Normal newt limb regeneration requires matrix metalloproteinase function

Abstract

Newts regenerate lost limbs through a complex process involving dedifferentiation, migration, proliferation, and redifferentiation of cells proximal to the amputation plane. To identify the genes controlling these cellular events, we performed a differential display analysis between regenerating and nonregenerating limbs from the newt Notophthalmus viridescens. This analysis, coupled with a direct cloning approach, identified a previously unknown Notophthalmus collagenase gene (nCol) and three known matrix metalloproteinase (MMP) genes, MMP3/10a, MMP3/10b, and MMP9, all of which are upregulated within hours of limb amputation. MMP3/10b exhibits the highest and most ubiquitous expression and appears to account for the majority of the proteolytic activity in the limb as measured by gel zymography. By testing purified recombinant MMP proteins against potential substrates, we show that nCol is a true collagenase, MMP9 is a gelatinase, MMP3/10a is a stromelysin, and MMP3/10b has an unusually broad substrate profile, acting both as a stromelysin and noncanonical collagenase. Exposure of regenerating limbs to the synthetic MMP inhibitor GM6001 produces either dwarfed, malformed limb regenerates or limb stumps with distal scars. These data suggest that MMPs are required for normal newt limb regeneration and that MMPs function, in part, to prevent scar formation during the regenerative process.

Macrophages are required for adult salamander limb regeneration

“…Inhibition of TGF-β1 signaling blocks successful limb regeneration in the salamander (25), and fibronectin forms part of the provisional matrix during scar-free repair (31) that may contribute to a regeneration permissive environment. The stunted activation of MMP9 and MMP3 on macrophage ablation is consistent with an essential role for matrix remodeling in successful limb regeneration (47) and implicates macrophages as a key regulator of matrix degrading enzymes. Other matrix components such as collagen were markedly altered in the absence of macrophages, leading to a fibrous cap.…”

Mechanisms of urodele limb regeneration

“…MMPs also prevent reassembly of a basement membrane, thereby ensuring contact between the wound epidermis and the underlying tissues. The importance of MMPs to histolysis is underscored by the failure of blastema formation in amputated newt limbs treated with the MMP inhibitor GM6001 (Vinarsky, Atkinson, Stevenson, Keating, & Odelberg, 2005). The wound epidermis and the AEC are major sources of MMPs (Godwin, Pinto, & Rosenthal, 2013) and also function to eliminate cellular and particulate debris generated by tissue destruction and the bactericidal activity of neutrophils and macrophages (Singer & Inoue, 1964; Singer & Salpeter, 1961).…”

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

“…Osteoclasts degrade bone matrix via hydrochloric acid, acid hydrolases, and MMPs. Up-regulated MMP transcripts include MMP-2 and À9 (gelatinases), and MMP-3/10a and b (stromelysins) (Grillo et al, 1968;Dresden and Gross, 1970;Yang and Bryant, 1994;Miyazaki et al, 1996;Ju and Kim, 1998;Yang et al, 1999;Park and Kim, 1999;Kato et al, 2003;Vinarsky et al, 2005). In the newt limb, the basal layer of the wound epidermis transcribes MMP3/ 10a and b, as well as a novel MMP with low homology to the others (Kato et al, 2003).…”

“…The regeneration-deficient limbs exhibit different enzyme levels and temporal patterns of enzyme expression than the regeneration-competent wild-type axolotl limb (Santosh et al, 2011), suggesting that these differences play a role in the abnormal histolysis and thus availability of cells for dedifferentiation noted in regeneration-deficient limbs (Wolfe et al, 2000). The importance of MMPs to regeneration was underscored by the failure of blastema formation in amputated newt limbs treated with the MMP inhibitor GM6001 (Vinarsky et al, 2005).…”