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David M Gardiner - One of the best experts on this subject based on the ideXlab platform.
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gene expression during the first 28 days of axolotl Limb Regeneration i experimental design and global analysis of gene expression
Regeneration (Oxford England), 2015Co-Authors: Randal S Voss, David M Gardiner, Ken Muneoka, Alex Palumbo, Radha Nagarajan, Arnold J Stromberg, Antony AthippozhyAbstract:While it is appreciated that global gene expression analyses can provide novel insights about complex biological processes, experiments are generally insufficiently powered to achieve this goal. Here we report the results of a robust microarray experiment of axolotl foreLimb Regeneration. At each of 20 post‐amputation time points, we estimated gene expression for 10 replicate RNA samples that were isolated from 1 mm of heterogeneous tissue collected from the distal Limb tip. We show that the Limb transcription program diverges progressively with time from the non‐injured state, and divergence among time adjacent samples is mostly gradual. However, punctuated episodes of transcription were identified for five intervals of time, with four of these coinciding with well‐described stages of Limb Regeneration—amputation, early bud, late bud, and pallet. The results suggest that Regeneration is highly temporally structured and regulated by mechanisms that function within narrow windows of time to coordinate transcription within and across cell types of the regenerating Limb. Our results provide an integrative framework for hypothesis generation using this complex and highly informative data set.
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the axolotl Limb blastema cellular and molecular mechanisms driving blastema formation and Limb Regeneration in tetrapods
Regeneration (Oxford England), 2015Co-Authors: Catherine D Mccusker, Susan V Bryant, David M GardinerAbstract:The axolotl is one of the few tetrapods that are capable of regenerating complicated biological structures, such as complete Limbs, throughout adulthood. Upon injury the axolotl generates a population of Regeneration-competent Limb progenitor cells known as the blastema, which will grow, establish pattern, and differentiate into the missing Limb structures. In this review we focus on the crucial early events that occur during wound healing, the neural-epithelial interactions that drive the formation of the early blastema, and how these mechanisms differ from those of other species that have restricted regenerative potential, such as humans. We also discuss how the presence of cells from the different axes of the Limb is required for the continued growth and establishment of pattern in the blastema as described in the polar coordinate model, and how this positional information is reprogrammed in blastema cells during Regeneration. Multiple cell types from the mature Limb stump contribute to the blastema at different stages of Regeneration, and we discuss the contribution of these types to the regenerate with reference to whether they are "pattern-forming" or "pattern-following" cells. Lastly, we explain how an engineering approach will help resolve unanswered questions in Limb Regeneration, with the goal of translating these concepts to developing better human regenerative therapies.
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understanding positional cues in salamander Limb Regeneration implications for optimizing cell based regenerative therapies
Disease Models & Mechanisms, 2014Co-Authors: Catherine D Mccusker, David M GardinerAbstract:Regenerative medicine has reached the point where we are performing clinical trials with stem-cell-derived cell populations in an effort to treat numerous human pathologies. However, many of these efforts have been challenged by the inability of the engrafted populations to properly integrate into the host environment to make a functional biological unit. It is apparent that we must understand the basic biology of tissue integration in order to apply these principles to the development of regenerative therapies in humans. Studying tissue integration in model organisms, where the process of integration between the newly regenerated tissues and the ‘old’ existing structures can be observed and manipulated, can provide valuable insights. Embryonic and adult cells have a memory of their original position, and this positional information can modify surrounding tissues and drive the formation of new structures. In this Review, we discuss the positional interactions that control the ability of grafted cells to integrate into existing tissues during the process of salamander Limb Regeneration, and discuss how these insights could explain the integration defects observed in current cell-based regenerative therapies. Additionally, we describe potential molecular tools that can be used to manipulate the positional information in grafted cell populations, and to promote the communication of positional cues in the host environment to facilitate the integration of engrafted cells. Lastly, we explain how studying positional information in current cell-based therapies and in regenerating Limbs could provide key insights to improve the integration of cell-based regenerative therapies in the future.
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retrotransposon long interspersed nucleotide element 1 line 1 is activated during salamander Limb Regeneration
Development Growth & Differentiation, 2012Co-Authors: Wei Zhu, Akira Satoh, Susan V Bryant, Gerald M Pao, Dwight Kuo, Jason L Nathanson, Gene W Yeo, Randal S Voss, David M GardinerAbstract:Salamanders possess an extraordinary capacity for tissue and organ Regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander Limb Regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during Regeneration. In addition, higher genomic LINE-1 content was also detected in the Limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during Regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the Limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl Limb Regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of Limb Regeneration.
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regulation of dermal fibroblast dedifferentiation and redifferentiation during wound healing and Limb Regeneration in the axolotl
Development Growth & Differentiation, 2008Co-Authors: Akira Satoh, Susan V Bryant, David M GardinerAbstract:Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The Accessory Limb Model (ALM) in the axolotl allows for the identification of signals from the wound epidermis, nerves and dermal fibroblasts that interact to regenerate a Limb. In the present study, we have used the ALM to identity the axolotl (Ambystoma mexicanum) orthologue of Twist (AmTwist), a basic helix-loop-helix transcription factor that is involved in the Regeneration of the dermis during Limb Regeneration. AmTwist is expressed during the blastema stages in Regeneration, but is inhibited by signals from the nerve during the early stages when dermal fibroblasts dedifferentiate to form blastema cells. As the dermis regenerates, AmTwist is expressed in association with the synthesis of type I collagen in the proximal region of the blastema. Exogenous bone morphogenetic protein-2 leads to an increase in AmTwist expression, and therefore may function as an endogenous regulator of AmTwist expression and dermis Regeneration. The nerve appears to have a dual function in Regeneration by coordinately regulating dedifferentiation and redifferentiation of dermal fibroblasts.
Jeremy P. Brockes - One of the best experts on this subject based on the ideXlab platform.
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mechanisms underlying vertebrate Limb Regeneration lessons from the salamander
Biochemical Society Transactions, 2014Co-Authors: Jeremy P. Brockes, Phillip B. GatesAbstract:Limb Regeneration in adult salamanders proceeds by formation of a mound of progenitor cells called the Limb blastema. It provides several pointers for regenerative medicine. These include the role of differentiated cells in the origin of the blastema, the role of regenerating axons of peripheral nerves and the importance of cell specification in conferring morphogenetic autonomy on the blastema. One aspect of Regeneration that has received less attention is the ability to undergo multiple episodes without detectable change in the outcome, and with minimal effect of aging. We suggest that, although such pointers are valuable, it is important to understand why salamanders are the only adult tetrapod vertebrates able to regenerate their Limbs. Although this remains a controversial issue, the existence of salamander-specific genes that play a significant role in the mechanism of Regeneration provides evidence for the importance of local evolution, rather than a purely ancestral mechanism. The three-finger protein called Prod1 is discussed in the present article as an exemplar of this approach.
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regulation of p53 is critical for vertebrate Limb Regeneration
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Maximina H Yun, Phillip B. Gates, Jeremy P. BrockesAbstract:Extensive Regeneration of the vertebrate body plan is found in salamander and fish species. In these organisms, Regeneration takes place through reprogramming of differentiated cells, proliferation, and subsequent redifferentiation of adult tissues. Such plasticity is rarely found in adult mammalian tissues, and this has been proposed as the basis of their inability to regenerate complex structures. Despite their importance, the mechanisms underlying the regulation of the differentiated state during Regeneration remain unclear. Here, we analyzed the role of the tumor-suppressor p53 during salamander Limb Regeneration. The activity of p53 initially decreases and then returns to baseline. Its down-regulation is required for formation of the blastema, and its up-regulation is necessary for the redifferentiation phase. Importantly, we show that a decrease in the level of p53 activity is critical for cell cycle reentry of postmitotic, differentiated cells, whereas an increase is required for muscle differentiation. In addition, we have uncovered a potential mechanism for the regulation of p53 during Limb Regeneration, based on its competitive inhibition by ΔNp73. Our results suggest that the regulation of p53 activity is a pivotal mechanism that controls the plasticity of the differentiated state during Regeneration.
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the aneurogenic Limb identifies developmental cell interactions underlying vertebrate Limb Regeneration
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Anoop Kumar, Phillip B. Gates, Jean Paul Delgado, Graham Neville, Andrew Forge, Jeremy P. BrockesAbstract:The removal of the neural tube in salamander embryos allows the development of nerve-free aneurogenic Limbs. Limb Regeneration is normally nerve-dependent, but the aneurogenic Limb regenerates without nerves and becomes nerve-dependent after innervation. The molecular basis for these tissue interactions is unclear. Anterior Gradient (AG) protein, previously shown to rescue Regeneration of denervated Limbs and to act as a growth factor for cultured Limb blastemal cells, is expressed throughout the larval Limb epidermis and is down-regulated by innervation. In an aneurogenic Limb, the level of AG protein remains high in the epidermis throughout development and Regeneration, but decreases after innervation following transplantation to a normal host. Aneurogenic epidermis also shows a fivefold difference in secretory gland cells, which express AG protein. The persistently high expression of AG in the epithelial cells of an aneurogenic Limb ensures that Regeneration is independent of the nerve. These findings provide an explanation for this classical problem, and identify regulation of the epidermal niche by innervation as a distinctive developmental mechanism that initiates the nerve dependence of Limb Regeneration. The absence of this regulation during anuran Limb development might suggest that it evolved in relation to Limb Regeneration.
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functional convergence of signalling by gpi anchored and anchorless forms of a salamander protein implicated in Limb Regeneration
Journal of Cell Science, 2011Co-Authors: Robert A Blassberg, Phillip B. Gates, Acely Garzagarcia, Azara Janmohamed, Jeremy P. BrockesAbstract:The GPI-anchor is an established determinant of molecular localisation and various functional roles have been attributed to it. The newt GPI-anchored three-finger protein (TFP) Prod1 is an important regulator of cell behaviour during Limb Regeneration, but it is unclear how it signals to the interior of the cell. Prod1 was expressed by transfection in cultured newt Limb cells and activated transcription and expression of matrix metalloproteinase 9 (MMP9) by a pathway involving ligand-independent activation of epidermal growth factor receptor (EGFR) signalling and phosphorylation of extracellular regulated kinase 1 and 2 (ERK1/2). This was dependent on the presence of the GPI-anchor and critical residues in the α-helical region of the protein. Interestingly, Prod1 in the axolotl, a salamander species that also regenerates its Limbs, was shown to activate ERK1/2 signalling and MMP9 transcription despite being anchorless, and both newt and axolotl Prod1 co-immunoprecipitated with the newt EGFR after transfection. The substitution of the axolotl helical region activated a secreted, anchorless version of the newt molecule. The activity of the newt molecule cannot therefore depend on a unique property conferred by the anchor. Prod1 is a salamander-specific TFP and its interaction with the phylogenetically conserved EGFR has implications for our view of Regeneration as an evolutionary variable.
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evidence for the local evolution of mechanisms underlying Limb Regeneration in salamanders
Integrative and Comparative Biology, 2010Co-Authors: Acely Garzagarcia, Paul C Driscoll, Jeremy P. BrockesAbstract:Synopsis The most extensive regenerative ability in adult vertebrates is found in the salamanders. Although it is often suggested that Regeneration is an ancestral property for vertebrates, our studies on the cell-surface three-finger-protein Prod 1 provide clear evidence for the importance of local evolution of Limb Regeneration in salamanders. Prod 1 is implicated in both patterning and growth in the Regeneration of Limbs. It interacts with well-conserved proteins such as the epidermal growth-factor receptor and the anterior gradient protein that are widely expressed in phylogeny. A detailed analysis of the structure and sequence of Prod 1 in relation to other vertebrate three-finger proteins in mammals and zebra fish supports the view that it is a salamander-specific protein. This is the first example of a taxon-specific protein that is clearly implicated in the mechanisms of Regeneration. We propose the hypothesis that Regeneration depends on the activity of taxon-specific components in orchestrating a cellular machinery that is extensively conserved between regenerating and non-regenerating taxa. This hypothesis has significant implications for our outlook on Regeneration in vertebrates, as well as for the strategies employed in extending regenerative ability in mammals.
Fulin Chen - One of the best experts on this subject based on the ideXlab platform.
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integrative analysis of micrornaome transcriptome and proteome during the Limb Regeneration of cynops orientalis
Journal of Proteome Research, 2019Co-Authors: Jie Tang, Jihong Cui, Xin Xie, Fulin ChenAbstract:Salamanders completely regenerate their Limbs after amputation. Thus, these animals are unique models to investigate the mechanisms modulating Regeneration in vertebrates. To investigate the influence of microRNAs (miRNAs) on newt Limb Regeneration, the miRNAs and mRNAs were simultaneously profiled using Illumina HiSeq 2500 System during Limb Regeneration of Cynops orientalis at 3, 7, 14, 30 and 42 days postamputation. A total of 203 miRNAs and 4230 mRNAs were identified to be differentially expressed. Together with the proteomic data obtained from our previous study, integrative analysis of multiple profiling data sets was performed to construct an interaction network of differentially expressed miRNAs, mRNAs and proteins. Results of GO and KEGG analyses showed that the differentially expressed miRNA targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism. The stage-specific regulation of miRNAs on their targets was analyzed by hierarchical clustering analysis and validated by qRT-PCR. The negative regulation of miR-223 and miR-133a on their targets was tested by performing dual luciferase reporter assay. The integration analysis will provide a powerful tool to identify the regulatory mechanisms of miRNAs and their targets. The results may have implications in understanding the complex mechanisms underlying newt Limb Regeneration.
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Integrative Analysis of MicroRNAome, Transcriptome, and Proteome during the Limb Regeneration of Cynops orientalis
2019Co-Authors: Jie Tang, Jihong Cui, Xin Xie, Fulin ChenAbstract:Salamanders completely regenerate their Limbs after amputation. Thus, these animals are unique models to investigate the mechanisms modulating Regeneration in vertebrates. To investigate the influence of microRNAs (miRNAs) on newt Limb Regeneration, the miRNAs and mRNAs were simultaneously profiled using Illumina HiSeq 2500 System during Limb Regeneration of Cynops orientalis at 3, 7, 14, 30 and 42 days postamputation. A total of 203 miRNAs and 4230 mRNAs were identified to be differentially expressed. Together with the proteomic data obtained from our previous study, integrative analysis of multiple profiling data sets was performed to construct an interaction network of differentially expressed miRNAs, mRNAs and proteins. Results of GO and KEGG analyses showed that the differentially expressed miRNA targets were mainly directed to cytoskeletal remodeling and carbohydrate metabolism. The stage-specific regulation of miRNAs on their targets was analyzed by hierarchical clustering analysis and validated by qRT-PCR. The negative regulation of miR-223 and miR-133a on their targets was tested by performing dual luciferase reporter assay. The integration analysis will provide a powerful tool to identify the regulatory mechanisms of miRNAs and their targets. The results may have implications in understanding the complex mechanisms underlying newt Limb Regeneration
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itraq based quantitative proteomic analysis of cynops orientalis Limb Regeneration
BMC Genomics, 2017Co-Authors: Jie Tang, Hanxue Zheng, Lu Yin, Mei Sun, Wenjun Wang, Jihong Cui, Wenguang Liu, Xin Xie, Fulin ChenAbstract:Salamanders regenerate their Limbs after amputation. However, the molecular mechanism of this unique Regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating Limbs 3, 7, 14, 30 and 42 days post amputation (dpa). Of 2636 proteins detected in total, 253 proteins were differentially expressed during different Regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in Limb Regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding. This work indicated that significant proteome alternations occurred during salamander Limb Regeneration. The results may provide fundamental knowledge to understand the mechanism of Limb Regeneration.
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Additional file 2: Table S2. of ITRAQ-based quantitative proteomic analysis of Cynops orientalis Limb Regeneration
2017Co-Authors: Jie Tang, Hanxue Zheng, Lu Yin, Mei Sun, Wenjun Wang, Jihong Cui, Wenguang Liu, Xin Xie, Fulin ChenAbstract:The differentially expression proteins (DEPs) identified during Cynops orientalis Limb Regeneration. (XLSX 194 kb
Akira Satoh - One of the best experts on this subject based on the ideXlab platform.
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stability and plasticity of positional memory during Limb Regeneration in ambystoma mexicanum
Developmental Dynamics, 2020Co-Authors: Reiko Iwata, Aki Makanae, Akira SatohAbstract:Background Urodele amphibians are capable of regenerating their organs after severe damage. During such Regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb Regeneration has been investigated as a representative phenomenon of organ Regeneration. Cells known as blastema cells are induced after Limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to Limb bud cells. Just like Limb bud cells, the induced blastema cells are positioned along the three Limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing Limb axes was not well described with molecular markers. Results In this study, we grafted skin from GFP-transgenic axolotls and traced the cell lineage with position-specific gene expressions in order to investigate the correlation of the newly established axes and cellular origin. Shh- and Lmx1b-expressing cells emerged from the posterior skin and dorsal skin, respectively, even though the skin was transplanted to an inconsistent position. Shox2, a posterior marker gene, could be activated in cells derived from distal skin. Conclusions Our results suggest that the location memories on anteroposterior and dorsoventral axes are relatively stable in a regenerating blastema though cellular differentiation is reprogrammed.
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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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Ectopic Fgf signaling induces the intercalary response in developing chicken Limb buds
BMC, 2018Co-Authors: Aki Makanae, Akira SatohAbstract:Abstract Background Intercalary pattern formation is an important regulatory step in amphibian Limb Regeneration. Amphibian Limb Regeneration is composed of multiple steps, including wounding, blastema formation, and intercalary pattern formation. Attempts have been made to transfer insights from Regeneration-competent animals to Regeneration-incompetent animalsat each step in the Regeneration process. In the present study, we focused on the intercalary mechanism in chick Limb buds. In amphibian Limb Regeneration, a proximodistal axis is organized as soon as a regenerating blastema is induced. Intermediate structures are subsequently induced (intercalated) between the established proximal and distal identities. Intercalary tissues are derived from proximal tissues. Fgf signaling mediates the intercalary response in amphibian Limb Regeneration. Results We attempted to transfer insights into intercalary Regeneration from amphibian models to the chick Limb bud. The zeugopodial part was dissected out, and the distal and proximal parts were conjunct at st. 24. Delivering ectopic Fgf2 + Fgf8 between the distal and proximal parts resulted in induction of zeugopodial elements. Examination of HoxA11 expression, apoptosis, and cell proliferation provides insights to compare with those in the intercalary mechanism of amphibian Limb Regeneration. Furthermore, the cellular contribution was investigated in both the chicken intercalary response and that of axolotl Limb Regeneration. Conclusions We developed new insights into cellular contribution in amphibian intercalary Regeneration, and found consistency between axolotl and chicken intercalary responses. Our findings demonstrate that the same principal of Limb Regeneration functions between Regeneration-competent and -incompetent animals. In this context, we propose the feasibility of the induction of the Regeneration response in amniotes
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FGF and BMP derived from dorsal root ganglia regulate blastema induction in Limb Regeneration in Ambystoma mexicanum.
Developmental Biology, 2016Co-Authors: Akira Satoh, Aki Makanae, Yurie Nishimoto, Kazumasa MitogawaAbstract:Urodele amphibians have a remarkable organ Regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in Limb and tail Regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating Regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful Limb Regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating Limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the Limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired Limb Regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ Regeneration ability.
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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.
Elly M Tanaka - One of the best experts on this subject based on the ideXlab platform.
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pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl Limb Regeneration
Developmental Biology, 2018Co-Authors: David N. Drechsel, Akira Tazaki, Catarina R Oliveira, Regis P Lemaitre, Prayag Murawala, Elly M TanakaAbstract:Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers Regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature Limb tissue and within the blastema. These cells remain competent to participate in Regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl Limb Regeneration.
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Efficient gene knockin in axolotl and its use to test the role of satellite cells in Limb Regeneration.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Ji-feng Fei, Dunja Knapp, Maritta Schuez, Yuka Taniguchi, David N. Drechsel, Elly M TanakaAbstract:Salamanders exhibit extensive regenerative capacities and serve as a unique model in Regeneration research. However, due to the lack of targeted gene knockin approaches, it has been difficult to label and manipulate some of the cell populations that are crucial for understanding the mechanisms underlying Regeneration. Here we have established highly efficient gene knockin approaches in the axolotl (Ambystoma mexicanum) based on the CRISPR/Cas9 technology. Using a homology-independent method, we successfully inserted both the Cherry reporter gene and a larger membrane-tagged Cherry-ERT2-Cre-ERT2 (∼5-kb) cassette into axolotl Sox2 and Pax7 genomic loci. Depending on the size of the DNA fragments for integration, 5-15% of the F0 transgenic axolotl are positive for the transgene. Using these techniques, we have labeled and traced the PAX7-positive satellite cells as a major source contributing to myogenesis during axolotl Limb Regeneration. Our work brings a key genetic tool to molecular and cellular studies of axolotl Regeneration.
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fgf8 and shh substitute for anterior posterior tissue interactions to induce Limb Regeneration
Nature, 2016Co-Authors: Eugeniu Nacu, David N. Drechsel, Elena Gromberg, Catarina R Oliveira, Elly M TanakaAbstract:In salamanders, grafting of a left Limb blastema onto a right Limb stump yields Regeneration of three Limbs, the normal Limb and two 'supernumerary' Limbs. This experiment and other research have shown that the juxtaposition of anterior and posterior Limb tissue plus innervation are necessary and sufficient to induce complete Limb Regeneration in salamanders. However, the cellular and molecular basis of the requirement for anterior-posterior tissue interactions were unknown. Here we have clarified the molecular basis of the requirement for both anterior and posterior tissue during Limb Regeneration and supernumerary Limb formation in axolotls (Ambystoma mexicanum). We show that the two tissues provide complementary cross-inductive signals that are required for Limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a Limb, but activation of hedgehog (HH) signalling was sufficient to drive Regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signalling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signalling was not sufficient to drive Regeneration; however, ectopic expression of FGF8 together with endogenous HH signalling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on sonic hedgehog (SHH) signalling from posterior tissue. Together, our findings identify key anteriorly and posteriorly localized signals that promote Limb Regeneration and show that these single factors are sufficient to drive non-regenerating blastemas to complete Regeneration with full elaboration of skeletal elements.
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progressive specification rather than intercalation of segments during Limb Regeneration
Science, 2013Co-Authors: Kathleen Roensch, Akira Tazaki, Osvaldo Chara, Elly M TanakaAbstract:An amputated salamander Limb regenerates the correct number of segments. Models explaining Limb Regeneration were largely distinct from those for Limb development, despite the presence of common patterning molecules. Intercalation has been an important concept to explain salamander Limb Regeneration, but clear evidence supporting or refuting this model was lacking. In the intercalation model, the first blastema cells acquire fingertip identity, creating a gap in positional identity that triggers Regeneration of the intervening region from the stump. We used HOXA protein analysis and transplantation assays to show that axolotl Limb blastema cells acquire positional identity in a proximal-to-distal sequence. Therefore, intercalation is not the primary mechanism for segment formation during Limb Regeneration in this animal. Patterning in development and Regeneration uses similar mechanisms.
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Connective tissue cells, but not muscle cells, are involved in establishing the proximo-distal outcome of Limb Regeneration in the axolotl.
Development, 2013Co-Authors: Eugen Nacu, Mareen Glausch, Huy Quang Le, Febriyani Fiain Rochel Damanik, Dunja Knapp, Tobias Richter, Shahryar Khattak, Maritta Schuez, Elly M TanakaAbstract:During salamander Limb Regeneration, only the structures distal to the amputation plane are regenerated, a property known as the rule of distal transformation. Multiple cell types are involved in Limb Regeneration; therefore, determining which cell types participate in distal transformation is important for understanding how the proximo-distal outcome of Regeneration is achieved. We show that connective tissue-derived blastema cells obey the rule of distal transformation. They also have nuclear MEIS, which can act as an upper arm identity regulator, only upon upper arm amputation. By contrast, myogenic cells do not obey the rule of distal transformation and display nuclear MEIS upon amputation at any proximo-distal level. These results indicate that connective tissue cells, but not myogenic cells, are involved in establishing the proximo-distal outcome of Regeneration and are likely to guide muscle patterning. Moreover, we show that, similarly to Limb development, muscle patterning in Regeneration is influenced by β-catenin signalling.