maculatum denuded of wound epidermis and grafted into dorsal fin tunnels (where they would presumably become innervated by axons of the fin connective tissue) form skeletal elements that are subnormal in size, suggesting that cell proliferation is reduced or halted in the absence of the AEC (Stocum and Dearlove, 1972).…”