Epimorphic regeneration in mice is p53-independent

Abstract

The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M "arrest," showed that p21(Cip1/Waf1) was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53(-/-) mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21(-/-) mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.

Figure 1

Mice were ear-punched using a 2 mm hole punch and hole diameter was read 2 and 4 weeks post-injury. Red columns = female mice, the blue columns = male mice; error bars show standard deviations. (A) MRL/MpJ mice were bred to p53^−/− mice and IC1 intercrosses were tested as WT, heterozygous and homozygous null for the p53 allele. (B) B6.p53^−/− mice were bred from B6.p53^+/− litters and WT, heterozygous and homozygous p53 null mice were examined.

Figure 2

Preliminary analysis of mutant mice derived from MMHCC for regeneration capability. The ear pinnae of mice (n = 2 to 5) were wounded by hole punching and followed for 6 weeks. Healer (regeneration-competent) controls (MRL/MpJ and p21^−/−) and non-healer (regeneration-incompetent) controls (B6, FVB) are included in this study. The experimental mice tested include GADD45^−/−, p16^−/− and tgfβ1, rag2^−/−.

Figure 3

Histological analysis of ear sections from ear-punched mice 42 days post-injury. Ears were fixed and embedded,and sections through the hole were stained with Alcian blue (cartilage). In (A) sections at low magnification (4X) show the degree of healing with hatched lines indicating the likely original hole cut. Above, there is a marker showing 2 mm (the original size of the hole). In (B) there are higher magnifications (20X) of the selected healing/regenerating ear tissue showing sections from (a) an MRL/MpJ mouse, (b) 3 different MRL.p53^−/− mice, (c) a TgfB1^−/−Rag2^−/− mouse and (d) a p21^−/− mouse. Arrows indicate adipocytes (red), chondrocytes (blue-green) and hair follicles (black).