Regeneration in Murphy Roths Large Mice

Introduction

Regeneration

Regeneration is the process of replacing damaged tissue with phenotypically and functionally identical tissue. Invertebrates and amphibians are the most proficient at regeneration in the animal kingdom, able to regenerate whole limbs and organs. Mammals do not display a great deal of proficiency at regeneration, with the exception of tissue in the liver.

For regeneration to take place there must be a source of highly proliferative cells able to be recruited to the wound site and differentiate into multiple cell types as the tissue regenerates. In invertebrate species this is accomplished by recruitment of multipotent stem cells spread throughout the body. Amphibians regenerate by de-differentiating local cells that reenter the cell cycle and divide rapidly, differentiating into their original identities as the tissue regenerates. Regeneration usually begins by the formation of an epithelial cap over the wound, which takes on a signaling role as proliferative cells gather ‘under’ the epithelial cap and begin dividing.

Characteristics of regenerative tissue

In mammalian, amphibian, and invertebrate tissues capable of regeneration, a high percentage of cells are seen arrested in the G2/M phase. Another common feature of regeneration capable tissue is heightened levels of DNA damage. As cells are constantly proliferating, mistakes in DNA synthesis accumulate and require heightened DNA repair mechanisms or rates of apoptosis to ensure the cells do not become cancerous. To effectively maintain the genetic health of cell populations, homologous recombination of damaged DNA would have to be utilized to repair DNA without mistakes. As homologous recombination requires sister chromatids to use as a template, this is a possible explanation for the high number of tetraploid (G2 phase) cells in regenerating tissue, as these cells have sister chromatids available for use as a template.

Mammalian tissue regeneration via cell cycle reentry.

The MRL mouse

The Murphy Roths Large (MRL) strain of Mus musculus garnered attention due to its ability to completely regenerate ear hole punches, including lost cartilage.[2] They have been shown to regenerate damaged areas of the heart with little scarring, and recovery of myocardial function.[3] Mice are known to partially regenerate amputated digits, however, the MRL mouse displays a markedly increased capacity that mimics that of amphibians, although it is not capable of the level of limb regeneration seen in amphibians, forming a blastema-like structure not found in any other mouse strain.[4] In another study, MRL mice were able to heal skin grafts more rapidly and with less scarring than B10.BR mice (haplotypically identical to MRL mice), in part due to their increased ability to recruit stem cells to the wound area.[5] MRL mice are prone to autoimmune conditions, although it is non known whether this is connected to their regenerative capacity.

Findings of  Heber-Katz et al.

Mouse Strains Used

There were several mouse strains used in the study. MRL mice, along with LG/J mice, a MRL ancestor strain that shares the regenerative ability of MRL mice, were used as the model regenerative organisms (healers). B6 and SM/J mice were used as controls (nonhealers). Additionally, B6129SF2/J mice, both wild type (WT) and a p21 knockout (p21-/-) were used to examine whether the phenotypical differences between healer and nonhealer mice were due to deletion of the gene that codes for p21.

Mechanism Under Investigation

Due to the high number of cells in the G2/M phasem a G1 checkpoint deficiency was hypothesized as a possible cause of the regenerative phenotype. If cells were not properly stopped from entering G2 at the G1 phase, then they would not undergo quiescence. The presence of a notable G1 checkpoint protein, the p53-dependent p21 (a cyclin-dependent kinase inhibitor), was tested, due to the fact that absence of p21 is commonly detected in embryonic stem cells.[6] If a defect or deletion of the gene responsible for coding p21 was the cause of the regenerative phenotype, then deletion of the p21 gene would potentially cause a regenerative phenotype in nonhealer mice.

p21 is recruited by p53 to arrest the cell in G1, inhibiting apoptosis as a result.

Results

Cells from healer and nonhealer strains were fluorescently labeled with propidium idodie (which binds to DNA with little sequence preference) and the level of fluorescence the cells exhibited measured with flow cytometry to gain a quantitative analysis of the DNA levels in the cell. This was used to determine the number of cells arrested in the G2/M phase. Both healer strains and p21-/- mice displayed an increased number of cells arrested in the G2/M phase, compared to nonhealer and p21+/+ mice, respectively.

Fig.1. Cell cycle analysis of nonhealer and healer ear-derived cells. A secondary peak indicates the relative number of cells in the G2/M phase.

To determine the levels of DNA damage, the phosphorylation of HA2X, a variant of the histone protein H2 that becomes phosphoroylated on its serine 129 residue in response to double strand breaks, was measured using immunofluorescence.  Healer cells displayed elevated levels of HA2X phosphorylation compared to nonhealer mice, indicating that healer mice experience high levels of DNA damage likely caused by increased replication stress.

Fig. 3. Detection of γH2AX and TopBP1 foci in cells and normal tissue. (A) Number of irridiated (black) and non-irradiated (white) dermal cells from healer and non-healer mice staining positively for γH2AX. (B) Ear and small intestine cells from healer and non-healer mice stained for γH2AX. (C) Western blot detection of γH2AX. (D) Number of irridiated (black) and non-irradiated (white) dermal cells from healer and non-healer mice staining positively for TopBP1, an enzyme involved in breakage of DNA strands.

To determine the levels of endogenous DNA damage, a Single Cell Gel Electrophoresis assay (also known as a comet assay) was performed on cells from healer and nonhealer mice. The higher the number of double strand breaks, the larger the ‘tail’ of the comet would be. Healer cells displayed a much greater number of double strand breaks than nonhealer cells. To test whether this DNA damage was being repaired in healer cells, the number of Rad51 foci, indicative of homologous recombination, was determined. Healer cells displayed greatly increased Rad51 foci relative to nonhealer cells. As homologous recombination may not always be sufficient for controlling DNA damage in tissue, markers of apoptosis were tested for. A TUNEL assay, which measures the amount of terminal DNA fragments by quantifying the levels of dUTP able to bind to the ends of the fragments, showed increased levels of DNA fragmentation. Western blot showed increased levels of caspace 3, a protein critical to apoptosis, in cells from healer mice.

Fig. 4. (A) Comet assay shows increased levels of DNA damage in MRL mice (c and f are after irraqdiation. (B) Increased levels of Rad 51 foci in MRL mice indicate heightened repair of DNA damage. (C) Higher levels of Caspase-3, as detected by Western blot and histological immunostaining, indicate a higher number of cells enter apoptosis in healer mice. (D) Increased amount of fragmented DNA detected by TUNEL assay support heightened levels of apoptosis in healer mice. (E) Western blotting fails to detect p21 in MRL mice even after irradiation to induce DNA damage.

To test whether the regenerative capabilities of the MRL mouse are due to p21 deletion, the results of a through and through ear puncture test of the p21 knockout (p21-/-) were compared to the WT control. If the ear tissue is non-regenerating, as in nonhealer mice, the tissue will scar around the hole formed by the punch and the hole will remain. In regenerating tissue, seen in healer mice, a blastema will form around the edge of the hole and cells will proliferate until the hole is closed. The p21 knockout mice displayed the ability to regeneratively heal the ear punches almost as efficiently as MRL mice, while the WT control was unable to close the hole.

Fig. 5. P21 knockout mouse ear hole closure. (A) Mean hole diameters of healer and nonhealer mice. Bars represent standard deviation. (B-C) Ear hole closure of p21 knockout and wild type mice after 5 days. (D-E) After 35 days. (F–H) Histological analysis shows p21−/− ear hole tissue on day 35. (F) Ear tissue extending from the cut cartilage. (G) Normal blastema-like tissue and (H) condensations extending from the cut cartilage can be seen. (I–L) Analysis of cells from the ear pinnae of p21+/+ mice and p21−/− mice for (I and J) γH2AX staining (DAPI = blue; anti-γH2AX = red), (K and L) comets. (M) Precentage of cells displaying γH2AX staining or comets (N) Cell cycle analysis (using propidium iodide) of p21−/− cells in red and p21+/+ cells in blue. (O) Model describing the links between cell cycle checkpoint control, proliferation, and the tissue regeneration phenotype. After tissue injury such as an ear hole punch, hyperproliferation ensues. In WT or p21-proficient cells, a G1 checkpoint is enacted leading to scar formation. In p21-defective cells such as those derived from the MRL strain and p21−/− mice, rounds of unscheduled DNA synthesis can occur, leading to DNA damage and an abundance of DNA damage markers. A G2 checkpoint can be enacted, leading to enhanced tissue regeneration.

Strengths and Weaknesses

This study is notable not only for determining a mechanisms behind mammalian tissue regeneration, but also for showing that it can be induced by modification of a single gene. While the study replicated the experiments done to determine DNA damage and cell cycle arrest, the mechanisms used to control DNA damage were not tested for in the knockout mice. It is therefore unknown whether deletion of p21 is able to replicate the increased homologous recombination and apoptosis seen in MRL mice. This is notable, as p21-/- mice were found in other studies to be defective in homologous recombination[7] and experience an elevated risk of tumorogensis.[8]

Additional information:

  1. Regeneration in Hydra: http://www.devbio.biology.gatech.edu/?page_id=683
  2. Regeneration in Planaria: http://www.devbio.biology.gatech.edu/?page_id=2526
  3. Regeneration in Axolotls: http://www.devbio.biology.gatech.edu/?page_id=2685
  4. Regeneration in Zebrafish: http://www.devbio.biology.gatech.edu/?page_id=6534
  5. Overview of regeneration in animals: http://www.hhmi.org/biointeractive/tissue-regeneration-animals
  6. Further reading on regeneration: http://www.us.elsevierhealth.com/media/us/samplechapters/9781437707922/Chapter%2003.pdf

References:

  1. Bedelbaeva K, Snyder A, Gourevitch D, et al. Lack of p21 expression links cell cycle control and appendage regeneration in mice. Proc Natl Acad Sci USA. 2010;107(13):5845-50. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2851923/
  2. Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB. Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthr Cartil. 2008;16(11):1319-26. http://www.ncbi.nlm.nih.gov/pubmed/18455447
  3. Leferovich JM, Bedelbaeva K, Samulewicz S, et al. Heart regeneration in adult MRL mice. Proc Natl Acad Sci USA. 2001;98(17):9830-5. http://www.pnas.org/content/98/17/9830.full
  4. Gourevitch DL, Clark L, Bedelbaeva K, Leferovich J, Heber-katz E. Dynamic changes after murine digit amputation: the MRL mouse digit shows waves of tissue remodeling, growth, and apoptosis. Wound Repair Regen. 2009;17(3):447-55. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645464/
  5. Tolba RH, Schildberg FA, Decker D, et al. Mechanisms of improved wound healing in Murphy Roths Large (MRL) mice after skin transplantation. Wound Repair Regen. 2010;18(6):662-70. http://onlinelibrary.wiley.com/doi/10.1111/j.1524-475X.2010.00631.x/pdf
  6. Dolezalova D, Mraz M, Barta T, et al. MicroRNAs regulate p21(Waf1/Cip1) protein expression and the DNA damage response in human embryonic stem cells. Stem Cells. 2012;30(7):1362-72. https://www.ncbi.nlm.nih.gov/pubmed/22511267
  7. Mauro M, Rego MA, Boisvert RA, et al. p21 promotes error-free replication-coupled DNA double-strand break repair. Nucleic Acids Res. 2012;40(17):8348-60. http://nar.oxfordjournals.org/content/early/2012/06/25/nar.gks612.full
  8. Gartel AL, Tyner AL. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther. 2002;1(8):639-49. http://mct.aacrjournals.org/content/1/8/639.abstract

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