Regulation of p53 is critical for vertebrate limb regeneration

Abstract

Extensive regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander limb regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during limb regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during regeneration.

Keywords: carcinogenesis; chondrogenesis; myogenesis; p73.

Fig. 1.

p53 activity is regulated during limb regeneration. (A) Representative longitudinal section of a blastema at 15 dpa or its corresponding limb, stained with antibodies against axolotl p53 and Hoechst 33258. (B) Schematic representation of the p53 luciferase reporter assay. (C) Luciferase activity assay in axolotl AL1 cells 48 h after transfection with the indicated constructs. Luciferase level was normalized to the level of Renilla luciferase, and expressed in relation to the pGL3 control vector. (D) Luciferase activity assay at different stages of limb regeneration. p53Luc was electroporated alongside a Renilla luciferase control vector into both limbs of an axolotl (0 dpa, p53Luc+axop53), or into a blastema and its contralateral limb (10, 15, 18, 25, 35 dpa; SV40, mLuc). Luciferase activity was normalized to the level of Renilla luciferase and expressed relative to the activity of a single intact limb. SV40 is a luciferase expression vector without p53 binding sites (**P < 0.01). (E) Representative longitudinal section of a blastema or limb following electroporation of pRFP-N2 and staining with antibodies against axolotl p53 and Hoechst 33258. (F) qRT-PCR analysis of Gadd45 levels in blastemas, normalized to Ef1-α or L-27, 72 h after electroporation with the indicated vectors. (G) qRT-PCR analysis of Gadd45, Mdm2, and p53 expression levels in blastemas at different stages of regeneration relative to a normal limb, normalized to those of Ef1-α. Similar results were obtained normalizing to L27. (H) Representative images of blastemal (I), muscle (II), epidermal (III), and cartilage (IV) cells stained with antibodies against axolotl Gadd45. Values are given as means ± SEM, n = 6 in C and F; n = 8 in D and G. Bl, blastema mesenchyme; C. cartilage E, epidermis; M, muscle; we, wound epidermis. (Magnification: A, 20×; E, 40×; H, I and II, 40×; H, III and IV, 60×.)

Fig. 2.

Regulation of p53 activity is necessary for limb regeneration. Schematic representation of axolotl treatment programs with nutlin3a (A and G) or α-pifithrin (D and J) during the blastema formation or redifferentiation phases of limb regeneration. Dotted arrow indicates the duration of each treatment and how they affect p53 activity. (B, E, H, and K) Representative images of blastemas 10 d after the treatment indicated in A, D, G, and J, and the equivalent experiment using vehicle alone (control). (C, F, I, and L) The number of axolotls in each stage of regeneration at the end of the corresponding treatment as indicated in A, D, G, and J.

Fig. 3.

p53 activity is essential for myogenesis. (A) Representative image of A1 cells before and 4 d after inducing myotube formation, stained with antibodies against axolotl p53 (red) and Hoechst 33258 (blue). (B) Luciferase activity assay in A1 myotubes (4 d postinduction) relative to that of uninduced A1 cells. A1 cells were transfected with p53Luc and Renilla luciferase. Firefly luciferase level was normalized to the level of Renilla luciferase and expressed as the ratio (A1 myotubes/A1 control), n = 10. (C, Upper) phase contrast micrograph of A1 cells 1 and 4 d after induction of myogenesis. Cells were induced in the presence of 1 µM nutlin3a or 10 µM α-pifithrin. (Lower) Quantification of myotube formation (number of nuclei within myotubes relative to total nuclei expressed as a percentage) in A1 cells were incubated for 4 d in 0.25% FCS in the presence of the indicated doses of nutlin3a or α-pifithrin. (**P < 0.01, ***P < 0.001). (D) Quantification of BrdU⁺ cells at 3 d after myogenesis induction in the presence of 1 µM nutlin3a, 10 µM α-pifithrin, or DMSO. Values represent the mean ± SEM, n = 6 (C and D).

Fig. 4.

Down-regulation of p53 levels is required for postmitotic cell cycle reentry. (A) In situ hybridization analysis of Gadd45 mRNA expression in A1 cells and myotubes following the indicated treatments. (B) Percentage of myotubes entering S-phase as measured by BrdU incorporation. Myotubes were induced under normal conditions and incubated for 2.5 d in 10% FCS in the presence of DMSO, 1 µM nutlin3a, or 10 µM α-pifithrin (**P < 0.01). (C) Quantification of p-RB^S807/811-positive cells. Myotubes were incubated in 0.25% FCS or 10% FCS with 1 µM nutlin3a or vehicle, then fixed and stained as described. (D) Western blot analysis of myotube extracts—including mononucleates—indicating levels of p-RB^S807/811 and β-actin. Values from B and C represent the mean ± SEM, n = 6.

Fig. 5.

ΔNp73 acts as a p53 dominant negative and is up-regulated during blastema formation. (A) qRT-PCR analysis of axolotl ΔNp73 or full-length p73 in several tissues relative to their levels in normal limbs, normalized to Ef1-α. (B) Western blot analysis of myc-tagged proteins and β-actin in AL1 cell extracts 48 h posttransfection with axolotl ΔNp73-myc or p53-myc constructs. (C) Luciferase activity assay in AL1 cells 48 h posttransfection with the indicated vectors, normalized to the level of Renilla luciferase and expressed relative to the activity of pGL3 control vector. (D) qRT-PCR analysis of axolotl ΔNp73 and Gadd45 expression levels in regenerating blastemas relative to a normal limb, normalized to those of Ef1-α. (E) Regenerated limbs, 3.5 wk after coelectroporation of pair-matched mid-bud blastemas with either N2-nGFP or ΔNp73 alongside N2-RFP. (F) Quantification of limbs exhibiting normal or delayed regeneration 3.5 wk following electroporation of the indicated constructs. (G) Representative longitudinal sections of regenerated limbs. Note characteristic cell foci in cartilage of ΔNp73-expressing cells. (Magnification: 30×.) (H) Percentage of electroporated cells incorporated into muscle, connective tissue and cartilage following electroporation as indicated in F. Values represent the mean ± SEM, n = 4 in A–C; n = 6 in D and H (*P < 0.05, **P < 0.01). E, epidermis; C cartilage; CT, connective tissue; M, muscle.