Learn about the current research in the field of regeneration by reading and discussing “Missing Limb? Clarke, and E.M. Tanaka. A multipotent satellite cell progeny clone. It’s not that they have special regeneration genes,” Gardiner says. 1987 Dec 1; 1(6):434-40. A few hours to a day following limb amputation, epidermal cells around the wound area are recruited in order to cover the wound (3). First, the local cells dedifferentiate at the wound site into progenitor to form a blastema. As we can distinguish between the process of cellularization of the syncytial myofiber on one hand and satellite cell activation on the other, the quantitative aspects of these two separate events can be examined. Video 1 shows the derivation of proliferating mononucleate cells from a 10–14-d-old newt myofiber in vitro. J Cell Biol 30 January 2006; 172 (3): 433–440. 2A). From there, the cells differentiate and create the appropriate body part. Myofiber-derived cells migrated onto the surrounding substrate and proliferated. Animals were left to recover overnight in an aqueous solution of 0.5% sulfamerazine (Sigma-Aldrich) before being placed back into a 25°C water environment. 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 speed of salamander limb regeneration varies among species and developmental stage, but is nevertheless impressive. Single cells were isolated with cloning cylinders (Sigma-Aldrich) and incubated for 30 s in trypsin-EDTA (0.05% trypsin and 0.53 mM EDTA; Invitrogen) at room temperature. (2004) reflect phylogenetic or ontogenetic differences, or are caused by dissimilarities in the experimental paradigms. Our data clearly show that satellite cells, which are comparable to mammalian skeletal muscle stem cells, exist in newt skeletal muscle as well. Single myofibers were placed in 35-mm Falcon culture dishes (BD Biosciences) coated with 1 mg/ml Matrigel (BD Biosciences) in DME supplemented with 13% FCS (Invitrogen), 1% Glutamax, 1% penicillin/streptomycin, and 1% insulin (Sigma-Aldrich) and cultured at 25°C. 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. (E and F) High resolution images of the circled area in C and D, showing significant amounts of BrdU-labeled cells in the blastema. Although no fluorescent dextran-containing progeny were seen in 69 of 70 single myofiber cultures, these images show the single occasion when two fluorescent dextran+ cells were detected (arrows and boxes), but these cells did not proliferate. As part of that positional memory in the fibroblast cells, the blastema knows to grow in the proper sequence to avoid defective regeneration. Archives of Medical Research. For immunofluorescence studies, primary antibodies were detected with appropriate species-specific Alexa Fluor–conjugated secondary antibodies (Invitrogen). Suzuki M, Yakushiji N, Nakada Y, Satoh A, Ide H, Tamura K. Limb regeneration in Xenopus laevis froglet. Photomicrographs showing that the fluorescent dextran exclusively labels myonuclei in the syncytium, but not the nuclei in satellite cells. At the medium bud stage, BrdU-labeled cells were found within both the blastema (Fig. Similar to the in vivo analyses, we found an additional basement membrane between the myofiber itself and the satellite cells, as indicated by the Pax7–collagen type IV double immunostaining (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. Because the fluorescent NLS-dextran signal was easily detectable in all of the myonuclei and we analyzed the myofiber-derived progeny at 12-h intervals, we can exclude the possibility that the NLS-dextran signal was diluted because of rapid proliferation. Bars, 50 μm. Myofiber cultures were analyzed using both brightfield and fluorescence microscopy at 12-h intervals before fixation or passaging of the myofiber-derived cells. (H–J) Pax7+ cells appear in an early bud stage blastema. Kumar, A., C.P. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. Research certain organisms and their regenerative processes. 2 μg of each cell lysate was separated on a 10% PAGE gel and transferred to nitrocellulose membrane. Paleontologists have discovered fossils of For clonal analyses, cells were cultured at a density of 0.5–1.0%, so that single cells were clearly discernible. In agreement with this, none of the Pax7+ satellite cells were labeled with NLS-dextran. Edgerton. Though salamanders do regulate their genes differently from other species. In addition,  histolysis of these cells prior to dedifferentiation is triggered by matrix metalloproteinases, (MMPs) (2). We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. The roles of injury, nerves, and the wound epidermis during the initiation of amphibian limb regeneration. 2009 Jul 31; 20(5):565-574. S2). S1). (C) Western blot analyses show the increased amount of M-cadherin and myosin heavy chain and the reduced amount of Pax7 proteins as a result of myogenic differentiation (lane a, proliferation medium; lane b, after 6 d in myogenic differentiation medium). 1, C and D). Objectives: Students will: 1. Although the potential role of stem cells in blastema formation has been suggested (Corcoran and Ferretti, 1999; Carlson, 2003; Odelberg, 2004), no such cells have been previously identified in the newt limb. Han M, Yang X, Taylor G, Burdsal CA, Anderson RA, Muneoka K. Limb regeneration in higher vertebrates: developing a roadmap. Furthermore, lineage shifting across germ layer boundaries has been shown to occur during salamander tail regeneration (Echeverri and Tanaka, 2002). No Pax7+ cells were detected outside the skeletal muscle tissue (unpublished data). In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. 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. Wada, M.R., M. Inagawa-Ogashiwa, S. Shimizu, S. Yasumoto, and N. Hashimoto. An LSM 510 Meta laser microscope with LSM 5 Image Browser software (both Carl Zeiss MicroImaging, Inc.) was used for confocal analyses. Regeneration may have been an ability we lost, rather than something salamanders gained. (B and C) The same clonal progeny population can enter an osteogenic pathway. Seale, P., L.A. Sabourin, A. Girgis-Gabardo, A. Mansouri, P. Gruss, and M.A. This lineage tracer cannot be transferred between cells and, therefore, should only label myonuclei. All experiments were performed according to European Community and local ethics committee guidelines. The best staging, based on meticulous histological series, is available for Notophthalmus, which completes adult limb regeneration within less than 2 months (Iten and Bryant, 1973) . (A–C) Photomicrographs showing that injected fluorescent NLS-dextran marks the nuclei of the myofiber. (C and D) Photomicrographs showing a typical Pax7+ cell being surrounded by basement membrane. Cells that were not cultured in adipogenic media were negative for Oil Red staining (Fig. We find that the salamander myofiber contains a satellite cell population. Broder, Y.C., A. Stanhill, N. Zakai, A. Friedler, C. Gilon, and A. Loyter. Simultaneously, Pax7 levels dropped in the protein extracts (Fig. Limb regeneration itself is not a unique feature of salamanders as all other organisms possess some degree of regenerative capability. A central question is whether the generation of progenitor cells during limb regeneration and mammalian tissue repair occur via separate or overlapping mechanisms. (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). (G–I) Photomicrographs showing that the vast majority of the myofiber progeny lack the NLS-dextran lineage tracer. Developmental Biology. 2006 May 3; 6:26-37. These proliferating satellite cells retained Pax7 expression and were also positive for MyoD for several generations (Fig. The upper and lower surfaces (A) Photomicrograph showing an isolated single newt skeletal muscle fiber directly after attachment. Tissue samples were mounted on cork using Gum Tragacanth (Sigma-Aldrich), snap frozen in isopentane (VWR), and cooled to freezing point in liquid nitrogen. However, both studies underpin the necessity to further assess the quantitative aspects and functional relevance of satellite cell activation that leads to multipotent progeny on one hand and cellularization and/or fragmentation of the syncytium on the other during limb regeneration. These data show that satellite cell activation, rather than cellularization of the syncytium, resulted in a proliferating cell progeny population in our culture system. 5-μm-thin frozen sections were thawed at room temperature and immediately fixed in acetone/methanol (1:1) for 5 min at –20°C. 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. Why is this so? Figure 1: The limb regeneration process over a 70 day period. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. (D) Satellite cell progeny can enter an adipogenic pathway, as revealed by Oil red staining in lipid droplets (arrowheads). Monitoring Plots• 1 m X 1 m plots• Cover boards are at least 5 m apart• Salamanders are captured and measured. 7. PDF | A research project on the regeneration of salamander with a special focus on Newt and Axolotl regeneration. Both static analyses and dynamic in vivo tracing showed that skeletal muscle fibers break up, the syncytium becomes fragmented as a response to limb or tail removal, and muscle-derived mononucleate progeny significantly contribute to the blastema (Thornton, 1938; Hay, 1959, 1962; Lentz, 1969; Echeverri et al., 2001). As a control, we injected the contralateral limbs with PBS before amputation at the same axial level. However, mammalian skeletal muscle regeneration does not involve cellularization of the syncytium. Newt satellite cell progeny are multipotent. Progeny cells bud off the myofiber and proliferate. In accordance with earlier observations on mammalian myofiber cultures (Zammit et al., 2004), Pax7 expression became heterogeneous in prolonged newt satellite cell progeny cultures (unpublished data). 3. S2 shows a multipotent satellite cell progeny clone. 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. (C) Time-lapse photomicrographs showing a sequence of one representative budding event, which leads to the derivation of a mononucleate cell. 1, H–J). Antibodies were diluted in blocking buffer and sections were mounted in mounting medium (DakoCytomation) containing 100 ng/ml DAPI (Sigma-Aldrich). (C and D) Arrows point to two myonuclei, and arrowheads point to an M-cadherin+ satellite cell. Many simple organisms such as Hydra have a high regenerative ability. Bars, 50 μm. 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). The regenerates were harvested at different time points and processed for immunohistochemistry. 2. Western blot analyses confirmed the up-regulation of myosin heavy chain and M-cadherin during myogenesis, which was concomitant with the increased number of myotubes and the decreased number of myoblasts in the culture (Fig. After 3 and 6 d in differentiation medium, cells were fixed with 2% PFA and processed for immunofluorescence studies. The re-expression of various developmental genes including HOX genes helps to direct the regeneration process of the limb structure, ensuring proper differentiation of blastemal cells until it is completely rebuilt (9). Tissues below the wound epidermis such as local cartilage, muscle, and Schwann cells then undergo histolysis which leads to cell dedifferentiation (3). 2003 Dec 31; 34(6):565-71. Sections were blocked with 20% normal goat serum (DakoCytomation) diluted in PBS for 30 min at room temperature. Arrows point to two visible nuclei, which could either be myonuclei or located in satellite cells. 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). 2005 Mar 1;279(1):86-98. Myofiber-derived proliferating cells are satellite cell progeny. Poss, K.D., M.T. The blastema grows distally over time via the proliferation of mesenchymal stem cells until the limb has fully regenerated. Scientific American. An alkaline phosphatase+ focus is shown in E, and Alizarin red marks calcium deposits produced by osteogenic cells in F. (G) Lack of Alizarin red staining in cells cultured in proliferation media. Epimorphic limb regeneration proceeds by rapid wound closure and is critically dependent on the formation of a multipotent mesenchymal growth zone, the blastema, which gives rise to the newly formed limb (Wallace, 1981). These results show that implanted satellite cell progeny can give rise to new tissues during limb regeneration and indicate that metaplasia may occur during salamander limb regeneration. Clearly, additional experiments are required to assess the plasticity of satellite cells in vivo and to establish whether metaplasia characterizes salamander limb regeneration. Satellite cell progeny were grown in the presence of 10 μM BrdU for 6 d before injection. Buckingham, T.A. This is mediated with the help of factors secreted by the AEC (2). Circulation Research. This process requires a sufficient amount of nerves to be present in the wound area. 4. 5, A and B). (J–M) WE3+ (Tassava et al., 1986)/BrdU-labeled cells are present in the epidermis (arrows). Video 1 illustrates the budding of single cells from the myofiber, and Fig. Images were taken at room temperature and were further processed using Photoshop (Adobe) according to the JCB guidelines. Mammalian satellite cells reside between the basal lamina and the sarcolemma of the myofiber (Seale et al., 2000). Primary antibodies were recognized with species-specific streptavidin-conjugated secondary antibodies (GE Healthcare). Salamander’s Genome Guards Secrets of Limb Regrowth. We also noted a contribution to the epidermis and detected satellite cell progeny within newly formed cartilage tissue. These structures include the jaws, spinal cord, heart ventricles, some eye structures, and most notably their limbs (1). (L–N) Satellite cell progeny express Pax7 (red) and MyoD (green) for several generations. Partridge. The observed multipotentiality of satellite cell progeny does not directly address the question of whether activated satellite cells adopt divergent fates without in vitro expansion. (A and B) Immunostaining of limb skeletal muscle identifies Pax7+ cells within skeletal muscle tissue. Some things you may want to know: Their morphs are … (E) Lack of alkaline phosphatase+ foci in cultures kept in proliferation media. Third, similar to the mammalian myofiber cultures, we observed that satellite cell activation occurred that was characterized by cell cycle reentry and proliferation of the satellite cell progeny population. Rudnicki. Time-lapse observation. For myogenic differentiation, satellite cell progeny were grown to 90–100% confluency and incubated in DME supplemented with 0.5% horse serum (Invitrogen), 1% Glutamax, 1% penicillin/streptomycin, and 1% insulin. In a study similar to our own, Kumar et al. Kelly, A. Wernig, M.E. Fig. Vinarsky V, Atkinson DL, Stevenson TJ, Keating MT, Odelberg SJ. 5 E) and that the cells produced calcium deposits stained by Alizarin red (Fig. Newts were anesthetized and decapitated. Gross. collagen) of the extracellular matrix which provides structural support to surrounding cells (8). Isolated salamander myotubes can also undergo a cellularization process by which the syncytium turns into mononucleate progeny after reimplantation into the regenerating limb (Lo et al., 1993; Kumar et al., 2000). Normal newt limb regeneration requires matrix metalloproteinase function. While it is currently not possible to induce limb regeneration in humans, by studying the genetic and molecular mechanisms at work during injury repair in early mammalian life and in other organisms which can fully regenerate limbs, we can make strides towards tapping into this suppressed ability in the future. Ein Salamander als Vorbild. Cells in adipogenic medium were stained with Oil red (Colter et al., 2001). 3 (A and B) shows a myofiber directly after attachment and with proliferating progeny after ∼15 d in culture. 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. (A and B) Arrows point to a Pax7+ satellite cell, and arrowheads point to the collagen IV+ basal lamina. C is an overlay of the fluorescent and light microscopy images. *, blastema. S1 shows that the progeny of injected BrdU-labeled satellite cells are found in the regenerate, but not in the contralateral regenerate. As the wound epidermis develops, it eventually forms another structure called the apical epithelial cap (AEC) which resembles a bud on the surface of the site of amputation. Search for other works by this author on: Ajiro, K., K. Yoda, K. Utsumi, and Y. Nishikawa. 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. 5 G). Gardiner. Hilgers, and T.J. Hinterberger. Arrowheads point to a Pax7/H3P double-positive cell. Progeny of injected BrdU-labeled satellite cells are found in the regenerate, but not in the contralateral regenerate. "We need to know exactly what salamanders … Note the visible striation demarking the sarcomeres. 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. These data show that skeletal muscle satellite cell progeny can adopt nonmyogenic fates and indicate that satellite cells could represent a multipotent blastema progenitor population. In 1955, Piatt made a comprehensive analysis of salamander spinal cord regeneration by transecting trunk spinal cord at different ages, embryonic, larval and adult (Piatt, 1955). During this process, cells of the blastema produce neurotrophic factors involved in the regeneration of sensory and motor nerves (3). 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. The membrane was blocked with 5% dry milk fat and 0.1% Tween 20 (Sigma-Aldrich) in PBS and subsequently probed with primary antibodies. 6, C–F). 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). This site uses cookies. However, when salamanders drop their tails, they lose not only flesh but also nerves. (A) A clonal progeny population can enter an adipogenic pathway, as revealed by Oil red staining in lipid droplets (arrowheads). 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.). Injected BrdU-labeled satellite cell progeny incorporate into new tissues during limb regeneration. 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. (C and D) Photomicrographs showing a late bud stage regenerate. The sanctuary of the blind cave salamander in Moulis, France on January 16, 2001 - Adult blind cave salamander in its natural surrounding. Membranes were developed using an ECL detection kit (GE Healthcare). MMPs are enzymes that have the ability to degrade proteins (e.g. Second, the blastemal cells will undergo cell proliferation, patterning, cell differentiation and tissue growth using similar genetic mechanisms that deployed during embryonic development. These 3 axes include the proximal-distal, anterior-posterior, and dorsal ventral axes. 2003 Jan 16; 226(2):280-94. That means that nerve axon regeneration is happening at the wound site in tandem with tissue, bone and muscle regeneration. Pax7+ cells are present in newt limb skeletal muscle. 3 A). An alkaline phosphatase+ focus is shown in B, and Alizarin red marks calcium deposits produced by osteogenic cells in C. (D) Lack of Alizarin red staining in cells cultured in proliferation media. Schnapp, E., M. Kragl, L. Rubin, and E.M. Tanaka. The pictures underneath G–I are enlarged images of the boxed area. Finally, we showed that the satellite cell progeny population in newts is multipotent, which has also been observed in mammals (Asakura et al., 2001; Wada et al., 2002; Shefer et al., 2004). 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. 8. doi: https://doi.org/10.1083/jcb.200509011. When cells were exposed to adipogenic media, we detected that at least 30% of the cells contained lipid droplets and displayed adipocyte morphology. Limb regeneration in salamanders occurs in several overlapping steps. Thomas KA. Number of mitotically active Pax7+ cells in amputated and non-amputated limbs. 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. 2003 May 2; 92(8):827-39. 2, A and B). 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 . The myofiber morphology has changed and several lobular structures are seen while mononucleate progeny has been produced. Salamander species vary in size, from 3.9 cm to 180 cm. For time-lapse observations, the salamanders were placed in a sealed observation chamber (Fig. 2, A and B) and M-cadherin+ cells (Fig. (2004) showed that limb myofibers isolated from axolotl larvae undergo cellularization and fragmentation. The cultures were counterstained by hematoxylin. With a fully sequenced genome in hand, scientists hope they are finally poised to learn how axolotls regenerate lost body parts After digestion, myofibers were disaggregated as previously described (Rosenblatt et al., 1995). Beauchamp, J.R., L. Heslop, D.S. BrdU-labeled cells were not detected in the contralateral regenerate, which was injected with PBS before amputation (Fig. “Salamanders are not special. B and C are high power magnifications of the boxed area in A. All other proliferating cells were NLS-dextran negative. Keating, and A. Nechiporuk. 2 E shows the location of newt satellite cells compared with their mammalian counterparts. 4, G–I), and these two cells did not proliferate. 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). And there are many other examples of limited critter regeneration of specific body tissues and parts. Shefer, G., M. Wleklinski-Lee, and Z. Yablonka-Reuveni. 20,000 cells were suspended in 4 μl PBS diluted with 24% water. As shown in Fig. 4, A–C). 5 D). Salamanders and many other related amphibians have a remarkable aptitude for the regeneration of various body structures when compared to other vertebrates. At all stages of development, the spinal cord was completely regenerated, anatomicaly and functionally, although the adult took longer time to regenerate. The arrow points to a myotube that is devoid of lipid droplets. The question is how blastema formation is induced in mammals and how it can be promoted. Excess fat and connective tissue was carefully removed from around the musculature. Nye HL, Cameron JA, Chernoff EA, Stocum DL. Maroto, B., N. Valle, R. Saffrich, and J.M. Zammit, P.S., J.P. Golding, Y. Nagata, V. Hudon, T.A. There was no difference in the speed and morphology of regeneration between cell- and PBS-injected limbs. Dotted line marks the level of amputation. RESULTS Note the protrusion of the myofiber in the circled area, which is concomitant with the appearance of a mononucleate progeny. At present, it is unclear whether the discrepancies between our observations and the model presented by Kumar et al. Bars, 50 μm. To start addressing these questions we combined histological analyses and in vitro culture of single newt myofibers, along with implantation and tracing of labeled myofiber-derived cells. (B) Photomicrograph showing the same 15-d-old myofiber in culture. 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). 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). This process is generally referred to as the dedifferentiation step leading to the formation of blastema progenitors (Iten and Bryant, 1973). 10. (D–F). Fig. Animals were anesthetized and cells were injected using a Hamilton syringe intramuscularly in the upper forelimb halfway between the elbow and shoulder. 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). Cameron, J.A., A.R. BrdU-labeled satellite cell progeny are found in the blastema (*) and, notably, also in the epidermis. Yakushiji N, Yokoyama H, Tamura K. Repatterning in amphibian limb regeneration: a model for study of genetic and epigenetic control of organ regeneration. 2, C and D), muscle fibers from the newt limb could be copurified with a satellite cell population after isolation and plating. Arrows point to mononuclei, arrowheads point to satellite cell. 3 C shows the single frame sequence of one budding event taken from the time-lapse movie capture in Video 1. The authors noted that only 3.5% of the myofibers contained the satellite type of cells and that these were not observed in their skeletal muscle fiber plasticity model. 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. However, a basement membrane surrounds the Pax7+ cells (Fig. Digirolamo, C.M., D. Stokes, D. Colter, D.G. To understand the cellular basis of the plasticity of skeletal muscle fibers, we established an ex vivo culture of living, intact single newt myofibers. When satellite cell progeny were cultured in osteogenic media, we saw that 10% of the cells produced alkaline phosphatase–positive foci (Fig. Fig. The contralateral PBS-injected regenerate was also devoid of BrdU-labeled cells at this stage (Fig. For example, adult newts can rebuild entire limbs, tails, and jaws through an epimorphic regeneration process that leads to the restoration of complete and functional tissue architecture (Brockes and Kumar, 2002). Echeverri, K., J.D. Lunt, D.J. Figure 3: The overall process of limb regeneration. The process of dedifferentiation is not yet well understood due to its complexity and the fact that it does not follow the same process for each tissue type. Seminars in Cell & Developmental Biology. We saw Pax7+ cells outside of skeletal muscle tissue 4 d after amputation, and detected Pax7+ cells within the blastema upon formation (Fig. Partridge, and P.S. Boxed area is shown at high magnification in H and I. Skeletal muscle is an important contributor to blastema formation (Brockes, 1997). Zammit, L. Heslop, A. Petrie, T.A. Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. 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. Is to prevent the entry of debris into the wound site in tandem with tissue bone... Shimizu, S. Yasumoto, and the M-cadherin+ ( MyHC ) myotubes in a ( 11 ) cells at. 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