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6068_02_p3-9 3/27/06 2:58 PM Page 3 REJUVENATION RESEARCH Volume 9, Number 1, 2006 © Mary Ann Liebert, Inc. Conjecture: Can Continuous Regeneration Lead to Immortality? Studies in the MRL Mouse ELLEN HEBER-KATZ, JOHN LEFEROVICH, KHAMILIA BEDELBAEVA, DMITRI GOUREVITCH, and LISE CLARK ABSTRACT A particular mouse strain, the MRL mouse, has been shown to have unique healing properties that show normal replacement of tissue without scarring. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivaling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. We propose this mouse as a model for continuous regeneration with possible life-extending properties. We will use the classical “immortal” organism, the hydra, for comparison and examine those key phenotypes that contribute to their immortality as they are expressed in the MRL mouse versus control mouse strains. The phenotypes to be examined include the rate of proliferation and the rate of cell death, which leads to a continual turnover in cells without an increase in mass. mouse has a profound capacity for regeneration in some ways rivaling the classic newt and axolotl species raises the possibility that humans, too, may have the capacity to regenerate. The authors propose this mouse as a model for continuous regeneration of tissue with or without intentional injury and with possible life-extending properties. This paper compares several properties of this mouse to those of the classical “immortal” organism, the hydra. INTRODUCTION I there could be no life. If everything regenerated there would be no death. All organisms exist between these two extremes. Other things being equal, they tend toward the latter end of the spectrum, never quite achieving immortality because this would be incompatible with reproduction. F THERE WERE NO REGENERATION —Richard J. Goss (1969)1 THE HYDRA The hydra is a multicellular organism with a very simple body plan. It is a member of the Cnidaria, which arose early in metazoan evolution. It has two cell layers, the endoderm and the ectoderm (which is made up of epithelial cells interspersed with interstitial cells); these The MRL mouse has been shown to have unique healing properties that show epimorphic regeneration with the formation of a blastema and the replacement of tissue with normal architecture and little or no scar formation. The serendipitous discovery that the MRL The Wistar Institute, Philadelphia, Pennsylvania. 3 6068_02_p3-9 3/27/06 2:58 PM Page 4 4 two layers are separated by a basement membrane.2 The epithelial cells have been shown to have a cell cycle time of approximately 3 days and are continuously cycling so that the tissue mass of the animal doubles every 3 to 4 days. These cycling cells are considered to be stem cells. However, the hydra does not change in size because there is a dynamic and equivalent gain and loss of cells.3 The cells that are lost are either sloughed off from the appendages such as the head, tentacles, and the foot and make up around 20% of the tissue, or are lost through their incorporation into newly forming buds4 making up 80% of the lost cell mass, as can been seen in Figure 1. Therefore, it is obvious that “old” cells do not persist for any length of time in the hydra but are constantly being replaced. The cells that migrate to the end of the appendages have already differentiated; these are the cells that are sloughed off. Thus, the hydra has been considered to be an immortal organism. However, until recently, there were little data to prove this claim. Experimental data on Hydra vulgaris were collected over a 4-year period and support this contention.5 Martinez essentially examined FIG. 1. Cross section of a hydra. The two layers of epithelial cells are seen though the cells of the interstitial cell lineage are not included. The arrows indicate the movement of cells to the extremities where they are sloughed off. Adapted from Ref. 2. HEBER-KATZ ET AL. senescence, an increase in age-specific mortality with increasing age, or lack thereof by determining mortality rates and reproductive rates. He raised the hydra individually and followed multiple animals to determine both mortality and budding rates over 4 years. During this time period, there was no increase in agespecific mortality. This was compared with drosophila, annelids, and other organisms, which clearly show changes over this period of time. Also, the budding rate showed little or no decline. Martinez calculated that over the 4 years of observation, the epithelial cells divided an average of 300 times and the whole body was replaced approximately 60 times. The hydra also has a high capacity for cell regeneration and re-aggregation after amputation, being cut into very small pieces, or separation into cells.6–9 Also, budding in the fully grown hydra could be considered an example of the regenerative response. Hydra is not the only organism that does this. Planaria as well have been shown to have similar characteristics, with stem cells that have rapid tissue dynamics, and displays a long or immortal life span. The stem cell population, or neoblasts, make up 30% of the cellular content of this flatworm and 99% of those neoblasts proliferate within 3 days, similar to the hydra stem cell populations.10 It has been shown also that at least during the regenerative response, the level of apoptosis is quite high.11 It has been suggested that planaria are also “almost . . . immortal.”10,12 This paper uses the hydra, which is a regenerating organism, as a model of continuous regeneration leading to immortality, which has been previously proposed by others. It is proposed here that the key to this, again as noted, is the cell dynamics in the organism, which include high levels of proliferation and cell death. The authors propose that these characteristics are also important in mammalian regeneration. A regenerating mouse model, the MRL mouse, is examined to test this proposal. THE MRL MOUSE RESPONSE TO INJURY The MRL mouse13 has been shown to respond to injury by a regenerative process that replaces 3/27/06 2:58 PM Page 5 5 IMMORTALITY AND CONTINUOUS REGENERATION injured tissue with new tissue and with the growth and differentiation of new structures. This was first shown in the unique closure of ear holes and the regrowth of cartilage in the ear pinna.14 Although this is not seen in mice other than this mouse strain and its parental line, the LG/J mouse strain, hole closure has been reported in both rabbit ears and bat wings.15,16 The MRL mouse has also been shown to heal cryoinjuries to the right ventricle of the heart through cell proliferation, little scar formation, and functional recovery.17 Recently, it has been shown also that MRL digit amputation in either of the two phalanges, shows both cellular proliferation and differentiation through chondrogenesis and osteogenesis. This response is distinct from other mouse studies examining digit tip regeneration through the nail bed of the claw,18–20 a response described in humans as well.21,22 The MRL-type healing has been shown to involve increases in amounts and length of time expressed in matrix metalloproteinases (MMP), especially MMP-2 and MMP-9,23 a response also shown to be important in both vertebrate and nonvertebrate regeneration.24–28 A key event in MRL healing is the early breakdown of the basement membrane leading to epithelial–mesenchymal interactions and blastema formation,22 again shown to be significant in the limb regeneration seen in amphibians.29–33 Inhibition of the ear hole closure response in the MRL has been seen with the use of minocycline, a molecule known to block MMPs.34 Also, reduced levels of MMPs in wounds in the MRL have been shown to be consistent with reduced regenerative responses, as noted in cortical brain stab injuries in the MRL mouse.35 Here, early elevated MMP responses accompanied significant differences in healing responses between the MRL and the control but reduction in MMP levels over time accompanied more scar formation in the MRL. Experiments in the authors’ and other laboratories have examined genetic loci36–39 that control ear hole closure in these mice using microsatellite mapping and F2 and backcross generation. The results have indicated that ear hole closure is indeed a complex and sexually dimorphic trait. Gene expression studies also have revealed significant candidate genes, in- cluding Pref-1 or dlk-1,40 collagen type I,17 MMP-2,23 and vimentin and keratin.41 The unique type of healing seen in this mouse provides an opportunity to test the hydra-derived theory of steady-state cell turnover with increased proliferation and elimination of cells and enhanced longevity. What is significant in the MRL? For a proliferative response, examine the incorporation of the thymidine analogue, bromodeoxyuridine (BrdU), which is incorporated into DNA and then detected using specific antibody. Are there more BrdU cells after BrdU pulsing in MRL mouse tissue compared with control mouse tissue? Is this true for both uninjured as well as injured tissue? For cell elimination or cell death, the tunnel assay is used to detect DNA fragmentation or examine the loss of given cell types in tissue using specific antibody. Again, is there is a difference in uninjured versus injured tissue? Either C57BL/6 or Swiss Webster mice are used for controls. Both proliferation and cell death are topics of interest in the MRL mouse because the MRL.lpr/lpr mouse has a mutation in the fas gene, a gene involved in cell death and shown to be caused by a retrotransposon insertion into the second intron of the fas gene,42,43 leading to an absence of cell death. Thus, as the MRL.lpr/lpr mouse ages, lymphocytes in lymphoid organs display unregulated lymphoproliferation. However, it must be pointed out that 8 % BrdU-positive cardiomyocytes 6068_02_p3-9 7 6 5 4 3 2 1 0 1 2 3 4 5 6 FIG. 2. BrdU labeling of cardiomyocytes. Mice were given BrdU in their drinking water over 30 days. Crosssections of hearts were stained with antibody to BrdU and DAPI and positive cardiomyocytes were identified, counted, and compared with the total number of cardiomyocytes from sequential H&E sections. 3/27/06 2:58 PM Page 6 6 HEBER-KATZ ET AL. FIG. 3. Tunnel assays on cardiac tissue. Tunnel assays were carried out on mouse heart tissue using CardioTACS (Trevigen, Inc., Gaithersburg, MD). Normal tissue from MRL (A) and B6 (B) show TdT-directed HRP-nucleotide additions, which stain the nuclei blue with the tissue being counterstained with nuclear fast red. Heart tissue 2 days after cryoinjury also was examined at and adjacent to the injury site in MRL (C) and B6 (D) tissue and show tunnel-positive cells. these studies have been done in the MRL/MpJ mouse, does not have the fas mutation. Cardiac tissue As stated, after cryoinjury to the right ventricle of the mouse heart, the MRL and the control C57BL/6 (B6) mouse show healing differences.17 Syngeneic bone marrow chimeras were generated as controls for future adoptive transfer experiments. Even after x-irradiation (9 Gy) and bone marrow reconstitution, these mice could still heal like normal mice. Thus, it was found that MRL chimeric mice closed ear holes and healed heart in an MRL-like fashion and B6 chimeric mice did not close ear holes and scarred in a B6-like fashion.44 These mice were examined pre- and post-injury for the degree of cardiomyocyte proliferation as compared with a normal heart from untreated mice. As shown in the following, differences in BrdU incorporation were seen in these two mouse strains (Fig. 2). Normal heart tissue from MRL (lane 1) showed a greater number of BrdU positive cardiomyocytes than normal B6 heart tissue (lane 4). After x-irradiation and syngeneic BM reconstitution, BrdU cardiomyocytes were higher in both MRL (lane 2) and % of cells that were BrdU+, within 0.3mm 14 MRL SWISS 12 10 % 6068_02_p3-9 8 6 4 2 0 2 4 7 14 Days post lesion FIG. 4. BrdU-positive cell counts. Mice were injured on day 0. Four hours before the injury site was examined, BrdU was injected intraperitoneally. The graph shows the percentage of cells positive for BrdU and prolonged cell division up to a distance of 3 mm at 2, 4, 7, and 14 days post lesioning. Adapted from Ref. 35. 6068_02_p3-9 3/27/06 2:58 PM Page 7 7 IMMORTALITY AND CONTINUOUS REGENERATION FIG. 5. GFAP-positive cells 2 days after cortical stab injury. Frozen sections of the cortical injury were prepared and stained with anti-GFAP antibody. Figures show the transient increased GFAP-negative zone in the MRL compared with the Swiss Webster mice adjacent to the injury site. The arrows show where the lesion is and the scale bars 1 mm. Adapted from Ref. 35. B6 (lane 5), but still showed significant differences between the two strains. Finally, after cryoinjury of the syngeneic chimeric heart, the number of BrdU cells almost doubled in the MRL (lane 3) but did not change in the B6 (lane 6). Thus, in all cases, the degree of proliferation was at least twice as great in the MRL. To examine the degree of cell death in heart tissue, a tunnel assay was used that measures fragmented 3 DNA ends which react with terminal deoxynucleotide transferase (TdT) and are end labeled with HRP-coupled nucleotides. In Figure 3, tunnel was carried out in normal and 2 day post-cryoinjured cardiac tissue. It can be seen clearly that there is a vast difference in the number of tunnel cells in the MRL injured heart compared with the B6 injured heart. There also appears to be a difference in uninjured heart, with more tunnel positive cells being present in the MRL. Which cells are undergoing apoptosis was not determined in these experiments. Brain cortical stabs As discussed, brain injuries in MRL versus Swiss Webster mice showed significant differences early after injury, but the healing seen at 14 days appeared similar in many ways, including scar formation. Two striking differences were seen.35 First, analysis of BrdU cells after a 4-hour BrdU pulse showed differences in the MRL, compared with the SW during the first 7 days (Fig. 4). This response shows twofold differences, as was seen in the heart. Second, extreme differences were seen in the tissue response after injury. In Figure 5, the arrow shows the stab wound in an area of tissue that is highly rich in astrocytes. In the Swiss Webster mice, the region around the wound site is depleted of astrocytes, as measured by GFAP staining, and appears dark. The area around the wound in the MRL also is depleted of astrocytes. However, in this case one cannot find astrocytes in the field shown, except at the extreme end distal to the injury site. One presumes that the response to injury in the MRL involves drastic cell death compared with the Swiss Webster mice. CONCLUSION From the limited data shown in the preceding in two MRL and control mice injury models, the differences seen in increased rates of proliferation and rates of cell death in the MRL mouse supports the comparison to the hydra. This indicates that the MRL mouse has a higher cell turnover rate and therefore a higher cell replacement rate with more new cells than seen in the control mice. If the hydra is an example of immortality that is fueled by its high cellular turnover rate, then one might expect that the MRL mouse also may 6068_02_p3-9 3/27/06 2:58 PM Page 8 8 HEBER-KATZ ET AL. show signs of extended longevity. These experiments are in progress. ACKNOWLEDGMENTS This work was generously supported from its inception by the G. Harold and Leila Y. Mathers Foundation, F.M. Kirby Foundation, and the W.W. Smith Foundation, and by a grant from the National Institutes of Health. REFERENCES 1. Goss RJ. Principles of Regeneration. New York: Academic Press, 1969. 2. Bode HR. The interstitial cell lineage of hydra: a stem cell system that arose early in evolution. J Cell Sci 1996;109:1155–1164. 3. David CN, Campbell RD. Cell cycle kinetics and development of Hydra attenuata. I. Epithelial cells. J Cell Sci 1972;11(2):557–568. 4. Otto JJ, Campbell RD. Tissue economics of hydra: regulation of cell cycle, animal size and development by controlled feeding rates. J Cell Sci 1977;28:117–132. 5. Martinez DE. Mortality patterns suggest lack of senescence in hydra. Exp Gerontol 1998;33:217–225. 6. Gierer A, Berking S, Bode H, et al. Regeneration of hydra from re-aggregated cells. Nat New Biol 1972; 239:98–101. 7. Muller WA. Pattern formation in the immortal Hydra. Trends Genet 1996;12:91–96. 8. Galliot B, Schmid V. Cnidarians as a model system for understanding evolution and regeneration. 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Epidermal-mesodermal interaction during morphogenesis of the limb regeneration blastema in larval salamanders. J Exp Zool 1972;181:49–62. 30. Repesh LA, Oberpriller JC. Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens. Am J Anat 1980;159:187–208. 31. Globus M, Vethamany-Globus S, Lee YCI. Effect of apical epidermal cap on mitotic cycle and cartilage differentiation in regeneration blastemata in the newt, Notophthalmus viridescens. Dev Biol 1980;75:358–372. 32. Stocum DL, Crawford K. Use of retinoids to analyze the cellular basis of positional memory in regenerating amphibian limbs. Biochem Cell Biol 1987;65:750–761. 33. Brockes JP. Amphibian limb regeneration: rebuilding a complex structure. Science 1997;276:81–87. 6068_02_p3-9 3/27/06 2:58 PM Page 9 IMMORTALITY AND CONTINUOUS REGENERATION 34. Ohishi K, Fujita N, Morinaga Y, et al. 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Identification of wound healing/regeneration quantitative trait loci (QTL) at multiple time points that explain seventy percent of variance in (MRL/MpJ and SJL/J) mice F2 population. Genome Res 2001;11:2027–2033. 40. Samulewicz SJ, Clark L, Seitz A, et al. Expression of Pref-1, a delta-like protein, in healing mouse ears. Wound Repair Regen 2002;10:215–221. 9 41. Masinde G, Li X, Baylink DJ, et al. Isolation of wound healing/regeneration genes using restrictive fragment differential display-PCR in MRL/MPJ and C57BL/6 mice. Biochem Biophys Res Commun 2005; 330:117–122. 42. Watanabe-Fukunaga R, Brannan C, Copeland NG, et al. Lymphoproliferation disorder in mice is explained by defects in Fas antigen that mediates apoptosis. Nature l992;356:314–316. 43. Adachi M, Watanabe-Fukunaga R, Nagata S. Aberrant transcription caused by the insertion of an endogenous retrovirus in an apoptosis gene. PNAS 1993;90:1756–1760. 44. Bedelbaeva K, Gourevitch D, Clark L, et al. The MRL mouse heart healing response shows donor dominance in allogeneic fetal liver chimeric mice. Cloning Stem Cells 2004;4:352–363. Address reprint requests to: Ellen Heber-Katz, Ph.D. 3601 Spruce Street Philadelphia, PA 19104 E-mail: [email protected]