Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

BACKGROUND: Axolotls can regenerate their limbs. In their limb regeneration process, developmental genes are re-expressed and reorganize the developmental axes, in which the position-specific genes are properly re-expressed. However, how such position specificity is reorganized in the regeneration processes has not been clarified. To address this issue, we focused on the reactivation process of Lmx1b, which determines the limb dorsal identity in many animals. RESULTS: Here, we show that Lmx1b expression is maintained in the dorsal skin before amputation and is activated after amputation. Furthermore, we demonstrate that only cells located in the dorsal side prior to limb amputation could reactivate Lmx1b after limb amputation. We also found that Lmx1b activation was achieved by nerve presence. The nerve factors, BMP2+FGF2+FGF8 (B2FF), consistently reactivate Lmx1b when applied to the dorsal skin. CONCLUSIONS: These results imply that the retained Lmx1b expression in the intact skin plays a role in positional memory, which instruct cells about the spatial positioning before amputation. This memory is reactivated by nerves or nerve factors that can trigger the entire limb regeneration process. Our findings highlight the role of nerves in amphibian limb regeneration, including both the initiation of limb regeneration and the reactivation of position-specific gene expression.

DOI： 10.1002/dvdy.476

PubMed

researchmap

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/dvdy.476

researchmap

Publisher：Cold Spring Harbor Laboratory  

Abstract

Limb regeneration in Ambystoma mexicanum occurs in various size fields and can recreate consistent limb morphology. The mechanism that supports such stable limb morphogenesis regardless of size is unknown. Sonic hedgehog (SHH) and fibroblasts growth factor 8 (FGF8) play important roles in anteroposterior limb patterning, similar to other tetrapods. Focusing on these two factors, we investigated the detailed expression pattern and function of Shh and Fgf8 in various blastema sizes in axolotl limb regeneration. We measured and functionally analyzed the expression domains of Shh and Fgf8 in regenerating limb blastema of various sizes, and found that, although the position and size of the Shh⁺ and Fgf8⁺ domains varied depending on the size of the blastema, the secretion of SHH was maintained at a relatively fixed working distance, regardless of blastema size. This stable secretory distance of SHH resulted in the formation of an active proliferative zone (aPZ) in the vicinity of SHH, regardless of blastema size. The aPZ was under the mitogenic influence of SHH and FGF8, resulting in high cell density in the aPZ. We also examined the impact of the aPZ on digit formation. We found that the first digit formation occurs in the aPZ. Next, the aPZ gradually shifts posteriorly as digits develop, which contributes to new digit formation at the site of the shifted aPZ. We also found that the exogenously formed aPZ caused extra digit formation even after the completion of autopod morphogenesis. Our findings suggest that the variable Shh-Fgf8 positioning in various blastema sizes causes various positioning of the aPZ, and that the aPZ leads to digit formation. The mechanism we propose here accounts for stable digit morphogenesis regardless of blastema sizes and urodele-specific digit formation.

One-Sentence Summary

A unique SHH-FGF8 spatial interaction compensates for robust limb morphogenesis in various limb sizes.

DOI： 10.1101/2022.01.04.475010

researchmap

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Amphibians have a very high capacity for regeneration among tetrapods. This superior regeneration capability in amphibians can be observed in limbs, the tail, teeth, external gills, the heart, and some internal organs. The mechanisms underlying the superior organ regeneration capability have been studied for a long time. Limb regeneration has been investigated as the representative phenomenon for organ-level regeneration. In limb regeneration, a prominent difference between regenerative and nonregenerative animals after limb amputation is blastema formation. A regeneration blastema requires the presence of nerves in the stump region. Thus, nerve regulation is responsible for blastema induction, and it has received much attention. Nerve regulation in regeneration has been investigated using the limb regeneration model and newly established alternative experimental model called the accessory limb model. Previous studies have identified some candidate genes that act as neural factors in limb regeneration, and these studies also clarified related events in early limb regeneration. Consistent with the nervous regulation and related events in limb regeneration, similar regeneration mechanisms in other organs have been discovered. This review especially focuses on the role of nerve-mediated fibroblast growth factor in the initiation phase of organ regeneration. Comparison of the initiation mechanisms for regeneration in various amphibian organs allows speculation about a fundamental regenerative process.

DOI： 10.1002/jez.b.23093

PubMed

researchmap

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: Axolotls have remarkable organ-level regeneration capability. They can regenerate their limbs, tail, brain, gills, and heart. The liver had been considered to be a regenerative organ in these highly regeneration-competent animals. Therefore, no research had been performed on liver regeneration in urodele amphibians. In the present study, we focused on axolotl liver regeneration and found a unique regeneration mechanism compared with other vertebrates. RESULTS: Partial hepatectomy (PH) was performed to assess axolotl liver regeneration. Regeneration was assessed using block-face imaging (CoMBi), histology, cell proliferation, weight gain, and Albumin (Alb) + area. Axolotl liver histology was compared with other vertebrates. Axolotl liver consists of Glisson's capsule, sinusoids, and hepatic cord with no apparent lobule structures. Hepatocytes were mononucleated or multinucleated. PH increased the multinucleated hepatocytes and the Alb + area, but there was no apparent liver shape recovery even 40 days after PH. Gene expression pattern suggests that no epimorphic regeneration takes place. We also found that the increase in the number of proliferating hepatocytes was regulated by ERK-signaling. CONCLUSION: Our findings suggest that axolotls, which have epimorphic regeneration ability, regenerate their liver via unique mechanisms, compensatory congestion.

DOI： 10.1002/dvdy.262

PubMed

researchmap

Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nerve-dependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration. The presence of nerves is an important factor in successful organ regeneration in amphibians. The Mexican salamander, Ambystoma mexicanum, is able to regenerate limbs, tail, and gills when nerves are present. However, the nervedependency of tooth regeneration has not been evaluated. Here, we reevaluated tooth regeneration processes in axolotls using a three-dimensional reconstitution method called CoMBI and found that tooth regeneration is nerve-dependent although the dentary bone is independent of nerve presence. The induction and invagination of the dental lamina were delayed by denervation. Exogenous Fgf2, Fgf8, and Bmp7 expression could induce tooth placodes even in the denervated mandible. Our results suggest that the role of nerves is conserved and that Fgf+Bmp signals play key roles in axolotl organ-level regeneration.

DOI： 10.1038/s41598-020-66142-2

PubMed

researchmap

Authorship：Last author,　Corresponding author  

DOI： 10.1002/dvdy.96

PubMed

researchmap

Authorship：Last author,　Corresponding author  

DOI： 10.1016/j.ydbio.2019.04.011

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BioMed Central Ltd.  

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.

DOI： 10.1186/s40851-018-0090-2

Scopus

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier Inc.  

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.

DOI： 10.1016/j.ydbio.2017.10.007

Scopus

PubMed

researchmap

Authorship：Lead author,　Corresponding author  

DOI： 10.1387/ijdb.180118as

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Limb regeneration is considered a form of limb redevelopment because of the molecular and morphological similarities. Forming a regeneration blastema is, in essence, creating a developing limb bud in an adult body. This reactivation of a developmental process in a mature body is worth studying. Xenopus laevis has a biphasic life cycle that involves distinct larval and adult stages. These distinct developmental stages are useful for investigating the reactivation of developmental processes in post -metamorphic frogs (froglets). In this study, we focused on the re-expression of a larval gene (krt62.L) during Xenopus froglet limb regeneration. Recently renamed krt62.L, this gene was known as the larval keratin (xlk) gene, which is specific to larval-tadpole stages. During limb regeneration in a froglet, krt62.L was re-expressed in a basal layer of blastema epithelium, where adult-specific keratin (Krt12.6.S) expression was also observable. Nerves produce important regulatory factors for amphibian limb regeneration, and also play a role in blastema formation and maintenance. The effect of nerve function on krt62.L expression could be seen in the maintenance of krt62.L expression, but not in its induction. When an epidermis-stripped limb bud was grafted in a froglet blastema, the grafted limb bud could reach the digit-forming stage. This suggests that krt62.L-positive froglet blastema epithelium is able to support the limb development process. These findings imply that the developmental process is locally reactivated in an postmetamorphic body during limb regeneration.

DOI： 10.1016/j.ydbio.2017.10.015

Web of Science

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2016.07.005

Web of Science

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Urodele amphibians have remarkable organ regeneration ability. They can regenerate not only limbs but also a tail throughout their life. It has been demonstrated that the regeneration of some organs are governed by the presence of neural tissues. For instance, limb regeneration cannot be induced without nerves. Thus, identifying the nerve factors has been the primary focus in amphibian organ regeneration research. Recently, substitute molecules for nerves in limb regeneration, Bmp and Fgfs, were identified. Cooperative inputs of Bmp and Fgfs can induce limb regeneration in the absence of nerves. In the present study, we investigated whether similar or same regeneration mechanisms control another neural tissue governed organ regeneration, i.e., tail regeneration, in Ambystoma mexicanum. Neural tissues in a tail, which is the spinal cord, could transform wound healing responses into organ regeneration responses, similar to nerves in limb regeneration. Furthermore, the identified regeneration inducer Fgf2+Fgf8+Bmp7 showed similar inductive effects. However, further analysis revealed that the blastema cells induced by Fgf2+Fgf8+Bmp7 could participate in the regeneration of several tissues, but could not organize a patterned tail. Regeneration inductive ability of Fgf2+Fgf8+Bmp7 was confirmed in another urodele, Pleurodeles waltl. These results suggest that the organ regeneration ability in urodele amphibians is controlled by a common mechanism. (C) 2015 The Authors. Published by Elsevier Inc.

DOI： 10.1016/j.ydbio.2015.12.012

Web of Science

PubMed

J-GLOBAL

researchmap

DOI： 10.1002/reg2.39

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publisher：WILEY-BLACKWELL  

Limb regeneration ability, which can be observed in amphibians, has been investigated as a representative phenomenon of organ regeneration. Recently, an alternative experimental system called the accessory limb model was developed to investigate early regulation of amphibian limb regeneration. The accessory limb model contributed to identification of limb regeneration inducers in urodele amphibians. Furthermore, the accessory limb model may be applied to other species to explore universality of regeneration mechanisms. This review aims to connect the insights recently gained to emboss universality of regeneration mechanisms among species. The defined molecules (BMP7 (or2)+FGF2+FGF8) can transform skin wound healing to organ (limb) regeneration responses. The same molecules can initiate regeneration responses in some species.

DOI： 10.1111/dgd.12230

Web of Science

PubMed

J-GLOBAL

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

DOI： 10.1371/journal.pone.0133375

Web of Science

PubMed

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

researchmap

Authorship：Last author,　Corresponding author  

DOI： 10.1007/978-1-4939-2495-0_8

PubMed

J-GLOBAL

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of beta-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BISK1). The BISK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BISK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BISK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2014.03.027

Web of Science

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Urodele amphibians can regenerate their limbs after amputation. After amputation, undifferentiated cells appear on the amputation plane and form regeneration blastema. A limb blastema recreates a complete replica of the original limb. It is well known that disturbance of the location of limb tissues prior to amputation perturbs limb patterning, suggesting that different intact limb tissues carry different location information despite their identical appearance. The cause of such differences in intact tissues remains unknown. In this study, we found that Lmx1b, Tbx2, and Tbx3 genes, which are expressed in developing limb in a region specific manner, remained detectable in a mature axolotl limb. Furthermore, those position-specific gene expression patterns were conserved in mature limbs. Treatment with retinoic acid ( RA), which is known to have ventralizing activity, changed Lmx1b expression in intact dorsal skin and dorsal character to ventral, indicating that conserved Lmx1b expression was due to the dorsal character and not leaky gene expression. Furthermore, we found that such conserved gene expression was rewritable in regeneration blastemas. These results suggest that axolotl limb cells can recognize their locations and maintain limbness via conserved expression profiles of developmental genes.

DOI： 10.2108/zsj.31.6

Web of Science

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

Urodele amphibians can regenerate their limbs. During limb regeneration, dermal fibroblasts are transformed into undifferentiated cells called blastema cells. These dermis-blastema cells show multipotency. Such so-called endogenous reprogramming of cell differentiation is one of the main targets of amphibian limb regeneration studies. It is well recognized that nerve presence controls the initiation of limb regeneration. Accordingly, nerve factors have been sought in amphibian limb regeneration. To investigate it, a relatively new study system called the accessory limb model (ALM) was developed. Using ALM, two signaling cascades (Fgf and Gdf5 signaling) came under focus. In the present study, Growth and differentiation factor-5 (Gdf5) application to wounded skin initiated limb regeneration responses and resulted in induction of a blastema-like structure in the absence of a nerve. However, the Gdf5-induced structure showed defects as a regeneration blastema, such as absence of detectable Prrx1 expression by in situ hybridization. The defects could be remedied by additional Fibroblasts growth factor (Fgf) inputs. These two inputs (Gdf5 and Fgfs) were sufficient to substitute for the nerve functions in the induction of limb regeneration. Indeed, Fgf2, Fgf8, and Gdf5 applications with the contralateral skin graft resulted in limb formation without nerve supply. Furthermore, acquisition of cartilage differentiation potential of dermal fibroblasts was tested in an in vivo and in vitro combination assay. Dermal fibroblasts cultured with Gdf5 were difficult to participate in cartilage formation when the cultured cells were grafted into cartilage forming region. In contrast, dermal fibroblasts cultured with Fgf2 and Fgf8 became easier to participate into cartilage formation in the same procedure. These results contribute to our understanding of molecular mechanisms of the early phase of amphibian limb regeneration. © 2013 The Authors.

DOI： 10.1016/j.ydbio.2013.05.010

Scopus

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Background: Urodele amphibians have high regeneration capability that has been studied for a long time. Recently, a new experimental system called the accessory limb model was developed and becomes alternative choice for amphibian limb regeneration study. Although the accessory limb model has many advantages, an improvement was needed for some specific analysis, such as studying muscle origin. For that purpose, an accessory limb induction on nonlimb regions was attempted. Results: Accessory limb induction on a nonlimb region (flank) was possible by nerve deviation and limb skin grafting. Retinoic acid injections improved the induction rate. The induced limb possessed the same tissue context as a normal limb. Muscle cells were also abundantly observed. It is speculated that the muscle cells are derived from flank muscle tissues, because limb muscle cells are a migratory cell population and the accessory limb was induced apart from the original limb. We also found that migration of the muscle cells was regulated by Hgf/cMet signaling as in other vertebrates. Conclusions: Accessory limb induction was possible even in the nonlimb flank region. The flank-induced limb would be useful for further analysis of limb regeneration, especially for migratory cell populations such as muscle cells. Developmental Dynamics 242:932-940, 2013. © 2013 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.23984

Scopus

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Amphibian limb regeneration has been studied for a long time. In amphibian limb regeneration, an undifferentiated blastema is formed around the region damaged by amputation. The induction process of blastema formation has remained largely unknown because it is difficult to study the induction of limb regeneration. The recently developed accessory limb model (ALM) allows the investigation of limb induction and reveals early events of amphibian limb regeneration. The interaction between nerves and wound epidermis/epithelium is an important aspect of limb regeneration. During early limb regeneration, neurotrophic factors act on wound epithelium, leading to development of a functional epidermis/epithelium called the apical epithelial cap (AEC). AEC and nerves create a specific environment that inhibits wound healing and induces regeneration through blastema formation. It is suggested that FGF-signaling and MMP activities participate in creating a regenerative environment. To understand why urodele amphibians can create such a regenerative environment and humans cannot, it is necessary to identify the similarities and differences between regenerative and nonregenerative animals. Here we focus on ALM to consider limb regeneration from a new perspective and we also reported that focal adhesion kinase (FAK)Src signaling controlled fibroblasts migration in axolotl limb regeneration. Anat Rec, 2012. (c) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/ar.22529

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2012.07.021

Web of Science

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Background: Amphibians have greater regeneration capability than higher vertebrates. They can regenerate their limbs after an amputation. As a limb is regenerated, a regeneration-specific epithelium called the apical epithelial cap (AEC) is induced. The AEC is an essential structure for limb regeneration. Despite the importance of the AEC, molecular marker genes have not been well studied at the molecular level. Results: In the present study, keratin5 (KRT5) and KRT17 were investigated in an axolotl-regenerating limb. KRT5 and KRT17 were expressed in a regenerating limb but down-regulated in a differentiating limb. KRT5 showed characteristic regulation in a regenerating blastema. KRT5 was suppressed in the basal layer of the AEC. This KRT5 suppression was correlated to the blastema differentiation and nerve presence. Simple skin wounding could also upregulate both KRT5 and KRT17 gene expression. But these genes were suppressed within a shorter time than in limb regeneration. Conclusions: The KRT5 and KRT17 gene profile can be a useful marker gene to investigate AEC in limb regeneration. Developmental Dynamics 241:16161624, 2012. (c) 2012 Wiley Periodicals, Inc.

DOI： 10.1002/dvdy.23839

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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.

DOI： 10.1111/j.1440-169X.2012.01368.x

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2012.03.022

Web of Science

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.

DOI： 10.2108/zsj.29.191

Web of Science

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2011.04.017

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.

DOI： 10.1111/j.1440-169X.2010.01214.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically < 11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.

DOI： 10.1111/j.1440-169X.2010.01208.x

Web of Science

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Little effort has been made to apply the insights gained from studies of amphibian limb regeneration to higher vertebrates. During amphibian limb regeneration, a functional epithelium called the apical ectodermal cap (AEC) triggers a regenerative response. As long as the AEC is induced, limb regeneration will take place. Interestingly, similar responses have been observed in chicken embryos. The AEC is an equivalent structure to the apical ectodermal ridge (AER) in higher vertebrates. When a limb bud is amputated it does not regenerate; however, if the AER is grafted onto the amputation surface, damage to the amputated limb bud can be repaired. Thus, the AER/AEC is able to induce regenerative responses in both amphibians and higher vertebrates. It is difficult, however, to induce limb regeneration in higher vertebrates. One reason for this is that re-induction of the AER after amputation in higher vertebrates is challenging. Here, we evaluated whether AER re-induction was possible in higher vertebrates. First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred. These results open up new possibilities for limb regeneration in higher vertebrates since AER re-induction, which is considered a key factor in limb regeneration, is now possible. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2010.03.018

Web of Science

PubMed

researchmap

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

The accessory limb model has become an alternative model for performing investigations of limb regeneration in an amputated limb. In the accessory limb model, a complete patterned limb can be induced as a result of an interaction between the wound epithelium, a nerve and dermal fibroblasts in the skin. Studies should therefore focus on examining these tissues. To date, however, a study of cellular contributions in the accessory limb model has not been reported. By using green fluorescent protein (GFP) transgenic axolotl tissues, we can trace cell fate at the tissue level. Therefore, in the present study, we transgrafted GFP skin onto the limb of a non-GFP host and induced an accessory limb to investigate cellular contributions. Previous studies of cell contribution to amputation-induced blastemas have demonstrated that dermal cells are the progenitors of many of the early blastema cells, and that these cells contribute to regeneration of the connective tissues, including cartilage. In the present study, we have determined that this same population of progenitor cells responds to signaling from the nerve and wound epithelium in the absence of limb amputation to form an ectopic blastema and regenerate the connective tissues of an ectopic limb. Blastema cells from dermal fibroblasts, however, did not differentiate into either muscle or neural cells, and we conclude that dermal fibroblasts are dedifferentiated along its developmental lineage.

DOI： 10.1111/j.1440-169X.2009.01165.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

During limb regeneration, anuran tadpoles and urodele amphibians generate pattern-organizing, multipotent, mesenchymal blastema cells, which give rise to a replica of the lost limb including patterning in three dimensions. To facilitate the regeneration of nonregenerative limbs in other vertebrates, it is important to elucidate the molecular differences between blastema cells that can regenerate the pattern of limbs and those that cannot. In Xenopus froglet (soon after metamorphosis), an amputated limb generates blastema cells that do not produce proper patterning, resulting in a patternless regenerate, a spike, regardless of the amputation level. We found that re-expression of hoxa11 and hoxa13 in the froglet blastema is initiated although the subsequent proximal-distal patterning, including separation of the hoxa11 and hoxa13 expression domains, is disrupted. We also observed an absence of EphA4 gene expression in the froglet blastema and a failure of position-dependent cell sorting, which correlated with the altered hoxa11 and hoxa13 expression. Quantitative analysis of hoxa11 and hoxa13 expression revealed that hoxa13 transcript levels were reduced in the froglet blastema compared with the tadpole blastema. Moreover, the expression of sox9, an important regulator of chondrogenic differentiation, was detected earlier in patternless blastemas than in tadpole blastemas. These results suggest that appropriate spatial, temporal, and quantitative gene expression is necessary for pattern regeneration by blastema cells. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2009.11.026

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a "critical-size defect" (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2009.11.023

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Regenerating limbs of urodele amphibians and Xenopus tadpole are reconstructed along proximal-distal, anterior-posterior (AP), and dorsal-ventral axes. In contrast, a regenerated limb of the Xenopus froglet does not have digits, and only a simple cartilaginous structure referred to as a "spike" is formed. This suggests that repatterning along the AP axis is absent in the froglet blastema. Previous studies have shown that Shh and its target genes are not expressed in the froglet blastema. In this study, we activated Hedgehog signaling in the froglet blastema and found that target genes of Shh were inducible in the mesenchyme of limb blastema. Furthermore, we found that activation of the signaling had effects on blastema cell proliferation and chondrogenesis and resulted in the formation of multiple cartilaginous structures. These findings indicate that activation of signaling that is absent in the froglet blastema is effective for improvement of limb regeneration ability in the Xenopus froglet. Developmental Dynamics 238:1887-1896, 2009. (C) 2009 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22011

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOURNAL BONE JOINT SURGERY INC  

DOI： 10.2106/JBJS.I.00159

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

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.

DOI： 10.1111/j.1440-169X.2008.01072.x

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium. (c) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2008.04.030

Web of Science

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Mitf is a transcription factor of the basic/helix-loop-helix/leucine-zipper family which is indispensable for development of melanocytes and the retinal pigment epithelium. Our previous work using Xenopus laevis as a model system suggested that Mitf regulates melanosome dispersal in vivo though whether this was via melanosome transport or melanophore dendricity was not obvious. To better understand the role of Mitf, we have now characterized neural tube cultures from wild-type Mitf-injected or a dominant-negative Mitf-injected embryos and compared them with controls. In vitro, lower levels of Mitf activity induced less dendritic melanophores with aggregated melanosomes, whereas melanophores overexpressing Mitf had an extensive dendritic morphology with dispersed melanosomes. Moreover, immunorfluoresence assays reveal that expression of a dominant-negative Mitf leads to decreased Rab27a expression. These results suggest that Mitf is involved in the regulation of melanosome transport and the level of dendricity in melanophores.

DOI： 10.1111/j.1755-148X.2007.00420.x

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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 repatteming) 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 repatteming 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. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.09.022

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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 blasterna, cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.09.021

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Understanding the mechanisms that control amphibian limb regeneration should allow us to decipher the critical differences between amphibians and humans, which have the limited ability of organ regeneration. However, many issues at the cellular and molecular levels still remain unresolved. We have generated a transgenic Xenopus laevis line that expresses green fluorescent protein (GFP) under the control of mouse prxI limb enhancer, which directs reporter gene expression in limb mesenchyme in mice, and found that GFP accumulated in blastemal mesenchymal cells of the transgenic froglets after limb amputation. Thus, this transgenic line should provide a new approach to gain insights into the cellular dynamics and signaling pathways involved in limb blastema formation. We have also developed a culture system for forelimb explants of froglets and found that treatment with inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase 1/2 (MEK1/2) and phosphatidylinositol 3-kinase (PI3K) repressed GFP expression. These effects were partially reversible, and down-regulation of GFP was associated with inhibition of cell-cycle progression and induction of ectopic apoptosis. In addition, we found that ERK1/2 and AKT, downstream mediators of MEK1/2 and PI3K pathways, were activated in amputated forelimb stumps. These results demonstrate that MEK/ERK and PI3K/AKT pathways regulate limb blastema formation in the X laevis froglet. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.01.019

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：THESCIENTIFICWORLD LTD  

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.

DOI： 10.1100/tsw.2006.325

Web of Science

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Xenopus has 4 and 5 digits in a forelimb and hindlimb, respectively. It is thought that their limbs and digits develop in Xenopus by mechanisms that are almost conserved from amphibians to higher vertebrates. This is supported by some molecular evidence. The 5'hoxd genes are convenient marker genes for characterizing digits in the chick and mouse. The anteriormost digit is characterized by being hoxd13-positive and hoxd12 (hoxd11)-negative in the chick and mouse. In this study, we revealed that the anteriormost digit of the Xenopus forelimb is hoxd13-positive and hoxd11-positive, that is, a more posterior character than digit I. The order of formation of digit cartilages also suggested that Xenopus forelimb digit identity is 11 to V, not I to IV. We have also been interested in the relationship between digit identity and shh. The anteriormost digit develops in a shh-independent way. A limb treated with cyclopamine (a shh inhibitor) has a gene expression pattern (hoxd11-negative) similar to that in shh-deficient mice, suggesting that a hindlimb treated with cyclopamine has a digit I character. However, a Xenopus froglet regenerate (spike), which lacks shh expression during its regeneration process, does not have such an expression pattern, being hoxd11-positive. We investigated hoxd11 transcriptions in blastemas that formed in the anteriormost and posteriormost digits, and we found that the blastemas have different hoxd11 expression levels. These findings suggest that the froglet limb blastema does not have a mere digit I character in spite of shh defectiveness and that the froglet limb blastema recognizes its positional differences along the anterior-posterior axis.

DOI： 10.1002/dvdy.20985

Web of Science

PubMed

researchmap

DOI： 10.1100/tsw.2006.325

PubMed

researchmap

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Xenopus laevis larvae can regenerate an exact replica of the missing part of a limb after amputation at an early limb bud stage. However, this regenerative capacity gradually decreases during metamorphosis, and a froglet is only able to regenerate hypomorphic cartilage, resulting in a spike-like structure (spike). It has been reported that the spike has tissue deformities, e.g., a muscleless structure. However, our previous study demonstrated that the muscleless feature of the spike can be improved. The existence of other kinds of tissue, such as tendon, has not been clarified. In this study, we focused on the tendon and dermis, and we isolated the scleraxis and dermo-1 genes, which are known to be marker genes for the tendon and dermis, respectively. The expressions of these genes were investigated in both the developmental and regenerating processes of a Xenopus limb. Although muscle was needed to maintain scleraxis expression, scleraxis transcription was detectable in the muscleless spike. Additionally, although grafting of matured skin, including dermal tissue, inhibited limb regeneration, the expression of dermo-1, a dermal marker gene, was detected from the early stage of the froglet blastema. These results indicate that tendon precursor cells and dermal cells exist in the regenerating froglet blastema. Our results support the idea that spike formation in postmetamorphic Xenopus limbs is epimorphic regeneration.

DOI： 10.1002/dvdy.20701

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In Xenopus laevis, limb buds start to develop at a later point of the larval stage, prior to metamorphosis. This onset of limb development in Xenopus is totally different from that in amniotes such as birds and mammals, in which limb buds emerge at an early stage of embryogenesis, in parallel with other organogenesis. We investigated limb myogenesis in Xenopus, focusing on myogenic gene expression, myogenic ability of limb bud cells in the early stage, and the origin of myogenic precursor cells in the limb bud. The Xenopus early limb bud contains myoD/cardiac actin-positive and pax3/pax7-negative cells. Interestingly, results of transplantation experiments have revealed that this early limb bud contains myogenic precursor cells. In order to know the contribution of myogenic cells in somites to myogenic precursor cells in the early limb bud, we used a Cre-LoxP system for tracing over a long period. The results of fate tracing for myogenic cells in somites of the Xenopus embryo suggested that early-specified myogenic cells in somites do not contribute to limb muscle in Xenopus. Taken together, the results suggest that limb muscle development in Xenopus has characteristics of initiation and early events distinct from those of other vertebrate clades.

DOI： 10.1002/dvdy.20573

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

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 fort-nation, 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 Pryl, 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 Msxl 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. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ybio.2005.08.021

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

In Xenopus laevis, amputation of the adult limb results in the formation of a simple (hypomorphic) spike-like structure without joints, although tadpole limb bud regenerates complete limb pattern. The expression of some joint marker genes was examined in limb development and regeneration. Bmp center dot 4 and gdf center dot 5 were expressed and sox-9 expression was decreased in the joint region. Although developing cartilages were well-organized and had bmp-4 expressing perichondrocytes, the spike cartilage did not have such a structure, but only showed sparse bmp-4 expression. Application of BMP4-soaked beads to the spike led to the induction of a joint-like structure. These results suggest that the lack of joints in the spike is due to the deficiency of the accumulation of the cells that express bmp-4. Improvement of regeneration in the Xenopus adult limb that we report here for the first time will give us important insights into epimorphic regeneration. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.20484

Web of Science

PubMed

researchmap

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

A spike, a resultant regenerate made after amputation of a Xenopus froglet limb, has no muscle tissue. This muscle-less phenotype was analyzed by molecular approaches, and the results of analysis revealed that the spike expresses no myosin heavy chain or Pax7, suggesting that neither mature muscle tissue nor satellite cells exist in the spike. The regenerating blastema in the froglet limb lacked some myogenesis-related marker genes, myoD and myf5, but allowed implanted muscle precursor cells to survive and differentiate into myofiber. Implantation of hepatocyte growth factor (HGF)-releasing cell aggregates rescued this muscle-less phenotype and induced muscle regeneration in Xenopus froglet limb regenerates. These results suggest that failure of regeneration of muscle is due to a disturbance of the early steps of myogenesis under a molecular cascade mediated by HGF/c-met. Improvement of muscle regeneration in the Xenopus adult limb that we report here for the first time will give us important insights into epimorphic tissue regeneration in amphibians and other vertebrates. (c) 2005 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.20349

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE BV  

Web of Science

researchmap

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER SCIENCE BV  

Web of Science

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Presentation type：Poster presentation  

researchmap

Presentation type：Poster presentation  

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

researchmap

researchmap

researchmap

Language：English   Presentation type：Symposium, workshop panel (nominated)  

researchmap

Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

File： SRSS-JSID 2022 8 Feb22.pdf

researchmap

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

researchmap

Presentation type：Poster presentation  

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Presentation type：Symposium, workshop panel (nominated)  

researchmap

Venue：岡崎カンファレンスホール  

researchmap

Venue：品川  

researchmap

Venue：長崎新聞文化ホール  

researchmap

Venue：船堀タワーホール  

researchmap

Venue：札幌コンベンションセンター  

researchmap

Venue：CDB  

researchmap

Venue：Edinburph  

researchmap

Venue：東京（船堀タワーホール）  

researchmap

Venue：富山大会  

researchmap

Venue：イイノホール  

researchmap

Venue：基礎生物学研究所  

researchmap

Venue：東京（船堀タワーホール）  

researchmap

Venue：仙台国際施センター  

researchmap

Venue：岡山大学  

researchmap

Venue：鳥取コンベンションセンター  

researchmap

Venue：Okazaki Japan NIBB  

researchmap

Venue：OIST  

researchmap

Venue：岡崎コンフェレンスセンター  

researchmap

Venue：パシフィコ横浜  

researchmap

Venue：北海道大学  

researchmap

Venue：神戸  

researchmap

Venue：新潟 朱鷺メッセ  

researchmap

Venue：Florida, Tampa  

researchmap

Venue：神戸  

researchmap

Venue：筑波国際会議場  

researchmap

Venue：熊本大学  

researchmap

Venue：仙台  

researchmap

Venue：スペイン  

researchmap

Venue：長野県 高遠 さくらホテル  

researchmap

Venue：札幌駅地下B１F  

researchmap

Venue：岡山理科大学  

researchmap

Venue：岡山大学 J.Fホール  

researchmap

researchmap

Venue：未来科学博物館  

researchmap

Venue：神戸しあわせの村  

researchmap

Venue：岡山大学 歯学部  

researchmap

Venue：パシフィコ横浜  

researchmap

Venue：岡山  

researchmap

Venue：岡山・牛窓  

researchmap

Venue：東京大学  

researchmap

Venue：松江（くにびきメッセ）  

researchmap

Venue：Suzhou Dushu Lake Conference Center (中国蘇州）  

researchmap

Venue：Taipei  

researchmap

Venue：神戸  

researchmap

Venue：ウインク愛知  

researchmap

researchmap

Author：Other 

researchmap

researchmap

Author：Other 

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

researchmap

Type：Academic society, research group, etc. 

researchmap

Type：Competition, symposium, etc. 

researchmap

Type：Academic society, research group, etc. 

researchmap

Type：Academic society, research group, etc. 

researchmap