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Akira Satoh - One of the best experts on this subject based on the ideXlab platform.
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Limb Regeneration in Xenopus laevis Froglet PROCESS OF LIMB REGENERATION IN VERTEBRATES
2020Co-Authors: Makoto Suzuki, Nayuta Yakushiji, Akira Satoh, Hiroyuki Ide, Yasuaki Nakada, Koji TamuraAbstract:Limb regeneration in amphibians is a representative process of epimorphosis. This type of organ regeneration, in which a mass of undifferentiated cells referred to as the "Blastema" proliferate to restore the lost part of the amputated organ, is distinct from morphallaxis as observed, for instance, in Hydra, in which rearrangement of pre-existing cells and tissues mainly contribute to regeneration. In contrast to complete limb regeneration in urodele amphibians, limb regeneration in Xenopus, an anuran amphibian, is restricted. In this review of some aspects regarding adult limb regeneration in Xenopus laevis, we suggest that limb regeneration in adult Xenopus, which is pattern/tissue deficient, also represents epimorphosis. KEYWORDS: Xenopus, epimorphosis, limb regeneration, dedifferentiation, Blastema, spike, nerve dependence, muscle regeneration, wound healing PROCESS OF LIMB REGENERATION IN VERTEBRATES Regenerative ability of appendages (limbs/fins) in vertebrates varies greatly In contrast, the potential for limb regeneration in amphibians, including urodeles such as the axolotl and newts in particular, is outstanding among vertebrates 1. Within 1 day after limb amputation, the amputated surface is rapidly covered with epithelial cells following formation of a specialized dermis-free epithelial structure referred to as "wound epidermis". As the regeneration process progresses, this epithelial structure produces an apical epithelial cap (AEC), a structure that is morphologically and functionally similar to the AER. The AER emanates a number of growth factors in order to allow morphogenesis for limb bud outgrowth and patterning Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 28 If a defect is caused by surgical operation, pharmacological treatment, or spontaneous accident at any step in the process, proper limb regeneration is prevented, resulting in the formation of a hypomorphic structure. Thus, examinations of various defects in the limb regeneration process and investigations at molecular and cellular levels are promising approaches in order to understand the nature of epimorphosis in vertebrates. Covering the amputated plane with fully mature skin flaps, for example, inhibits formation of the wound epidermis and Blastema by preventing epithelial-mesenchymal interactions PROCESS OF LIMB REGENERATION IN XENOPUS The South African clawed frog, Xenopus laevis, is an anuran amphibian in which limb regeneration has been studied in detail. This frog was named from its strange (xeno-) limbs (-pus), which have a claw in some anterior digits of the hindlimbs. The strange character of the limb in Xenopus is also true of its developmental stage-dependent ability of limb regeneration Bryant and colleagues examined the regenerative ability of Xenopus limbs by experiments in which various amounts of autopodial tissues were removed There has been debate as to whether spike formation of the Xenopus froglet is based on simple tissue regeneration or epimorphosis. From histological observations, some researchers argued that spikes do not develop from the epimorphic Blastema containing dedifferentiated mesenchymal cells, but only from proliferative differentiated cells such as fibroblasts and chondrocytes within connective or skeletal tissues Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL DEDIFFERENTIATION AND NERVE DEPENDENCE IN THE XENOPUS LIMB REGENERATION Nerve dependency is a characteristic feature of epimorphic limb regeneration in amphibians. In urodeles, axons in the limb, which originate from spinal cord and dorsal root ganglia of the vertebral column, secrete neurotrophic factors into the Blastema that are essential for its growth. Therefore, surgical removal of these axons inhibits proper development of the Blastema and results in the simple restoration of wounded tissue[reviewed by 40]. These neurotrophic factors have been shown to mediate Blastema formation as mitogenic Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 30 FIGURE 2. Histological observations of early limb Blastema in the Xenopus froglet. Longitudinal sections of the froglet forelimb stumps were stained with hematoxylin, eosin and Alcian blue (A-D) or with anti-BrdU antibody and DAB (brown) after incorporation of BrdU for 1 h (E-H). (A,E) 0 days, (B,F) 1 day, (C,G) 2 days, (D,H) 4 days after amputation. Specimens were fixed in Bouin's fixative, embedded in paraffin, and sectioned at a thickness of 6 μm. The procedures for the BrdU treatment and immunohistochemistry were essentially the same as that of Suzuki et al. Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 31 conclude that nerve dependency is a key event for epimorphosis that is common to urodeles and anurans. Furthermore, our recent study REPATTERNING OF AMPUTATED FROGLET LIMBS Whereas urodeles can reconstruct an exact replica of the amputated limb, the Xenopus froglet cannot but regenerate a cartilaginous spike that has no skeletal pattern as described above. Histological observations showing that this hypomorphic structure has neither segmentation nor bifurcation in the cartilage suggest that froglet Blastemas have some deficiencies in the ability for pattern formation. Pattern formation of the limb has been extensively studied in the system of limb development in amniotes. This patterning involves morphogenesis along three axes (antero-posterior (AP), dorso-ventral (DV) and proximo-distal (PD) axes) Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 33 REDIFFERENTIATION OF AMPUTATED FROGLET LIMBS Since pattern formation of the limb bud is accompanied and even influenced by tissue differentiation, temporally and spatially coordinated tissue differentiation should be important for successful regeneration of vertebrate limbs. Urodele limb Blastemas can regenerate all tissue components, including epithelial tissues (skin glands), skeletal tissues (bone and cartilage), soft connective tissues (dermis, tendon and ligament), and muscles with satellite cells. In addition, proper angiogenesis In summary, the froglet Blastema is likely to contain many types of progenitor cells (epithelia, cartilage, blood vessels, dermis, and tendon/ligament cells), and at least two major deficiencies of the froglet spike (muscleless and jointless) are improvable by therapeutic treatments. Taken together with previous reports that mutual interaction between muscle and tendon cells is required for correct tendon development PERSPECTIVE In this article, we have reviewed old and recent information on Xenopus limb regeneration, and we would like to emphasize that limb regeneration in the Xenopus froglet represents epimorphosis and is comparable to that in urodele amphibians, although this process gives rise to an incomplete regenerate. Thus, this useful animal serves as a good model system to investigate epimorphic limb regeneration. The considerable accumulation of molecular information, sources, and techniques, including transgenesis in Xenopus in particular (as shown also in this review i
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activation of germline specific genes is required for limb regeneration in the mexican axolotl
Developmental Biology, 2012Co-Authors: Wei Zhu, Akira Satoh, James R Monaghan, Susan V Bryant, Gerald M Pao, Gillian M C Cummings, Timothy T Harkins, Randal S VossAbstract:The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and Blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb Blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the Blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.
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Blastema induction in aneurogenic state and prrx 1 regulation by mmps and fgfs in ambystoma mexicanum limb regeneration
Developmental Biology, 2011Co-Authors: Akira Satoh, Ayako Hirata, Aki Makanae, Yutaka SatouAbstract:Urodele amphibians can regenerate amputated limbs. It has been considered that differentiated dermal tissues generate multipotent and undifferentiated cells called Blastema cells during limb regeneration. In early phases of limb regeneration, Blastema cells are induced by nerves and the apical epithelial cap (AEC). We had previously investigated the role of neurotrophic factors in Blastema or Blastema-like formation consisting of Prrx-1 positive cells. A new system suitable for investigating early phases of limb regeneration, called the accessory limb model (ALM), was recently developed. In this study, we performed a comparative transcriptome analysis between a Blastema and wound using ALM. Matrix metalloproteinase (MMP) and fibroblast growth factor (FGF) signaling components were observed to be predominantly expressed in ALM Blastema cells. Furthermore, we found that MMP activity induced a Blastema marker gene, Prrx-1, in vitro, and FGF signaling pathways worked in coordination to maintain Prrx-1 expression and ALM Blastema formation. Furthermore, we demonstrated that these two activities were sufficient to induce an ALM Blastema in the absence of a nerve in vivo.
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correlation between shh expression and dna methylation status of the limb specific shh enhancer region during limb regeneration in amphibians
Developmental Biology, 2007Co-Authors: Nayuta Yakushiji, Makoto Suzuki, Akira Satoh, Tomoko Sagai, Toshihiko Shiroishi, Hisato Kobayashi, Hiroyuki Sasaki, Hiroyuki IdeAbstract:The Xenopus adult limb has very limited regeneration ability, and only a simple cartilaginous spike structure without digits is formed after limb amputation. We found that expression of Shh and its downstream genes is absent from the regenerating Blastema of the Xenopus froglet limb. Moreover, we found that a limb enhancer region of the Shh gene is highly methylated in the froglet, although the sequence is hypomethylated in the Xenopus tadpole, which has complete limb regeneration ability. These findings, together with the fact that the promoter region of Shh is hardly methylated in Xenopus, suggest that regenerative failure (deficiency in repatterning) in the Xenopus adult limb is associated with methylation status of the enhancer region of Shh and that a target-specific epigenetic regulation is involved in gene re-activation for repatterning during the Xenopus limb regeneration process. Because the methylation level of the enhancer region was low in other amphibians that have Shh expression in the Blastemas, a low methylation status may be the basic condition under which transcriptional regulation of Shh expression can progress during the limb regeneration process. These findings provide the first evidence for a relationship between epigenetic regulation and pattern formation during organ regeneration in vertebrates.
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nerve induced ectopic limb Blastemas in the axolotl are equivalent to amputation induced Blastemas
Developmental Biology, 2007Co-Authors: Akira Satoh, David M Gardiner, Susan V Bryant, Tetsuya EndoAbstract:Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control Blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic Blastema is equivalent to an amputation-induced Blastema. In the present study, we have determined that formation of both ectopic Blastemas and amputation-induced Blastemas is regulated by the same molecular mechanisms, and that both types of Blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and Blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced Blastemas. In addition, ectopic Blastema cells interact coordinately with amputation-induced Blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.
David M Gardiner - One of the best experts on this subject based on the ideXlab platform.
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regulation of axolotl ambystoma mexicanum limb Blastema cell proliferation by nerves and bmp2 in organotypic slice culture
PLOS ONE, 2015Co-Authors: Jeffrey Lehrberg, David M GardinerAbstract:We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb Blastemas. Blastema cells maintain many of the behaviors that are characteristic of Blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the Blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of Blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of Blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the Blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.
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position specific induction of ectopic limbs in non regenerating Blastemas on axolotl forelimbs
Regeneration (Oxford England), 2014Co-Authors: Catherine D Mccusker, Jeffrey Lehrberg, David M GardinerAbstract:Ectopic retinoic acid (RA) has been hypothesized to reprogram the positional identity of cells in developing and regenerating limbs to a single positional value corresponding to the posterior-ventral-proximal (PVPr) position on the limb. We tested this hypothesis by using RA to reprogram the information of Blastema cells that were induced to form at different positions around the limb circumference. We observed that RA treatment of Blastemas in anterior and dorsal locations, but not posterior and ventral locations, resulted in the induction of complete ectopic limbs. These position-specific differences in limb induction are probably due to differences in the positional disparity between the RA-reprogrammed Blastema cells and the cells at the periphery of the wound. These observations are consistent with the hypothesis that RA treatment reprograms the information in Blastema cells to the PVPr position on the limb, since anterior and dorsal positions have the largest disparity and posterior and ventral have the smallest disparity from the PVPr identity.
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positional information is reprogrammed in Blastema cells of the regenerating limb of the axolotl ambystoma mexicanum
PLOS ONE, 2013Co-Authors: Catherine D Mccusker, David M GardinerAbstract:The regenerating region of an amputated salamander limb, known as the Blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of Blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage Blastemas, as well as cells at the tip of late stage Blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud Blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the Blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the Blastema microenvironment.
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nerve induced ectopic limb Blastemas in the axolotl are equivalent to amputation induced Blastemas
Developmental Biology, 2007Co-Authors: Akira Satoh, David M Gardiner, Susan V Bryant, Tetsuya EndoAbstract:Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control Blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic Blastema is equivalent to an amputation-induced Blastema. In the present study, we have determined that formation of both ectopic Blastemas and amputation-induced Blastemas is regulated by the same molecular mechanisms, and that both types of Blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and Blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced Blastemas. In addition, ectopic Blastema cells interact coordinately with amputation-induced Blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.
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a stepwise model system for limb regeneration
Developmental Biology, 2004Co-Authors: Tetsuya Endo, Susan V Bryant, David M GardinerAbstract:The amphibian limb is a model that has provided numerous insights into the principles and mechanisms of tissue and organ regeneration. While later stages of limb regeneration share mechanisms of growth control and patterning with limb development, the formation of a regeneration Blastema is controlled by early events that are unique to regeneration. In this study, we present a stepwise experimental system based on induction of limb regeneration from skin wounds that will allow the identification and functional analysis of the molecules controlling this early, critical stage of regeneration. If a nerve is deviated to a skin wound on the side of a limb, an ectopic Blastema is induced. If a piece of skin is grafted from the contralateral side of the limb to the wound site concomitantly with nerve deviation, the ectopic Blastema continues to grow and forms an ectopic limb. Our analysis of dermal cell migration, contribution, and proliferation indicates that ectopic Blastemas are equivalent to Blastemas that form in response to limb amputation. Signals from nerves are required to induce formation of both ectopic and normal Blastemas, and the diversity of positional information provided by Blastema cells derived from opposite sides of the limb induces outgrowth and pattern formation. Hence, this novel and convenient stepwise model allows for the discovery of necessary and sufficient signals and conditions that control Blastema formation, growth, and pattern formation during limb regeneration.
Mark T Keating - One of the best experts on this subject based on the ideXlab platform.
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heat shock protein 60 is required for Blastema formation and maintenance during regeneration
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Shinji Makino, Geoffrey G Whitehead, Chingling Lien, Soo Joong Kim, Payal Jhawar, Akane Kono, Yasushi Kawata, Mark T KeatingAbstract:Zebrafish fin regeneration requires the formation and maintenance of Blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of Blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for Blastema formation and maintenance. We used a chemical mutagenesis screen to identify no Blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective Blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of Blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue.
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heat shock protein 60 is required for Blastema formation and maintenance during regeneration
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Shinji Makino, Geoffrey G Whitehead, Chingling Lien, Soo Joong Kim, Payal Jhawar, Akane Kono, Yasushi Kawata, Mark T KeatingAbstract:Zebrafish fin regeneration requires the formation and maintenance of Blastema cells. Blastema cells are not derived from stem cells but behave as such, because they are slow-cycling and are thought to provide rapidly proliferating daughter cells that drive regenerative outgrowth. The molecular basis of Blastema formation is not understood. Here, we show that heat-shock protein 60 (hsp60) is required for Blastema formation and maintenance. We used a chemical mutagenesis screen to identify no Blastema (nbl), a zebrafish mutant with an early fin regeneration defect. Fin regeneration failed in nbl due to defective Blastema formation. nbl also failed to regenerate hearts. Positional cloning and mutational analyses revealed that nbl results from a V324E missense mutation in hsp60. This mutation reduced hsp60 function in binding and refolding denatured proteins. hsp60 expression is increased during formation of Blastema cells, and dysfunction leads to mitochondrial defects and apoptosis in these cells. These data indicate that hsp60 is required for the formation and maintenance of regenerating tissue.
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positional cloning of a temperature sensitive mutant emmental reveals a role for sly1 during cell proliferation in zebrafish fin regeneration
Developmental Biology, 2003Co-Authors: Alex Nechiporuk, Kenneth D Poss, Stephen L Johnson, Mark T KeatingAbstract:Here, we used classical genetics in zebrafish to identify temperature-sensitive mutants in caudal fin regeneration. Gross morphological, histological, and molecular analyses revealed that one of these strains, emmental (emm), failed to form a functional regeneration Blastema. Inhibition of emm function by heat treatment during regenerative outgrowth rapidly blocked regeneration. This block was associated with reduced proliferation in the proximal Blastema and expansion of the nonproliferative distal Blastemal zone. Positional cloning revealed that the emm phenotype is caused by a mutation in the orthologue of yeast sly1, a gene product involved in protein trafficking. sly1 is upregulated in the newly formed Blastema as well as during regenerative outgrowth. Thus, sly1 is essential for Blastemal organization and proliferation during two stages of fin regeneration.
Koji Tamura - One of the best experts on this subject based on the ideXlab platform.
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Limb Regeneration in Xenopus laevis Froglet PROCESS OF LIMB REGENERATION IN VERTEBRATES
2020Co-Authors: Makoto Suzuki, Nayuta Yakushiji, Akira Satoh, Hiroyuki Ide, Yasuaki Nakada, Koji TamuraAbstract:Limb regeneration in amphibians is a representative process of epimorphosis. This type of organ regeneration, in which a mass of undifferentiated cells referred to as the "Blastema" proliferate to restore the lost part of the amputated organ, is distinct from morphallaxis as observed, for instance, in Hydra, in which rearrangement of pre-existing cells and tissues mainly contribute to regeneration. In contrast to complete limb regeneration in urodele amphibians, limb regeneration in Xenopus, an anuran amphibian, is restricted. In this review of some aspects regarding adult limb regeneration in Xenopus laevis, we suggest that limb regeneration in adult Xenopus, which is pattern/tissue deficient, also represents epimorphosis. KEYWORDS: Xenopus, epimorphosis, limb regeneration, dedifferentiation, Blastema, spike, nerve dependence, muscle regeneration, wound healing PROCESS OF LIMB REGENERATION IN VERTEBRATES Regenerative ability of appendages (limbs/fins) in vertebrates varies greatly In contrast, the potential for limb regeneration in amphibians, including urodeles such as the axolotl and newts in particular, is outstanding among vertebrates 1. Within 1 day after limb amputation, the amputated surface is rapidly covered with epithelial cells following formation of a specialized dermis-free epithelial structure referred to as "wound epidermis". As the regeneration process progresses, this epithelial structure produces an apical epithelial cap (AEC), a structure that is morphologically and functionally similar to the AER. The AER emanates a number of growth factors in order to allow morphogenesis for limb bud outgrowth and patterning Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 28 If a defect is caused by surgical operation, pharmacological treatment, or spontaneous accident at any step in the process, proper limb regeneration is prevented, resulting in the formation of a hypomorphic structure. Thus, examinations of various defects in the limb regeneration process and investigations at molecular and cellular levels are promising approaches in order to understand the nature of epimorphosis in vertebrates. Covering the amputated plane with fully mature skin flaps, for example, inhibits formation of the wound epidermis and Blastema by preventing epithelial-mesenchymal interactions PROCESS OF LIMB REGENERATION IN XENOPUS The South African clawed frog, Xenopus laevis, is an anuran amphibian in which limb regeneration has been studied in detail. This frog was named from its strange (xeno-) limbs (-pus), which have a claw in some anterior digits of the hindlimbs. The strange character of the limb in Xenopus is also true of its developmental stage-dependent ability of limb regeneration Bryant and colleagues examined the regenerative ability of Xenopus limbs by experiments in which various amounts of autopodial tissues were removed There has been debate as to whether spike formation of the Xenopus froglet is based on simple tissue regeneration or epimorphosis. From histological observations, some researchers argued that spikes do not develop from the epimorphic Blastema containing dedifferentiated mesenchymal cells, but only from proliferative differentiated cells such as fibroblasts and chondrocytes within connective or skeletal tissues Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL DEDIFFERENTIATION AND NERVE DEPENDENCE IN THE XENOPUS LIMB REGENERATION Nerve dependency is a characteristic feature of epimorphic limb regeneration in amphibians. In urodeles, axons in the limb, which originate from spinal cord and dorsal root ganglia of the vertebral column, secrete neurotrophic factors into the Blastema that are essential for its growth. Therefore, surgical removal of these axons inhibits proper development of the Blastema and results in the simple restoration of wounded tissue[reviewed by 40]. These neurotrophic factors have been shown to mediate Blastema formation as mitogenic Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 30 FIGURE 2. Histological observations of early limb Blastema in the Xenopus froglet. Longitudinal sections of the froglet forelimb stumps were stained with hematoxylin, eosin and Alcian blue (A-D) or with anti-BrdU antibody and DAB (brown) after incorporation of BrdU for 1 h (E-H). (A,E) 0 days, (B,F) 1 day, (C,G) 2 days, (D,H) 4 days after amputation. Specimens were fixed in Bouin's fixative, embedded in paraffin, and sectioned at a thickness of 6 μm. The procedures for the BrdU treatment and immunohistochemistry were essentially the same as that of Suzuki et al. Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 31 conclude that nerve dependency is a key event for epimorphosis that is common to urodeles and anurans. Furthermore, our recent study REPATTERNING OF AMPUTATED FROGLET LIMBS Whereas urodeles can reconstruct an exact replica of the amputated limb, the Xenopus froglet cannot but regenerate a cartilaginous spike that has no skeletal pattern as described above. Histological observations showing that this hypomorphic structure has neither segmentation nor bifurcation in the cartilage suggest that froglet Blastemas have some deficiencies in the ability for pattern formation. Pattern formation of the limb has been extensively studied in the system of limb development in amniotes. This patterning involves morphogenesis along three axes (antero-posterior (AP), dorso-ventral (DV) and proximo-distal (PD) axes) Suzuki et al.: Limb Regeneration in Xenopus laevis Froglet TheScientificWorldJOUNRAL (2006) 6(S1), 26-37 33 REDIFFERENTIATION OF AMPUTATED FROGLET LIMBS Since pattern formation of the limb bud is accompanied and even influenced by tissue differentiation, temporally and spatially coordinated tissue differentiation should be important for successful regeneration of vertebrate limbs. Urodele limb Blastemas can regenerate all tissue components, including epithelial tissues (skin glands), skeletal tissues (bone and cartilage), soft connective tissues (dermis, tendon and ligament), and muscles with satellite cells. In addition, proper angiogenesis In summary, the froglet Blastema is likely to contain many types of progenitor cells (epithelia, cartilage, blood vessels, dermis, and tendon/ligament cells), and at least two major deficiencies of the froglet spike (muscleless and jointless) are improvable by therapeutic treatments. Taken together with previous reports that mutual interaction between muscle and tendon cells is required for correct tendon development PERSPECTIVE In this article, we have reviewed old and recent information on Xenopus limb regeneration, and we would like to emphasize that limb regeneration in the Xenopus froglet represents epimorphosis and is comparable to that in urodele amphibians, although this process gives rise to an incomplete regenerate. Thus, this useful animal serves as a good model system to investigate epimorphic limb regeneration. The considerable accumulation of molecular information, sources, and techniques, including transgenesis in Xenopus in particular (as shown also in this review i
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limb Blastema cell a stem cell for morphological regeneration
Development Growth & Differentiation, 2009Co-Authors: Koji Tamura, Shiro Ohgo, Hitoshi YokoyamaAbstract:The limb Blastema cell, which is a major source of mesenchymal components in the limb regenerate, serves as a stem cell that possesses an undifferentiated state and multipotency. A remarkable property of the limb Blastema cell can be seen in its capability for morphogenesis. Elucidation of the molecular basis for morphological regeneration is essential for success in organ regeneration in humans, and characterization of limb Blastema cells will provide many insights into how to create three-dimensional morphology during the regeneration process. In this review, we deal with positional memory, a key trait of the limb Blastema cell in regard to morphological regeneration, making reference to classic surgical experiments, comparative descriptions of limb and fin Blastemas, and genetic/epigenetic regulation of gene transcription. Urodele amphibians, anuran amphibians, and teleosts are likely to share fundamental mechanisms for morphological regeneration, but there are several differences in the process of regeneration, including the epigenetic conditions. Accumulation of knowledge of the molecular mechanisms and epigenetic modifications of gene activation in morphological regeneration of the model organisms for which an overview is provided in this review will lead to successful stimulation of regenerative capacity in amniotes, which only have a limited capability for morphological regeneration.
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nerve dependent and independent events in Blastema formation during xenopus froglet limb regeneration
Developmental Biology, 2005Co-Authors: Makoto Suzuki, Akira Satoh, Hiroyuki Ide, Koji TamuraAbstract:Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of Blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during Blastema formation. We also investigated the lateral wound healing in comparison to Blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during Blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the Blastema were affected by denervation, suggesting that these events specific for Blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated Blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including Blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.
Catherine D Mccusker - One of the best experts on this subject based on the ideXlab platform.
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fgf bmp and ra signaling are sufficient for the induction of complete limb regeneration from non regenerating wounds on ambystoma mexicanum limbs
Developmental Biology, 2019Co-Authors: Warren A Vieira, Kaylee M Wells, Michael J Raymond, Larissa De Souza, Erik J Garcia, Catherine D MccuskerAbstract:Some organisms, such as the Mexican axolotl, have the capacity to regenerate complicated biological structures throughout their lives. Which molecular pathways are sufficient to induce a complete endogenous regenerative response in injured tissue is an important question that remains unanswered. Using a gain-of-function regeneration assay, known as the Accessory Limb Model (ALM), we and others have begun to identify the molecular underpinnings of the three essential requirements for limb regeneration; wounding, neurotrophic signaling, and the induction of pattern from cells that retain positional memory. We have previously shown that treatment of Mexican axolotls with exogenous retinoic acid (RA) is sufficient to induce the formation of complete limb structures from Blastemas that were generated by deviating a nerve bundle into an anterior-located wound site on the limb. Here we show that these ectopic structures are capable of regenerating and inducing new pattern to form when grafted into new anterior-located wounds. We additionally found that the expression of Alx4 decreases, and Shh expression increases in these anterior located Blastemas, but not in the mature anterior tissues, supporting the hypothesis that RA treatment posteriorizes Blastema tissue. Based on these and previous observations, we used the ALM assay to test the hypothesis that a complete regenerative response can be generated by treating anterior-located superficial limb wounds with a specific combination of growth factors at defined developmental stages. Our data shows that limb wounds that are first treated with a combination of FGF-2, FGF-8, and BMP-2, followed by RA treatment of the resultant mid-bud stage Blastema, will result in the generation of limbs with complete proximal/distal and anterior/posterior limb axes. Thus, the minimal signaling requirements from the nerve and a positional disparity are achieved with the application of this specific combination of signaling molecules.
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position specific induction of ectopic limbs in non regenerating Blastemas on axolotl forelimbs
Regeneration (Oxford England), 2014Co-Authors: Catherine D Mccusker, Jeffrey Lehrberg, David M GardinerAbstract:Ectopic retinoic acid (RA) has been hypothesized to reprogram the positional identity of cells in developing and regenerating limbs to a single positional value corresponding to the posterior-ventral-proximal (PVPr) position on the limb. We tested this hypothesis by using RA to reprogram the information of Blastema cells that were induced to form at different positions around the limb circumference. We observed that RA treatment of Blastemas in anterior and dorsal locations, but not posterior and ventral locations, resulted in the induction of complete ectopic limbs. These position-specific differences in limb induction are probably due to differences in the positional disparity between the RA-reprogrammed Blastema cells and the cells at the periphery of the wound. These observations are consistent with the hypothesis that RA treatment reprograms the information in Blastema cells to the PVPr position on the limb, since anterior and dorsal positions have the largest disparity and posterior and ventral have the smallest disparity from the PVPr identity.
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positional information is reprogrammed in Blastema cells of the regenerating limb of the axolotl ambystoma mexicanum
PLOS ONE, 2013Co-Authors: Catherine D Mccusker, David M GardinerAbstract:The regenerating region of an amputated salamander limb, known as the Blastema, has the amazing capacity to replace exactly the missing structures. By grafting cells from different stages and regions of Blastemas induced to form on donor animals expressing Green Fluorescent Protein (GFP), to non-GFP host animals, we have determined that the cells from early stage Blastemas, as well as cells at the tip of late stage Blastemas are developmentally labile such that their positional identity is reprogrammed by interactions with more proximal cells with stable positional information. In contrast, cells from the adjacent, more proximal stump tissues as well as the basal region of late bud Blastemas are positionally stable, and thus form ectopic limb structures when grafted. Finally, we have found that a nerve is required to maintain the Blastema cells in a positionally labile state, thus indicating a role for reprogramming cues in the Blastema microenvironment.