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1.
Biol Open ; 13(2)2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38319134

RESUMO

Limb regeneration in salamanders is achieved by a complex coordination of various biological processes and requires the proper integration of new tissue with old. Among the tissues found inside the limb, the skeleton is the most prominent component, which serves as a scaffold and provides support for locomotion in the animal. Throughout the years, researchers have studied the regeneration of the appendicular skeleton in salamanders both after limb amputation and as a result of fracture healing. The final outcome has been widely seen as a faithful re-establishment of the skeletal elements, characterised by a seamless integration into the mature tissue. The process of skeletal integration, however, is not well understood, and several works have recently provided evidence of commonly occurring flawed regenerates. In this Review, we take the reader on a journey through the course of bone formation and regeneration in salamanders, laying down a foundation for critically examining the mechanisms behind skeletal integration. Integration is a phenomenon that could be influenced at various steps of regeneration, and hence, we assess the current knowledge in the field and discuss how early events, such as tissue histolysis and patterning, influence the faithful regeneration of the appendicular skeleton.


Assuntos
Locomoção , Osteogênese , Animais , Humanos , Pesquisadores , Urodelos
2.
Nat Commun ; 14(1): 6346, 2023 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-37816738

RESUMO

Humans and other tetrapods are considered to require apical-ectodermal-ridge (AER) cells for limb development, and AER-like cells are suggested to be re-formed to initiate limb regeneration. Paradoxically, the presence of AER in the axolotl, a primary model organism for regeneration, remains controversial. Here, by leveraging a single-cell transcriptomics-based multi-species atlas, composed of axolotl, human, mouse, chicken, and frog cells, we first establish that axolotls contain cells with AER characteristics. Further analyses and spatial transcriptomics reveal that axolotl limbs do not fully re-form AER cells during regeneration. Moreover, the axolotl mesoderm displays part of the AER machinery, revealing a program for limb (re)growth. These results clarify the debate about the axolotl AER and the extent to which the limb developmental program is recapitulated during regeneration.


Assuntos
Ambystoma mexicanum , Galinhas , Humanos , Animais , Camundongos , Extremidades , Ectoderma , Regulação da Expressão Gênica no Desenvolvimento
3.
Nat Methods ; 20(8): 1117-1119, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37553398
4.
Methods Mol Biol ; 2562: 155-163, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272073

RESUMO

The axolotl is a great model for studying cartilage, bone and joint regeneration, fracture healing, and evolution. Stainings such as Alcian Blue/Alizarin Red have become workhorses in skeletal analyses, but additional methods complement the detection of different skeletal matrices. Here we describe protocols for studying skeletal biology in axolotls, particularly Alcian Blue/Alizarin Red staining, microcomputed tomography (µCT) scan and live staining of calcified tissue. In addition, we describe a method for decalcification of skeletal elements to ease sectioning.


Assuntos
Ambystoma mexicanum , Biologia , Animais , Azul Alciano , Microtomografia por Raio-X , Coloração e Rotulagem
5.
Methods Mol Biol ; 2562: 235-247, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272080

RESUMO

Embryo grafts have been an experimental pillar in developmental biology, and particularly, in amphibian biology. Grafts have been essential in constructing fate maps of different cell populations and migratory patterns. Likewise, autografts and allografts in older larvae or adult salamanders have been widely used to disentangle mechanisms of regeneration. The combination of transgenesis and grafting has widened even more the application of this technique.In this chapter, we provide a detailed protocol for embryo transplants in the axolotl (Ambystoma mexicanum ). The location and stages to label connective tissue, muscle, or blood vessels in the limb and blood cells in the whole animal. However, the potential of embryo transplants is enormous and impossible to cover in one chapter. Furthermore, we provide a protocol for blastema transplantation as an example of allograft in older larvae.


Assuntos
Ambystoma mexicanum , Extremidades , Animais , Ambystoma mexicanum/fisiologia , Extremidades/fisiologia , Tecido Conjuntivo , Larva/fisiologia
6.
Methods Mol Biol ; 2562: 321-333, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272085

RESUMO

The axolotl (Ambystoma mexicanum ) has been widely used as an animal model for studying development and regeneration. In recent decades, the use of genetic engineering to alter gene expression has advanced our knowledge on the fundamental molecular and cellular mechanisms, pointing us to potential therapeutic targets. We present a detailed, step-by-step protocol for axolotl transgenesis using either I-SceI meganuclease or the mini Tol2 transposon system, by injection of purified DNA into one-cell stage eggs. We add useful tips on the site of injection and the viability of the eggs.


Assuntos
Ambystoma mexicanum , Desoxirribonucleases de Sítio Específico do Tipo II , Animais , Ambystoma mexicanum/genética , Ambystoma mexicanum/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Técnicas de Transferência de Genes , DNA/genética , Injeções
7.
Elife ; 112022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36218256

RESUMO

Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.


Assuntos
Ambystoma mexicanum , Osteoclastos , Animais , Ambystoma mexicanum/fisiologia , Ácido Zoledrônico , Extremidades/fisiologia , Esqueleto
8.
Open Biol ; 12(6): 220078, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35728623

RESUMO

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.


Assuntos
Ambystoma mexicanum , Animais , Microscopia Confocal/métodos
9.
Wound Repair Regen ; 30(6): 652-664, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35596643

RESUMO

The vast majority of species that belong to the plant or animal kingdom evolved with two main strategies to counter tissue damage-scar formation and regeneration. Whereas scar formation provides a fast and cost-effective repair to exit life-threatening conditions, complete tissue regeneration is time-consuming and requires vast resources to reinstall functionality of affected organs or structures. Local environments in wound healing are widely studied and findings have provided important biomedical applications. Less well understood are organismic physiological parameters and signalling circuits essential to maintain effective tissue repair. Here, we review accumulated evidence that positions the interplay of local and systemic changes in metabolism as essential variables modulating the injury response. We particularly emphasise the role of lipids and lipid-like molecules as significant components long overlooked.


Assuntos
Cicatriz , Cicatrização , Animais , Cicatrização/fisiologia , Cicatriz/patologia , Transdução de Sinais
10.
Dev Dyn ; 251(6): 906-910, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35451159

RESUMO

The third annual meeting on "Salamander Models in Cross-disciplinary Biological Research" took place online on August 2021, bringing together over 200 international researchers using salamanders as research models and encompassing diverse fields, ranging from Development and Regeneration through to Immunology, Pathogenesis, and Evolution. The event was organized by Maximina H. Yun (Center for Regenerative Therapies Dresden, Germany) and Tatiana Sandoval-Guzmán (TU Dresden, Germany) with the generous support of the Deutsche Forschungsgemeinschaft, the Center for Regenerative Therapies Dresden, Technische Universität Dresden, and the Company of Biologists. Showcasing a number of emerging salamander models, innovative techniques and resources, and providing a platform for sharing both published and ongoing research, this meeting proved to be an excellent forum for exchanging ideas and moving research forwards. Here, we discuss the highlights stemming from this exciting scientific event.


Assuntos
Urodelos , Animais , Alemanha
11.
Dev Dyn ; 251(6): 1015-1034, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34322944

RESUMO

BACKGROUND: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.


Assuntos
Ambystoma mexicanum , Osso e Ossos , Envelhecimento , Animais , Desenvolvimento Ósseo , Osteogênese
12.
Nat Commun ; 12(1): 3354, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099670

RESUMO

Barrett's esophagus in gastrointestinal reflux patients constitutes a columnar epithelium with distal characteristics, prone to progress to esophageal adenocarcinoma. HOX genes are known mediators of position-dependent morphology. Here we show HOX collinearity in the adult gut while Barrett's esophagus shows high HOXA13 expression in stem cells and their progeny. HOXA13 overexpression appears sufficient to explain both the phenotype (through downregulation of the epidermal differentiation complex) and the oncogenic potential of Barrett's esophagus. Intriguingly, employing a mouse model that contains a reporter coupled to the HOXA13 promotor we identify single HOXA13-positive cells distally from the physiological esophagus, which is mirrored in human physiology, but increased in Barrett's esophagus. Additionally, we observe that HOXA13 expression confers a competitive advantage to cells. We thus propose that Barrett's esophagus and associated esophageal adenocarcinoma is the consequence of expansion of this gastro-esophageal HOXA13-expressing compartment following epithelial injury.


Assuntos
Esôfago de Barrett/genética , Carcinogênese/genética , Proteínas de Homeodomínio/genética , Oncogenes/genética , Adulto , Animais , Esôfago de Barrett/metabolismo , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/metabolismo , Trato Gastrointestinal/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Família Multigênica/genética , RNA-Seq/métodos
13.
Biol Open ; 8(7)2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31278164

RESUMO

The heterogeneous properties of dermal cell populations have been posited to contribute toward fibrotic, imperfect wound healing in mammals. Here we characterize an adult population of dermal fibroblasts that maintain an active Prrx1 enhancer which originally marked mesenchymal limb progenitors. In contrast to their abundance in limb development, postnatal Prrx1 enhancer-positive cells (Prrx1enh+) make up a small subset of adult dermal cells (∼0.2%) and reside mainly within dermal perivascular and hair follicle niches. Lineage tracing of adult Prrx1enh+ cells shows that they remain in their niches and in small numbers over a long period of time. Upon injury however, Prrx1enh+ cells readily migrate into the wound bed and amplify, on average, 16-fold beyond their uninjured numbers. Additionally, following wounding dermal Prrx1enh+ cells are found out of their dermal niches and contribute to subcutaneous tissue. Postnatal Prrx1enh+ cells are uniquely injury-responsive despite being a meager minority in the adult skin.

14.
Curr Opin Cell Biol ; 55: 36-41, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30031323

RESUMO

The process of building an organ, appendage, or organism requires the precise coordination of cells in space and time. Regeneration of those same tissues adds an additional element of complexity, emerging from the chaos of disease or injury to build a mass of progenitors from mature tissue. Translating insights from natural examples of tissue regeneration into engineered regenerative therapies requires a deep understanding of the journey of a cell directly following injury to its contribution to functional, scaled replacement tissue. Here we step through the chronological phases of regeneration and highlight emerging work that brings us closer to elucidating the unique intrinsic and extrinsic properties of cells during epimorphic regeneration.


Assuntos
Células/metabolismo , Regeneração , Animais , Desdiferenciação Celular , Movimento Celular , Transdução de Sinais , Cicatrização
15.
Nat Protoc ; 9(3): 529-40, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24504478

RESUMO

The axolotl (Mexican salamander, Ambystoma mexicanum) has become a very useful model organism for studying limb and spinal cord regeneration because of its high regenerative capacity. Here we present a protocol for successfully mating and breeding axolotls in the laboratory throughout the year, for metamorphosing axolotls by a single i.p. injection and for axolotl transgenesis using I-SceI meganuclease and the mini Tol2 transposon system. Tol2-mediated transgenesis provides different features and advantages compared with I-SceI-mediated transgenesis, and it can result in more than 30% of animals expressing the transgene throughout their bodies so that they can be directly used for experimentation. By using Tol2-mediated transgenesis, experiments can be performed within weeks (e.g., 5-6 weeks for obtaining 2-3-cm-long larvae) without the need to establish germline transgenic lines (which take 12-18 months). In addition, we describe here tamoxifen-induced Cre-mediated recombination in transgenic axolotls.


Assuntos
Ambystoma mexicanum/fisiologia , Criação de Animais Domésticos/métodos , Cruzamento/métodos , Técnicas de Transferência de Genes , Metamorfose Biológica/fisiologia , Modelos Animais , Animais , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Recombinação Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Tamoxifeno , Transposases/metabolismo
17.
Cell Stem Cell ; 14(2): 174-87, 2014 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-24268695

RESUMO

Salamanders regenerate appendages via a progenitor pool called the blastema. The cellular mechanisms underlying regeneration of muscle have been much debated but have remained unclear. Here we applied Cre-loxP genetic fate mapping to skeletal muscle during limb regeneration in two salamander species, Notophthalmus viridescens (newt) and Ambystoma mexicanum (axolotl). Remarkably, we found that myofiber dedifferentiation is an integral part of limb regeneration in the newt, but not in axolotl. In the newt, myofiber fragmentation results in proliferating, PAX7(-) mononuclear cells in the blastema that give rise to the skeletal muscle in the new limb. In contrast, myofibers in axolotl do not generate proliferating cells, and do not contribute to newly regenerated muscle; instead, resident PAX7(+) cells provide the regeneration activity. Our results therefore show significant diversity in limb muscle regeneration mechanisms among salamanders and suggest that multiple strategies may be feasible for inducing regeneration in other species, including mammals.


Assuntos
Ambystoma mexicanum/fisiologia , Desdiferenciação Celular , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Salamandridae/fisiologia , Células-Tronco/citologia , Animais , Animais Geneticamente Modificados , Proliferação de Células , Extremidades/fisiologia , Genes Reporter , Células Germinativas/citologia , Células Germinativas/metabolismo , Larva/fisiologia , Mesoderma/citologia , Mesoderma/transplante , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Fator de Transcrição PAX7/metabolismo
18.
Stem Cell Reports ; 1(1): 90-103, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24052945

RESUMO

The salamander is the only tetrapod that regenerates complex body structures throughout life. Deciphering the underlying molecular processes of regeneration is fundamental for regenerative medicine and developmental biology, but the model organism had limited tools for molecular analysis. We describe a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. We demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. Furthermore, we demonstrate the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. Finally, we inducibly overexpress the cell-cycle inhibitor p16 (INK4a) , which negatively regulates spinal cord regeneration. These tissue-specific germline axolotl lines and tightly inducible Cre drivers and LoxP reporter lines render this classical regeneration model molecularly accessible.


Assuntos
Rastreamento de Células/métodos , Marcação de Genes/métodos , Células Germinativas/metabolismo , Regeneração Nervosa , Ambystoma mexicanum , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Mutação em Linhagem Germinativa , Neurônios/citologia , Neurônios/metabolismo , Recombinação Genética
19.
BMC Dev Biol ; 13: 17, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23641815

RESUMO

BACKGROUND: Molecular studies of appendage regeneration have been hindered by the lack of a stable and efficient means of transferring exogenous genes. We therefore sought an efficient integrating virus system that could be used to study limb and tail regeneration in salamanders. RESULTS: We show that replication-deficient foamy virus (FV) vectors efficiently transduce cells in two different regeneration models in cell culture and in vivo. Injection of EGFP-expressing FV but not lentivirus vector particles into regenerating limbs and tail resulted in widespread expression that persisted throughout regeneration and reamputation pointing to the utility of FV for analyzing adult phenotypes in non-mammalian models. Furthermore, tissue specific transgene expression is achieved using FV vectors during limb regeneration. CONCLUSIONS: FV vectors are efficient mean of transferring genes into axolotl limb/tail and infection persists throughout regeneration and reamputation. This is a nontoxic method of delivering genes into axolotls in vivo/ in vitro and can potentially be applied to other salamander species.


Assuntos
Técnicas de Transferência de Genes , Regeneração/genética , Spumavirus/genética , Animais , Linhagem Celular , Vetores Genéticos , Humanos , Urodelos
20.
FASEB J ; 26(12): 4966-76, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22954591

RESUMO

Activins are critical components of the signaling network that controls female reproduction. However, their roles in hypothalamus, and the specific functions of their different receptors, have not been elucidated. Here, we investigated the expression and function of the activin receptor ALK7 in the female reproductive axis using Alk7-knockout mice. ALK7 was found in subsets of SF1-expressing granulosa cells in the ovary, FSH gonadotrophs in the pituitary, and NPY-expressing neurons in the arcuate nucleus of the hypothalamus. Alk7-knockout females showed delayed onset of puberty and abnormal estrous cyclicity, had abnormal diestrous levels of FSH and LH in serum, and their ovaries showed premature depletion of follicles, oocyte degeneration, and impaired responses to exogenous gonadotropins. In the arcuate nucleus, mutant mice showed reduced expression of Npy mRNA and lower numbers of Npy-expressing neurons than wild-type controls. Alk7 knockouts showed a selective loss of arcuate NPY/AgRP innervation in the medial preoptic area, a key central regulator of reproduction. These results indicate that ALK7 is an important regulator of female reproductive function and reveal a new role for activin signaling in the control of hypothalamic gene expression and wiring. Alk7 gene variants may contribute to female reproductive disorders in humans, such as polycystic ovary syndrome.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Hipófise/metabolismo , Reprodução/fisiologia , Transdução de Sinais/fisiologia , Receptores de Ativinas Tipo I/genética , Animais , Ciclo Estral/genética , Ciclo Estral/fisiologia , Feminino , Hormônio Foliculoestimulante/sangue , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hormônio Luteinizante/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Sistemas Neurossecretores/metabolismo , Ovário/metabolismo , Ovário/patologia , Reprodução/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Maturidade Sexual/genética , Maturidade Sexual/fisiologia , Transdução de Sinais/genética , Técnicas de Cultura de Tecidos
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