In addition, it has not been established whether dedifferentiation of skeletal muscle leads to the activation of a stem cell population within the tissue and if such cells could contribute to the new limb. Some things you may want to know: Their morphs are … Phinney, R. Class, and D.J. Because the blastema is a multipotent tissue, we tested whether newt satellite cells were able to adopt anything other than myogenic fates. There was no difference in the speed and morphology of regeneration between cell- and PBS-injected limbs. (2004) showed that limb myofibers isolated from axolotl larvae undergo cellularization and fragmentation. Salamanders are much better at regeneration, in every way, but at least we know mammals aren’t completely left out of the regeneration game. Colter, D.C., I. Sekiya, and D.J. "We need to know exactly what salamanders … Search for other works by this author on: Ajiro, K., K. Yoda, K. Utsumi, and Y. Nishikawa. What sets salamanders apart from the rest is that fact that they can fully regenerate amputated limbs at any time during their life cycle (2). 1975 Oct 31; 4(1):23-4. Myofiber cultures were analyzed using both brightfield and fluorescence microscopy at 12-h intervals before fixation or passaging of the myofiber-derived cells. For time-lapse observations, the salamanders were placed in a sealed observation chamber (Fig. These data show that quiescent satellite cell activation is a response to limb removal and the findings suggest that satellite cells leave their niche to incorporate into the blastema. Cells in osteogenic medium were stained with Alizarin red (Digirolamo et al., 1999), and alkaline phosphatase was detected using kit 85 (Sigma-Aldrich) according to the manufacturer's instructions. Partridge. Zammit, P.S., J.P. Golding, Y. Nagata, V. Hudon, T.A. 2, A and B) and M-cadherin+ cells (Fig. In addition, clonal analysis also indicated that the progeny are multipotent, displaying myogenic (not depicted), adipogenic, and osteogenic potential (Fig. collagen) of the extracellular matrix which provides structural support to surrounding cells (8). Limb regeneration depends on the formation of a blastema, from which the new appendage develops. These factors include fibroblast growth factors (FGFs) which are found in many organisms including humans, and are typically involved in tissue repair in adult organisms (3,5). MPPs are also involved in the prevention of scar formation, and contribute to the overall maintenance and growth of the blastema (1). 10. For immunoblotting, cells were lysed with RIPA buffer supplemented with a protease inhibitor cocktail (Roche). Roy, and V.R. Second, the blastemal cells will undergo cell proliferation, patterning, cell differentiation and tissue growth using similar genetic mechanisms that deployed during embryonic development. 3 A). A multipotent satellite cell progeny clone. 1. But in contrast to mammals, these cells were shown to be completely encapsulated by a basement membrane (Popiela, 1976; Cameron et al., 1986), and it has remained unsettled whether adult newts possess a cellular population that is equal to mammalian satellite cells. Proliferating clonal cells were maintained at a confluency of no more than 60% to avoid spontaneous differentiation before being subjected to differentiation studies. The following primary antibodies were used: mouse monoclonal anti-Pax7 IgG (Developmental Studies Hybridoma Bank), mouse monoclonal anti–myosin heavy chain IgG (MF20; Developmental Studies Hybridoma Bank), mouse monoclonal anti–M-cadherin IgG (Clone 1B11 used for immunofluorescence; nanotools GmbH), rabbit polyclonal anti–collagen type IV antibody (Rockland Immunochemicals, Inc.), rabbit polyclonal anti–M-cadherin antibody (used for immunoblotting; Invitrogen), rabbit polyclonal anti-H3P antibody (Upstate Biotechnology), rat monoclonal anti-BrdU IgG (Trichem ApS), rabbit polyclonal anti-MyoD antibody (Santa Cruz Biotechnology, Inc.), anti-WE3 monoclonal IgG (Developmental Studies Hybridoma Bank), mouse monoclonal anti–collagen type II IgG (CHEMICON International, Inc.). Abbreviation used in this paper: H3P, phosphorylated histone 3. This somewhat serendipitous turn took me on a journey of discoveries in the field of regeneration, and exposed me for the first time to salamanders, the vertebrates evolutionarily closest to us that are able to regenerate full limbs as adults. Differentiation. The type of salamander called axolotl, with its frilly gills and widely spaced eyes, looks like an alien and has other-worldly powers of regeneration. This is mediated with the help of factors secreted by the AEC (2). In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. First, we found that newt satellite cells or their progeny express molecular markers, such as Pax7, M-cadherin, and MyoD, all of which are expressed by mammalian satellite cells or their progeny as well (Zammit and Beauchamp, 2001). 2003 Jan 16; 226(2):280-94. Salamanders and many other related amphibians have a remarkable aptitude for the regeneration of various body structures when compared to other vertebrates. 20,000 cells were suspended in 4 μl PBS diluted with 24% water. Regeneration may have been an ability we lost, rather than something salamanders gained. Samulewicz, and E. Heber-Katz. The AEC is essential to regeneration as it secretes various growth factors which aid in limb outgrowth (2). The roles of injury, nerves, and the wound epidermis during the initiation of amphibian limb regeneration. However, the onset of tissue-specific molecular differentiation programs and the large number of satellite cell progeny within various tissues, which did not alter the speed and mode of regeneration, suggest that the integrated satellite cell progeny are functional. Images were taken at room temperature and were further processed using Photoshop (Adobe) according to the JCB guidelines. Tamaki, T., A. Akatsuka, K. Ando, Y. Nakamura, H. Matsuzawa, T. Hotta, R.R. The exact identity of signals that link tissue injury to blastema formation needs to be elucidated, as it may reveal key aspects of blastema formation involving both myofiber fragmentation and concomitant stem cell activation. In the case of salamander regeneration, they are reverted into mesenchymal stem cells which can differentiate into a number of cell types including bone, fat, and cartilage cells (6). Limb regeneration itself is not a unique feature of salamanders as all other organisms possess some degree of regenerative capability. Salamander limb regeneration occurs in two main steps. 7. (A and B) Photomicrographs showing a medium bud stage regenerate. As indicated by the presence of Pax7+ (Fig. (D) Satellite cell progeny can enter an adipogenic pathway, as revealed by Oil red staining in lipid droplets (arrowheads). Arrowheads point to a Pax7+ cell nucleus. Rosenblatt, J.D., A.I. If this condition is met, it results in the formation of a structure called the wound epidermis. Edgerton. Satellite cell activation prevails in our model of skeletal muscle plasticity, leading to the production of a multipotent progeny population. This work was supported by the Swedish Research Council (grant 20021937784641), the Swedish Foundation for Strategic Research, the Wenner-Gren Foundations, the Åke Wibergs Foundation, the Magnus Begvalls Foundation, Stiftelsen Lars Hiertas Minne, and the Karolinska Institute to A. Simon. 5 C). "We can look to salamanders as a template of what perfect regeneration looks like," lead study author James Godwin said in a statement. Shefer, G., M. Wleklinski-Lee, and Z. Yablonka-Reuveni. However, the additional basement membrane that separates newt satellite cells from the sarcolemma may reflect that newt satellite cells are in some respect evolutionary intermediates between interstitial stem cells and satellite cells, which were found to be separate populations in mammals (Asakura et al., 2002; Tamaki et al., 2002). Time points indicate the duration of the one specific budding event. For clonal analyses, cells were cultured at a density of 0.5–1.0%, so that single cells were clearly discernible. Limb regeneration in salamanders occurs in several overlapping steps. 2. Though salamanders do regulate their genes differently from other species. We saw that 86% of the isolated myofibers contained satellite cells and that only satellite cell progeny proliferated in our culture system, although we could not detect any sign of proliferating progeny that could have been derived by cellularization of the myofiber. These observations further suggest an important role of satellite cells in the regeneration of missing body parts in vertebrates. Amputation or tissue removal can lead to the regeneration of lost structures in some vertebrate species, such as the salamanders (e.g., the newt and the axolotl; Stocum, 1997; Tanaka, 2003; Brockes and Kumar, 2005). 2003 Dec 31; 34(6):565-71. Clarke, and E.M. Tanaka. Myofiber-derived proliferating cells are satellite cell progeny. These structures include the jaws, spinal cord, heart ventricles, some eye structures, and most notably their limbs (1). At specified time-points, the regenerating limbs were collected after anesthetization. Bars, 50 μm. Collins, C.A., I. Olsen, P.S. Formation of a blastema-like structure, although a rare event, is possible in mammals, as exemplified by the healing capacity of MRL mice and by the seasonal regeneration of deer antlers (Gourevitch et al., 2003; Price et al., 2005). Suggested Time Allowance: 45 minutes- 1 hour. Simultaneous to the development of the blastema, its cells begin to re-differentiate into tissue cells specific to the regenerating limb and limb structural repatterning proceeds. Budding of cells continued until the myofiber hypercontracted and detached from the substrate. 2005 Mar 1;279(1):86-98. A longer movie capture is shown in Video 1. (E) Schematic model of mouse and newt myofibers. We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. BrdU-labeled satellite cell progeny are found in the blastema (*) and, notably, also in the epidermis. 3. 5. Myofiber-derived cells migrated onto the surrounding substrate and proliferated. Cell dedifferentiation results in a population of mesenchymal stem cells which migrate to the wound surface and form a cone-shaped mass of cells known as the regeneration blastema (2). 2003 May 2; 92(8):827-39. Cell dedifferentiation is a process in which cells that have already changed into a specialized cell type are reverted back to an undifferentiated form, the opposite of differentiation. Video 1 illustrates the budding of single cells from the myofiber, and Fig. Hence, it is still not clear whether the term dedifferentiation solely refers to the reversal of the differentiated state of mature cells, to the activation of stem cells in the disorganizing tissues, or to a combination of these two definitions. Satellite cells can be copurified with isolated single skeletal muscle fibers. The purpose of the wound epidermis is to prevent the entry of debris into the wound site (4). On average, after 7 d in culture the myofibers started to produce proliferating progeny cells. Ein Salamander als Vorbild. With the knowledge of this ability, one might ask if human limb regeneration may be feasible in the future. An intriguing aspect of the regenerating salamander appendages is the reversal of differentiation. Developmental Biology. Progeny cells bud off the myofiber and proliferate. Rudnicki. PDF | A research project on the regeneration of salamander with a special focus on Newt and Axolotl regeneration. We propose skeletal muscle satellite cells as a potential target in the promotion of mammalian blastema formation. However, when salamanders drop their tails, they lose not only flesh but also nerves. A microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc.) with Openlab 3.1.7 software (Improvision Ltd.) was used for brightfield and fluorescence microscopy analyses. Identification of further stem cell populations in newt skeletal muscle, along with functional studies, could address this issue. In contrast, the few myotubes that were visible in the adipogenic media did not contain lipid droplets (Fig. Satellite cell progeny were grown in the presence of 10 μM BrdU for 6 d before injection. We describe a multipotent Pax7+ satellite cell population located within the skeletal muscle of the salamander limb. Almendral. (H–J) Pax7+ cells appear in an early bud stage blastema. Fibroblast growth factors. Excess fat and connective tissue was carefully removed from around the musculature. Earlier studies identified a cell population that is closely apposed to the myofiber in the adult newt limb as well. Buckingham, T.A. A group of muscles located between the elbow and wrist were isolated with forceps and carefully dissected away from the bone, handling only the tip of the muscle to prevent damage. (A) Photomicrograph showing an isolated single newt skeletal muscle fiber directly after attachment. Bars, 50 µm. Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. When satellite cell progeny were cultured in osteogenic media, we saw that 10% of the cells produced alkaline phosphatase–positive foci (Fig. Photomicrographs showing a medium bud stage (A and B) and late bud stage regenerate (E and F) with BrdU-labeled cells in the blastema and the epidermis. (F) Lack of Alizarin red staining in cultures kept in proliferation media. (J and K) Most of the myofiber-derived progeny remain Pax7+ (red) directly after activation, but the intensity of the staining is strongest closest to the hypercontracted myofiber (arrowheads). As shown in Fig. C is an overlay of the fluorescent and light microscopy images. A total of 25 minutes of recording time of my 3 male axolotls eating pellets and running around. Pax7+ cells are present in newt limb skeletal muscle. 5 D). S1). (2001), which showed that amputation as such was not sufficient to produce blastema progenitors. Injected BrdU-labeled satellite cell progeny incorporate into new tissues during limb regeneration. 6, C–F). And there are many other examples of limited critter regeneration of specific body tissues and parts. 5, A and B). To distinguish between these two events, we injected a fluorescein-conjugated nuclear-localizing dextran (NLS-dextran) into the myofibers directly after the attachment of the myofiber to the substrate (Fig. Gross. 5-μm-thin frozen sections were thawed at room temperature and immediately fixed in acetone/methanol (1:1) for 5 min at –20°C. The blastema grows distally over time via the proliferation of mesenchymal stem cells until the limb has fully regenerated. Furthermore, lineage shifting across germ layer boundaries has been shown to occur during salamander tail regeneration (Echeverri and Tanaka, 2002). Satellite cells (blue nuclei) are tightly attached to both mouse and newt myofibers, but an additional basement membrane (maroon) separates the satellite cells from the sarcolemma. Satellite cell progeny were labeled with BrdU before injection, during their in vitro expansion. Regrowing human limbs. 4, J–N). A unique feature of blastema formation in salamanders is the process of dedifferentiation of stump tissues that follows appendage removal. This is especially valid for skeletal muscle tissue because dedifferentiating skeletal muscle is a significant source of blastema progenitors. The time-lapse records suggested that the changes in the mitotic activity of supporting cells might have been linked to the presence of phagocytic leukocytes in the vicinity of the neuromast that was nearest to the wound. But they have been thwarted in the attempt by another peculiarity of the axolotl — it has the largest genome of any animal yet sequenced, 10 times larger than that of humans. 6, J–M). Mammalian skeletal muscle responds to various challenges, such as stretching or mechanical damage, by activating a proliferation program in satellite cells that is followed by differentiation and fusion into myotubes and into myofibers. Figure 1: The limb regeneration process over a 70 day period. Limb regeneration in salamanders occurs in several overlapping steps. 2008 Apr 1; 298(4):56-63. After 3 and 6 d in differentiation medium, cells were fixed with 2% PFA and processed for immunofluorescence studies. Dotted line marks the level of amputation. Newt satellite cell progeny are multipotent. (A) A clonal progeny population can enter an adipogenic pathway, as revealed by Oil red staining in lipid droplets (arrowheads). Sections were incubated with a relevant primary antibody overnight and with secondary antibodies for 1 h at room temperature. The pictures underneath G–I are enlarged images of the boxed area. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. The ability to form a regeneration blastema, which leads to the epimorphic regeneration of complex body structures, is restricted to some amphibians and fish among vertebrates (Poss et al., 2003). Although adult mammals do not form a blastema after limb amputation, their skeletal muscle tissue regenerates after injury (Charge and Rudnicki, 2004). (A and B) Immunostaining of limb skeletal muscle identifies Pax7+ cells within skeletal muscle tissue. We also noted a contribution to the epidermis and detected satellite cell progeny within newly formed cartilage tissue. 2 E shows the location of newt satellite cells compared with their mammalian counterparts. Out of the 70 single myofibers that we observed, we were only able to detect two mononucleate cells at one occasion that appeared to contain NLS-dextran (Fig. Gourevitch, D., L. Clark, P. Chen, A. Seitz, S.J. Salamander species vary in size, from 3.9 cm to 180 cm. The cultures were counterstained by hematoxylin. That means that nerve axon regeneration is happening at the wound site in tandem with tissue, bone and muscle regeneration. However, a basement membrane surrounds the Pax7+ cells (Fig. The original limb cells at the site of injury are thought to possess positional memory of their placement along the 3 axes of the limb which are inherited by cells of the blastema as they re-differentiate, allowing them to migrate to the appropriate position in the growing limb. To test whether newt skeletal muscle in the limb contains a satellite cell population, we used a monoclonal antibody against Pax7, which is a specific marker of skeletal muscle satellite cells. (E) Lack of alkaline phosphatase+ foci in cultures kept in proliferation media. Der Axolotl ist ein mexikanischer Schwanzlurch und kann abgetrennte Gliedmaßen und sogar Organe nachwachsen lassen. A few hours to a day following limb amputation, epidermal cells around the wound area are recruited in order to cover the wound (3). Instead, a stem cell population called satellite cells, which express markers such as Pax7, M-cadherin, and Myf5, reenters the cell cycle, proliferates, and incorporates into nascent or into preexisting myofibers during mammalian muscle regeneration (Cornelison and Wold, 1997; Collins et al., 2005). 4, G–I), and these two cells did not proliferate. Now Flowers and colleagues have found an ingenious way to circumvent the animal’s complex genome to identify at least two genes involved in regeneration, they report Jan. 28 in the journal eLife . Number of mitotically active Pax7+ cells in amputated and non-amputated limbs. 3 C shows the single frame sequence of one budding event taken from the time-lapse movie capture in Video 1. (E and F) High resolution images of the circled area in C and D, showing significant amounts of BrdU-labeled cells in the blastema. We isolated and plated single myofibers that were viable and displayed characteristic morphology, such as Z band striation marking the boundaries of the sarcomeres (Fig. Archives of Medical Research. Video 1 shows the derivation of proliferating mononucleate cells from a 10–14-d-old newt myofiber in vitro. In addition,  histolysis of these cells prior to dedifferentiation is triggered by matrix metalloproteinases, (MMPs) (2). After digestion, myofibers were disaggregated as previously described (Rosenblatt et al., 1995). BrdU-labeled cells were not detected in the contralateral regenerate, which was injected with PBS before amputation (Fig. Pax7+ cells are also found in the blastema of the regenerating axolotl tail (Schnapp et al., 2005) and tail regeneration in the Xenopus laevis tadpole also involves satellite cell activation (Gargioli and Slack, 2004). Mammals including humans have poor limb regenerative capacity as adults, but during the early stages of life such as the embryonic or fetal stages, they have a limited ability to regenerate the digit tips (2). It’s not that they have special regeneration genes,” Gardiner says. In this context, it is interesting to note the study by Echeverri et al. Hilgers, and T.J. Hinterberger. Limb regeneration itself is not a unique feature of salamanders as all other organisms possess some degree of regenerative capability. As blastema formation and regeneration occurred we saw that a large number of the injected, BrdU-labeled cells appeared in clusters within the blastema at all analyzed stages of the regeneration. The protocols for immunofluorescent staining of cells and newt single myofibers were followed as previously described (Beauchamp et al., 2000), with the exception that cells and myofibers were fixed with 2% PFA. The contralateral PBS-injected regenerate was also devoid of BrdU-labeled cells at this stage (Fig. All satellite cells were encased by basement membrane directly after attachment, and 99% of the cells in satellite cell positions were Pax7+. “Salamanders are not special. Echeverri, K., J.D. 1 (A and B), similar to mammalian muscle, Pax7+ cells are present in newt limb skeletal muscle. Bars, 50 μm. Digestion with type I collagenase (Sigma-Aldrich) solution (0.2% wt/vol in DME; Invitrogen) supplemented with 1% Glutamax (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) was performed in a water bath at 25°C for 3–4 h. All media used in this and subsequent cell cultures were diluted 24% with distilled water. All other proliferating cells were NLS-dextran negative. Video 1 is available at http://ww.jcb.org/cgi/content/full/jcb.200509011. Our data clearly show that satellite cells, which are comparable to mammalian skeletal muscle stem cells, exist in newt skeletal muscle as well. The FASEB Journal. 8. Why is this so? We saw Pax7+ cells outside of skeletal muscle tissue 4 d after amputation, and detected Pax7+ cells within the blastema upon formation (Fig. For adipogenic and osteogenic differentiation, cells were grown to 90–100% confluency and incubated in adipogenic and osteogenic media as described previously (Colter et al., 2001). 5 F). With a fully sequenced genome in hand, scientists hope they are finally poised to learn how axolotls regenerate lost body parts (E–G) Photomicrographs showing a mitotic Pax7+ cell 4 d after amputation. All four injected limbs developed cartilage at this stage, and BrdU-labeled cells were detected within newly formed cartilage tissue in all four cases (Fig. Second, when we isolated single myofibers a satellite cell population was copurified, despite the presence of an additional basal lamina between the satellite cell and sarcolemma. Photomicrographs showing that the fluorescent dextran exclusively labels myonuclei in the syncytium, but not the nuclei in satellite cells. (L–N) Satellite cell progeny express Pax7 (red) and MyoD (green) for several generations. What sets salamanders apart from the rest is that fact that they can fully regenerate amputated limbs at any time during their life cycle (2). These data show that satellite cell activation, rather than cellularization of the syncytium, resulted in a proliferating cell progeny population in our culture system. (A–D) Satellite cells are attached to the myofiber after isolation and plating. Cells in adipogenic medium were stained with Oil red (Colter et al., 2001). However, mammalian skeletal muscle regeneration does not involve cellularization of the syncytium. The peptide was covalently linked to fluorescein-conjugated dextran (70 kD; Invitrogen) via the COOH-terminal cysteine residue, using the heterobifunctional cross-linker sulfo-SMCC (Pierce Chemical Co.) as described previously (Broder et al., 1997; Maroto et al., 2004).

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