S2 shows a multipotent satellite cell progeny clone. Three-dimensional confocal microscopic analyses showed the complete absence of cells outside of the basal lamina, indicating that the myofiber cultures did not contain contaminating cells from elsewhere. 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). Collins, C.A., I. Olsen, P.S. 6, C–F). 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). (C and D) Photomicrographs showing a typical Pax7+ cell being surrounded by basement membrane. Satellite cell progeny were labeled with BrdU before injection, during their in vitro expansion. Way back in our evolutionary past, the common ancestors of people and salamanders could have been regenerators, since at least one distant relative of modern-day salamanders could do it. (A and B) Photomicrographs showing a medium bud stage regenerate. 9. Progeny of injected BrdU-labeled satellite cells are found in the regenerate, but not in the contralateral regenerate. 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. Suzuki M, Yakushiji N, Nakada Y, Satoh A, Ide H, Tamura K. Limb regeneration in Xenopus laevis froglet. (A–C) Photomicrographs showing that injected fluorescent NLS-dextran marks the nuclei of the myofiber. Buckingham, T.A. 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). 4. We saw Pax7+ cells outside of skeletal muscle tissue 4 d after amputation, and detected Pax7+ cells within the blastema upon formation (Fig. Myofibers were injected with NLS-conjugated fluorescein-dextran directly after their attachment, using a Femtojet in combination with an Injectman (Eppendorf AG). 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. (E and F) High resolution images of the circled area in C and D, showing significant amounts of BrdU-labeled cells in the blastema. RESULTS Abbreviation used in this paper: H3P, phosphorylated histone 3. 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. The question is how blastema formation is induced in mammals and how it can be promoted. The myofiber morphology has changed and several lobular structures are seen while mononucleate progeny has been produced. A total of 25 minutes of recording time of my 3 male axolotls eating pellets and running around. Video 1 shows the derivation of proliferating mononucleate cells from a 10–14-d-old newt myofiber in vitro. Salamander limb regeneration occurs in two main steps. 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). 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. Salamanders and many other related amphibians have a remarkable aptitude for the regeneration of various body structures when compared to other vertebrates. On the other hand, lizards are not able to do so despite having the natural capacity to regrow their tails. 2008 Apr 1; 298(4):56-63. Tamaki, T., A. Akatsuka, K. Ando, Y. Nakamura, H. Matsuzawa, T. Hotta, R.R. With the knowledge of this ability, one might ask if human limb regeneration may be feasible in the future. Newt satellite cell progeny are multipotent. (H–J) Pax7+ cells appear in an early bud stage blastema. All satellite cells were encased by basement membrane directly after attachment, and 99% of the cells in satellite cell positions were Pax7+. Phinney, R. Class, and D.J. 5 G). (C) Time-lapse photomicrographs showing a sequence of one representative budding event, which leads to the derivation of a mononucleate cell. To understand the cellular basis of the plasticity of skeletal muscle fibers, we established an ex vivo culture of living, intact single newt myofibers. Yu, S. Tajbakhsh, R.G. All experiments were performed according to European Community and local ethics committee guidelines. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. Clarke, and E.M. Tanaka. Ein gesunder Organismus ist zur Regeneration fähig. Rudnicki. Bars, 50 μm. Salamander’s Genome Guards Secrets of Limb Regrowth. In addition, clonal analysis also indicated that the progeny are multipotent, displaying myogenic (not depicted), adipogenic, and osteogenic potential (Fig. 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. MPPs are also involved in the prevention of scar formation, and contribute to the overall maintenance and growth of the blastema (1). This site uses cookies. Antibodies were diluted in blocking buffer and sections were mounted in mounting medium (DakoCytomation) containing 100 ng/ml DAPI (Sigma-Aldrich). 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. 1. (G–I) Photomicrographs showing that the vast majority of the myofiber progeny lack the NLS-dextran lineage tracer. BrdU-labeled cells are still seen in the blastema and in the epidermis. 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. Myofiber cultures were analyzed using both brightfield and fluorescence microscopy at 12-h intervals before fixation or passaging of the myofiber-derived cells. Identification of further stem cell populations in newt skeletal muscle, along with functional studies, could address this issue. Shefer, G., M. Wleklinski-Lee, and Z. Yablonka-Reuveni. Budding of cells continued until the myofiber hypercontracted and detached from the substrate. Zammit, P.S., J.P. Golding, Y. Nagata, V. Hudon, T.A. Note the visible striation demarking the sarcomeres. Edgerton. Pax7+ cells are present in newt limb skeletal muscle. Number of mitotically active Pax7+ cells in amputated and non-amputated limbs. After 3 and 6 d in differentiation medium, cells were fixed with 2% PFA and processed for immunofluorescence studies. Department of Cell and Molecular Biology, Karolinska Institute, 17177 Stockholm, Sweden. Zammit, L. Heslop, A. Petrie, T.A. Clearly, additional experiments are required to assess the plasticity of satellite cells in vivo and to establish whether metaplasia characterizes salamander limb regeneration. 1, E–G; and Table I). 5. Nevertheless, in light of the available observations, a plausible hypothesis is that skeletal muscle dedifferentiation results in a significant contribution by satellite cells to the blastema and to the regenerate. Time-lapse observation. (C and D) Arrows point to two myonuclei, and arrowheads point to an M-cadherin+ satellite cell. Starting at around two minutes and 20 seconds into the time-lapse video, or around five days of development, Hurney points to the passage of individual cells migrating across the … 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. 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). (H and I) Collagen type II+/BrdU-labeled cells are present in cartilage. 5 E) and that the cells produced calcium deposits stained by Alizarin red (Fig. (G) Photomicrographs showing cartilage. 6, G–I). We demonstrate that skeletal muscle dedifferentiation involves satellite cell activation and that these cells can contribute to new limb tissues. A synthetic polypeptide containing the NLS of the polyomavirus large T antigen, CGYGVSRKRPRPGC, was synthesized by Thermo Electron Corporation. (A) Photomicrograph showing an isolated single newt skeletal muscle fiber directly after attachment. 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. (C, D, G, and H) The lack of BrdU-labeled cells in the contralateral regenerates, which were harvested at the same time points. Yakushiji N, Yokoyama H, Tamura K. Repatterning in amphibian limb regeneration: a model for study of genetic and epigenetic control of organ regeneration. Keating, and A. Nechiporuk. Figure 3: The overall process of limb regeneration. Almendral. Limb regeneration itself is not a unique feature of salamanders as all other organisms possess some degree of regenerative capability. 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). The upper and lower surfaces 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) . If this condition is met, it results in the formation of a structure called the wound epidermis. 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. Conversely, none of the NLS-dextran–labeled myonuclei were Pax7+ (Fig. 1, C and D). S1). In contrast to mammals, salamanders can regenerate complex structures after injury, including entire limbs. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases structure, function, and biochemistry. The Anatomical Record Part B: The New Anatomist. Of particular interest is whether the generation of progenitor cells during epimorphic regeneration in salamander and during mammalian tissue repair proceeds by the activation of different or overlapping mechanisms. 4, D–F). Maroto, B., N. Valle, R. Saffrich, and J.M. 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. Some studies mark them using Visible Implant Elastomers (VIE)• Search cover boards every 2-3 weeks from April through October/early November• Other data can include vegetation identification, litter levels, decomposition, soil temperature and moisture• Leaf litter sample to identify invertebrates … collagen) of the extracellular matrix which provides structural support to surrounding cells (8). 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). (2001), which showed that amputation as such was not sufficient to produce blastema progenitors. (E) Lack of alkaline phosphatase+ foci in cultures kept in proliferation media. 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. 4, G–I), and these two cells did not proliferate. Tassava, R.A., B. Johnson-Wint, and J. 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. 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. Activation of salamander satellite cells occurs in an analogous manner to how the mammalian myofiber mobilizes stem cells during skeletal muscle tissue repair. A unique feature of blastema formation in salamanders is the process of dedifferentiation of stump tissues that follows appendage removal. 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). Prockop. B and C are high power magnifications of the boxed area in A. "We need to know exactly what salamanders … Circulation Research. The model in Fig. Kelly, A. Wernig, M.E. The skin was removed from the underside of the forelimbs, exposing the musculature. For example, if a salamander … Animals were anesthetized by placing them in an aqueous solution of 0.1% ethyl 3-aminobenzoate methanesulfonate salt (Sigma-Aldrich) for 15 min. 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. (E and F) Satellite cell progeny can enter an osteogenic pathway. As indicated by the presence of Pax7+ (Fig. Nye HL, Cameron JA, Chernoff EA, Stocum DL. During this process, cells of the blastema produce neurotrophic factors involved in the regeneration of sensory and motor nerves (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. Han M, Yang X, Taylor G, Burdsal CA, Anderson RA, Muneoka K. Limb regeneration in higher vertebrates: developing a roadmap. Bars, 50 μm. 10. 2003 May 2; 92(8):827-39. 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. , lineage shifting across germ layer boundaries has been produced ( B and C are power. 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