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Circulation Research. Roy, and V.R. An LSM 510 Meta laser microscope with LSM 5 Image Browser software (both Carl Zeiss MicroImaging, Inc.) was used for confocal analyses. Because the blastema is a multipotent tissue, we tested whether newt satellite cells were able to adopt anything other than myogenic fates. (E and F) Satellite cell progeny can enter an osteogenic pathway. Fibroblast growth factors. It is unclear to what extent differentiated cells reverse mature phenotypes and to what extent undifferentiated cells, such as stem cells, residing within differentiated tissues become activated, followed by their incorporation into the blastema. This suggests that a higher regenerative capacity was an ancestral trait which was eventually lost in mammals (11). C is an overlay of the fluorescent and light microscopy images. 4, G–I), and these two cells did not proliferate. 2, C and D), muscle fibers from the newt limb could be copurified with a satellite cell population after isolation and plating. The purpose of the wound epidermis is to prevent the entry of debris into the wound site (4). Partridge. the fire salamander, salamandra salamandra depositing the eggs in a forest puddle - salamander stock pictures, royalty-free photos & images 2. During this process, cells of the blastema produce neurotrophic factors involved in the regeneration of sensory and motor nerves (3). 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). 4, J–N). Tassava RA, Mescher AL. 4, A–C). MPPs are also involved in the prevention of scar formation, and contribute to the overall maintenance and growth of the blastema (1). Yu, S. Tajbakhsh, R.G. Myofiber cultures were analyzed using both brightfield and fluorescence microscopy at 12-h intervals before fixation or passaging of the myofiber-derived cells. Injected BrdU-labeled satellite cell progeny incorporate into new tissues during limb regeneration. The lack of molecular markers has also obstructed the prospective isolation of blastema progenitors. 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. The regenerates were harvested at different time points and processed for immunohistochemistry. Developmental Dynamics. With the knowledge of this ability, one might ask if human limb regeneration may be feasible in the future. 5 D). BrdU-labeled cells were also clearly visible in the late bud stage regenerate, although the intensity of the BrdU label varied more, compared with the medium bud stage regenerate (Fig. A few hours to a day following limb amputation, epidermal cells around the wound area are recruited in order to cover the wound (3). Note the visible striation demarking the sarcomeres. (D) Satellite cell progeny can enter an adipogenic pathway, as revealed by Oil red staining in lipid droplets (arrowheads). After 3 and 6 d in differentiation medium, cells were fixed with 2% PFA and processed for immunofluorescence studies. Partridge, and P.S. Limb regeneration in salamanders occurs in several overlapping steps. All experiments were performed according to European Community and local ethics committee guidelines. 1, C and D). Thus, limb regeneration and mammalian tissue repair share common cellular and molecular programs. Parry, and T.A. Cameron, J.A., A.R. Maroto, B., N. Valle, R. Saffrich, and J.M. (H–J) Pax7+ cells appear in an early bud stage blastema. There was no difference in the speed and morphology of regeneration between cell- and PBS-injected limbs. (C and D) Photomicrographs showing a typical Pax7+ cell being surrounded by basement membrane. 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. Progeny cells bud off the myofiber and proliferate. These proliferating satellite cells retained Pax7 expression and were also positive for MyoD for several generations (Fig. Excess fat and connective tissue was carefully removed from around the musculature. doi: https://doi.org/10.1083/jcb.200509011. Almendral. Velloso, Y. Imokawa, and J.P. Brockes. 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. Prockop. 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). 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. Search for other works by this author on: Ajiro, K., K. Yoda, K. Utsumi, and Y. Nishikawa. 3. We thank A. Lindquist for help with injections of myofibers, members of the Simon laboratory for discussions, and J. Frisén, O. Hermanson, and U. Lendahl for critical reading of the manuscript. The contralateral PBS-injected regenerate was also devoid of BrdU-labeled cells at this stage (Fig. Dedifferentiation of stump tissues, such as skeletal muscle, precedes blastema formation, but it was not known whether dedifferentiation involves stem cell activation. Wada, M.R., M. Inagawa-Ogashiwa, S. Shimizu, S. Yasumoto, and N. Hashimoto. (J–M) WE3+ (Tassava et al., 1986)/BrdU-labeled cells are present in the epidermis (arrows). The possibility to induce blastema formation and regeneration in mammals through the activation of a comparable dedifferentiation program has been proposed (Hughes, 2001; Bryant et al., 2002; Stocum, 2004). Han M, Yang X, Taylor G, Burdsal CA, Anderson RA, Muneoka K. Limb regeneration in higher vertebrates: developing a roadmap. Conversely, none of the NLS-dextran–labeled myonuclei were Pax7+ (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). In this context, it is interesting to note the study by Echeverri et al. BrdU-labeled cells were not detected in the contralateral regenerate, which was injected with PBS before amputation (Fig. (A) A clonal progeny population can enter an adipogenic pathway, as revealed by Oil red staining in lipid droplets (arrowheads). Gross. Budding of cells continued until the myofiber hypercontracted and detached from the substrate. (2004) showed that limb myofibers isolated from axolotl larvae undergo cellularization and fragmentation. Myofiber-derived proliferating cells are satellite cell progeny. (G–I) Photomicrographs showing that the vast majority of the myofiber progeny lack the NLS-dextran lineage tracer. J Cell Biol 30 January 2006; 172 (3): 433–440. 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). Hilgers, and T.J. Hinterberger. Beauchamp, J.R., L. Heslop, D.S. Asakura, A., P. Seale, A. Girgis-Gabardo, and M.A. Bryant, S.V., T. Endo, and D.M. 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. "We can look to salamanders as a template of what perfect regeneration looks like," lead study author James Godwin said in a statement. Our findings also identify satellite cells as potential targets in promoting mammalian blastema formation. Bars, 50 µm. 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