Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 48
Filtrar
1.
Development ; 149(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35266986

RESUMO

Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals.


Assuntos
Ambystoma mexicanum , Mitose , Animais , Ciclo Celular , Extremidades/fisiologia , Hibridização in Situ Fluorescente , Ubiquitinação
2.
FASEB J ; 38(19): e70101, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39387181

RESUMO

Bone is a three-dimensional (3D) highly dynamic tissue under constant remodeling. Commonly used tools to investigate bone biology require sample digestion for biomolecule extraction or provide only two-dimensional (2D) spatial information. There is a need for 3D tools to investigate spatially preserved biomarker expression in osteocytes. In this work, we present a new method, WISH-BONE, to label osteocyte messenger RNA (mRNA) and protein in whole-mount mouse bone. For mRNA labeling, we used hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) to label genes of interest in osteocytes. For protein labeling, samples were preserved using an epoxy-based solution that protects tissue structure and biomolecular components. Then an enzymatic matrix permeabilization step was performed to enable antibody penetration. Immunostaining was used to label various proteins involved in bone homeostasis. We also demonstrate the use of customized fluorescent nanobodies to target and label proteins in the cortical bone (CB). However, the relatively dim signal observed from nanobodies' staining limited detection. mRNA and protein labeling were performed in separate samples. In this study, we share protocols, highlight opportunities, and identify the challenges of this novel 3D labeling method. They are the first protocols for whole-mount osteocyte 3D labeling of mRNA and protein in mature mouse bones. WISH-BONE will allow the investigation of molecular signaling in bone cells in their 3D environment and could be applied to various bone-related fields of research.


Assuntos
Osteócitos , RNA Mensageiro , Animais , Osteócitos/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Hibridização in Situ Fluorescente/métodos , Osso e Ossos/metabolismo , Camundongos Endogâmicos C57BL , Masculino
3.
Proc Natl Acad Sci U S A ; 118(14)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33795516

RESUMO

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.


Assuntos
Acetilcolina/metabolismo , Técnicas Biossensoriais/métodos , Gânglios Parassimpáticos/metabolismo , Nanotecnologia/métodos , Acetilcolina/análise , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Bungarotoxinas/farmacologia , Carbocianinas/química , Antagonistas Colinérgicos/farmacologia , DNA/química , Feminino , Corantes Fluorescentes/química , Gânglios Parassimpáticos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Colinérgicos/metabolismo
4.
Gastroenterology ; 160(3): 755-770.e26, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33010250

RESUMO

BACKGROUND & AIMS: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODS: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction. RESULTS: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes. CONCLUSIONS: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.


Assuntos
Diferenciação Celular/genética , Sistema Nervoso Entérico/fisiologia , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Especificidade da Espécie , Adolescente , Adulto , Animais , Núcleo Celular/metabolismo , Colo/citologia , Colo/inervação , Modelos Animais de Doenças , Duodeno/citologia , Duodeno/inervação , Feminino , Gastroenteropatias/diagnóstico , Gastroenteropatias/genética , Gastroenteropatias/fisiopatologia , Motilidade Gastrointestinal , Humanos , Íleo/citologia , Íleo/inervação , Microdissecção e Captura a Laser , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/citologia , RNA-Seq , Fatores Sexuais , Análise de Célula Única , Adulto Jovem
5.
Proc Biol Sci ; 289(1975): 20220621, 2022 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-35582804

RESUMO

Movement-induced forces are critical to correct joint formation, but it is unclear how cells sense and respond to these mechanical cues. To study the role of mechanical stimuli in the shaping of the joint, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a transient receptor potential vanilloid 4 (TRPV4) agonist during joint morphogenesis. We quantified growth and shape in regrown humeri from whole-mount light sheet fluorescence images of the regenerated limbs. Results revealed significant differences in morphology and cell proliferation between groups, indicating that TRPV4 desensitization has an effect on joint shape. To link TRPV4 desensitization with impaired mechanosensitivity, we developed a finite element model of a regenerating humerus. Local tissue growth was the sum of a biological contribution proportional to chondrocyte density, which was constant, and a mechanical contribution proportional to fluid pressure. Computational predictions of growth agreed with experimental outcomes of joint shape, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.


Assuntos
Ambystoma mexicanum , Urodelos , Animais , Membro Anterior/anatomia & histologia , Morfogênese , Canais de Cátion TRPV
6.
Bioconjug Chem ; 33(11): 2223-2233, 2022 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-36327428

RESUMO

The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.


Assuntos
Química Click , Receptor Muscarínico M2 , Animais , Camundongos , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Corantes Fluorescentes/química , Ligantes , Alcinos
7.
Dev Dyn ; 250(6): 866-879, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33587313

RESUMO

BACKGROUND: Ambystoma mexicanum, the axolotl salamander, is a classic model organism used to study vertebrate regeneration. It is assumed that axolotls regenerate most tissues, but the exploration of lung regeneration has not been performed until now. RESULTS: Unlike the blastema-based response used during appendage regeneration, lung amputation led to organ-wide proliferation. Pneumocytes and mesenchymal cells responded to injury by increased proliferation throughout the injured lung, which led to a recovery in lung mass and morphology by 56 days post-amputation. Receptors associated with the Neuregulin signaling pathway were upregulated at one and 3 weeks post lung amputation. We show expression of the ligand, neuregulin, in the I/X cranial nerve that innervates the lung and cells within the lung. Supplemental administration of Neuregulin peptide induced widespread proliferation in the lung similar to an injury response, suggesting that neuregulin signaling may play a significant role during lung regeneration. CONCLUSION: Our study characterizes axolotl lung regeneration. We show that the lung responds to injury by an organ-wide proliferative response of multiple cell types, including pneumocytes, to recover lung mass.


Assuntos
Ambystoma mexicanum/fisiologia , Proliferação de Células/fisiologia , Lesão Pulmonar/fisiopatologia , Pulmão/fisiologia , Regeneração/fisiologia , Animais , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Neurregulinas/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima
8.
Development ; 144(4): 601-611, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087637

RESUMO

Salamanders are capable of regenerating amputated limbs by generating a mass of lineage-restricted cells called a blastema. Blastemas only generate structures distal to their origin unless treated with retinoic acid (RA), which results in proximodistal (PD) limb duplications. Little is known about the transcriptional network that regulates PD duplication. In this study, we target specific retinoic acid receptors (RARs) to either PD duplicate (RA treatment or RARγ agonist) or truncate (RARß antagonist) regenerating limbs. RARE-EGFP reporter axolotls showed divergent reporter activity in limbs undergoing PD duplication versus truncation, suggesting differences in patterning and skeletal regeneration. Transcriptomics identified expression patterns that explain PD duplication, including upregulation of proximal homeobox gene expression and silencing of distal-associated genes, whereas limb truncation was associated with disrupted skeletal differentiation. RARß antagonism in uninjured limbs induced a loss of skeletal integrity leading to long bone regression and loss of skeletal turnover. Overall, mechanisms were identified that regulate the multifaceted roles of RARs in the salamander limb including regulation of skeletal patterning during epimorphic regeneration, skeletal tissue differentiation during regeneration, and homeostatic regeneration of intact limbs.


Assuntos
Ambystoma mexicanum/fisiologia , Padronização Corporal , Receptores do Ácido Retinoico/metabolismo , Regeneração/fisiologia , Animais , Osso e Ossos/metabolismo , Diferenciação Celular , Extremidades/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Homeostase , Transcriptoma , Tretinoína/metabolismo , Microtomografia por Raio-X , Receptor gama de Ácido Retinoico
9.
Development ; 143(15): 2724-31, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27317805

RESUMO

The Mexican axolotl (Ambystoma mexicanum) is capable of fully regenerating amputated limbs, but denervation of the limb inhibits the formation of the post-injury proliferative mass called the blastema. The molecular basis behind this phenomenon remains poorly understood, but previous studies have suggested that nerves support regeneration via the secretion of essential growth-promoting factors. An essential nerve-derived factor must be found in the blastema, capable of rescuing regeneration in denervated limbs, and its inhibition must prevent regeneration. Here, we show that the neuronally secreted protein Neuregulin-1 (NRG1) fulfills all these criteria in the axolotl. Immunohistochemistry and in situ hybridization of NRG1 and its active receptor ErbB2 revealed that they are expressed in regenerating blastemas but lost upon denervation. NRG1 was localized to the wound epithelium prior to blastema formation and was later strongly expressed in proliferating blastemal cells. Supplementation by implantation of NRG1-soaked beads rescued regeneration to digits in denervated limbs, and pharmacological inhibition of NRG1 signaling reduced cell proliferation, blocked blastema formation and induced aberrant collagen deposition in fully innervated limbs. Taken together, our results show that nerve-dependent NRG1/ErbB2 signaling promotes blastemal proliferation in the regenerating limb and may play an essential role in blastema formation, thus providing insight into the longstanding question of why nerves are required for axolotl limb regeneration.


Assuntos
Ambystoma mexicanum/metabolismo , Regeneração Nervosa/fisiologia , Neuregulina-1/metabolismo , Ambystoma mexicanum/fisiologia , Animais , Western Blotting , Extremidades/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Regeneração Nervosa/efeitos dos fármacos , Regeneração Nervosa/genética , Neuregulina-1/genética , Oxazóis/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Triazóis/farmacologia
10.
Glycoconj J ; 36(2): 165-174, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30963354

RESUMO

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.


Assuntos
Sulfatos de Condroitina/análise , Heparitina Sulfato/análise , Ácido Hialurônico/análise , Sulfato de Queratano/análise , Retina/química , Ambystoma mexicanum , Animais , Sulfatos de Condroitina/metabolismo , Heparitina Sulfato/metabolismo , Ácido Hialurônico/metabolismo , Sulfato de Queratano/metabolismo , Retina/metabolismo , Suínos
11.
Genesis ; 56(6-7): e23214, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29956448

RESUMO

Analysis of cell cycle entry/exit and progression can provide fundamental insights into stem cell propagation, maintenance, and differentiation. The neural crest is a unique stem cell population in vertebrate embryos that undergoes long-distance collective migration and differentiation into a wide variety of derivatives. Using traditional techniques such as immunohistochemistry to track cell cycle changes in such a dynamic population is challenging, as static time points provide an incomplete spatiotemporal picture. In contrast, the fluorescent, ubiquitination-based cell cycle indicator (Fucci) system provides in vivo readouts of cell cycle progression and has been previously adapted for use in zebrafish. The most commonly used Fucci systems are ubiquitously expressed, making tracking of a specific cell population challenging. Therefore, we generated a transgenic zebrafish line, Tg(-4.9sox10:mAG-gmnn(1/100)-2A-mCherry-cdt1(1/190)), in which the Fucci system is specifically expressed in delaminating and migrating neural crest cells. Here, we demonstrate validation of this new tool and its use in live high-resolution tracking of cell cycle progression in the neural crest and derivative populations.


Assuntos
Ciclo Celular/fisiologia , Rastreamento de Células/métodos , Crista Neural/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Engenharia Genética/métodos , Proteínas Luminescentes/genética , Microscopia de Fluorescência/métodos , Crista Neural/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
12.
Dev Biol ; 424(1): 1-9, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28235582

RESUMO

Matching appendage size to body size is fundamental to animal function. Generating an appropriately-sized appendage is a robust process executed during development which is also critical for regeneration. When challenged, larger animals are programmed to regenerate larger limbs than smaller animals within a single species. Understanding this process has important implications for regenerative medicine. To approach this complex question, models with altered appendage size:body size ratios are required. We hypothesized that repeatedly challenging axolotls to regrow limb buds would affect their developmental program resulting in altered target morphology. We discovered that after 10 months following this experimental procedure, limbs that developed were permanently miniaturized. This altered target morphology was preserved upon amputation and regeneration. Future experiments using this platform should provide critical information about how target limb size is encoded within limb progenitors.


Assuntos
Ambystoma mexicanum/embriologia , Amputação Cirúrgica , Botões de Extremidades/embriologia , Botões de Extremidades/patologia , Animais , Ectromelia/patologia , Botões de Extremidades/anormalidades , Botões de Extremidades/inervação , Tecido Nervoso/patologia , Tamanho do Órgão , Regeneração
13.
Stem Cells ; 35(1): 236-247, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28028909

RESUMO

Some animals have the ability to generate large numbers of oocytes throughout life. This raises the question whether persistent adult germline stem cell populations drive continuous oogenesis and whether they are capable of mounting a regenerative response after injury. Here we demonstrate the presence of adult oogonial stem cells (OSCs) in the adult axolotl salamander ovary and show that ovarian injury induces OSC activation and functional regeneration of the ovaries to reproductive capability. Cells that have morphological similarities to germ cells were identified in the developing and adult ovaries via histological analysis. Genes involved in germ cell maintenance including Vasa, Oct4, Sox2, Nanog, Bmp15, Piwil1, Piwil2, Dazl, and Lhx8 were expressed in the presumptive OSCs. Colocalization of Vasa protein with H3 mitotic marker showed that both oogonial and spermatogonial adult stem cells were mitotically active. Providing evidence of stemness and viability of adult OSCs, enhanced green fluorescent protein (EGFP) adult OSCs grafted into white juvenile host gonads gave rise to EGFP OSCs, and oocytes. Last, the axolotl ovaries completely regenerated after partial ovariectomy injury. During regeneration, OSC activation resulted in rapid differentiation into new oocytes, which was demonstrated by Vasa+ /BrdU+ coexpression. Furthermore, follicle cell proliferation promoted follicle maturation during ovarian regeneration. Overall, these results show that adult oogenesis occurs via proliferation of endogenous OSCs in a tetrapod and mediates ovarian regeneration. This study lays the foundations to elucidate mechanisms of ovarian regeneration that will assist regenerative medicine in treating premature ovarian failure and reduced fertility. Stem Cells 2017;35:236-247.


Assuntos
Células-Tronco de Oogônios/citologia , Ovário/lesões , Ovário/fisiopatologia , Regeneração , Envelhecimento , Ambystoma mexicanum , Animais , Biomarcadores/metabolismo , RNA Helicases DEAD-box/metabolismo , Feminino , Células Germinativas/citologia , Proteínas de Fluorescência Verde/metabolismo , Masculino , Mitose , Oócitos/citologia , Células-Tronco de Oogônios/metabolismo , Ovário/patologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Testículo/citologia
14.
J Theor Biol ; 454: 345-356, 2018 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-29653160

RESUMO

Joints connect the skeletal components and enable movement. The appearance and development of articulations is due to different genetic, biochemical, and mechanical factors. In the embryonic stage, controlled biochemical processes are critical for organized growth. We developed a computational model, which predicts the appearance, location, and development of joints in the embryonic stage. Biochemical events are modeled with reaction diffusion equations with generic molecules representing molecules that 1) determine the site where the articulation will appear, 2) promote proliferation, and matrix synthesis, and 3) define articular cartilage. Our model accounts for cell differentiation from mesenchymal cells to pre-cartilaginous cells, then cartilaginous cells, and lastly articular cartilage. These reaction-diffusion equations were solved using the finite elements method. From a mesenchymal 'bud' of a phalanx, the model predicts growth, joint cleavage, joint morphology, and articular cartilage formation. Our prediction of the gene expression during development agrees with molecular expression profiles of joint development reported in literature. Our computational model suggests that initial rudiment dimensions affect diffusion profiles result in Turing patterns that dictate sites of cleavage thereby determining the number of joints in a rudiment.


Assuntos
Desenvolvimento Ósseo/fisiologia , Cartilagem Articular/embriologia , Simulação por Computador , Articulações/embriologia , Animais , Biomarcadores/metabolismo , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/fisiologia , Comunicação Celular/fisiologia , Diferenciação Celular , Proliferação de Células , Condrogênese/fisiologia , Biologia Computacional , Falanges dos Dedos da Mão/embriologia , Falanges dos Dedos da Mão/crescimento & desenvolvimento , Falanges dos Dedos da Mão/metabolismo , Fator 5 de Diferenciação de Crescimento/administração & dosagem , Fator 5 de Diferenciação de Crescimento/farmacocinética , Humanos , Articulações/citologia , Articulações/crescimento & desenvolvimento , Articulações/metabolismo , Modelos Teóricos , Morfogênese/fisiologia
15.
Development ; 141(10): 2165-71, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24764077

RESUMO

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability.


Assuntos
Ambystoma mexicanum/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Desoxirribonucleases/metabolismo , Marcação de Genes/métodos , Mutagênese Sítio-Dirigida/métodos , Ambystoma mexicanum/embriologia , Animais , Sequência de Bases , Desoxirribonucleases/genética , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação INDEL , Dados de Sequência Molecular , Regeneração/genética , Homologia de Sequência do Ácido Nucleico , Pequeno RNA não Traduzido
16.
Biochim Biophys Acta ; 1849(4): 454-61, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25088176

RESUMO

Injury is an inescapable phenomenon of life that affects animals at every physiological level. Yet, some animals respond to injury by rebuilding the damaged tissues whereas others are limited to scarring. Elucidating how a tissue insult from wounding leads to a regenerative response at the genetic level is essential to make regenerative advantages translational. It has become clear that animals with regenerative abilities recycle developmental programs after injury, reactivating genes that have lied dormant throughout adulthood. The question that is critical to our understanding of regeneration is how a specific set of developmentally important genes can be reactivated only after an acute tissue insult. Here, we review how injury-induced cellular stresses such as hypoxic, oxidative, and mechanical stress may contribute to the genomic and epigenetic changes that promote regeneration in animals. This article is part of a Special Issue entitled: Stress as a fundamental theme in cell plasticity.


Assuntos
Regeneração , Estresse Fisiológico/fisiologia , Ferimentos e Lesões , Animais , Reprogramação Celular/fisiologia , Epigênese Genética/fisiologia , Genoma/fisiologia , Crescimento e Desenvolvimento/genética , Humanos , Regeneração/genética , Transdução de Sinais/genética , Estresse Fisiológico/genética , Ferimentos e Lesões/genética , Ferimentos e Lesões/fisiopatologia
17.
Blood ; 124(8): 1232-41, 2014 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-24802774

RESUMO

Hematopoietic stem cell (HSC)-derived cells are involved in wound healing responses throughout the body. Unfortunately for mammals, wound repair typically results in scarring and nonfunctional reparation. Among vertebrates, none display such an extensive ability for adult regeneration as urodele amphibians, including 1 of the more popular models: the axolotl. However, a lack of knowledge of axolotl hematopoiesis hinders the use of this animal for the study of hematopoietic cells in scar-free wound healing and tissue regeneration. We used white and cytomegalovirus:green fluorescent protein(+) transgenic white axolotl strains to map sites of hematopoiesis and develop hematopoietic cell transplant methodology. We also established a fluorescence-activated cell sorter enrichment technique for major blood lineages and colony-forming unit assays for hematopoietic progenitors. The liver and spleen are both active sites of hematopoiesis in adult axolotls and contain transplantable HSCs capable of long-term multilineage blood reconstitution. As in zebrafish, use of the white axolotl mutant allows direct visualization of homing, engraftment, and hematopoiesis in real time. Donor-derived hematopoiesis occurred for >2 years in recipients generating stable hematopoietic chimeras. Organ segregation, made possible by embryonic microsurgeries wherein halves of 2 differently colored embryos were joined, indicate that the spleen is the definitive site of adult hematopoiesis.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Regeneração/fisiologia , Ambystoma mexicanum , Animais , Animais Geneticamente Modificados , Sobrevivência de Enxerto/fisiologia , Transplante de Células-Tronco Hematopoéticas
18.
Curr Top Microbiol Immunol ; 367: 53-74, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23239234

RESUMO

Many vertebrates have the amazing ability to regenerate all or portions of appendages including limbs, tails, fins, and digits. Unfortunately, our understanding of the cellular and molecular basis of appendage regeneration is severely lacking. However, recent technological advances that facilitate the tracking of cell lineages in vivo through space and time are allowing us to address the unknowns of regeneration, such as characterizing the cells that contribute to regeneration and identifying the tissues these cells differentiate into during regeneration. Here, we describe the experiments and the surprisingly uniform results that have emerged across diverse vertebrate species when specific cell lineages have been tracked during vertebrate appendage regeneration. These investigations show that vertebrates, from zebrafish to salamanders to mammals, utilize a limited amount of cellular plasticity to regenerate missing appendages. The universal approach to appendage regeneration is not to generate pluripotent cells that then differentiate into the new organ, but instead to generate lineage-restricted cells that are propagated in a progenitor-like state. Lessons learned from these natural cases of complex tissue regeneration might inform regenerative medicine on the best approach for re-growing complex tissues.


Assuntos
Nadadeiras de Animais/fisiologia , Extremidades/fisiologia , Regeneração , Cauda/fisiologia , Anfíbios/fisiologia , Animais , Desdiferenciação Celular , Linhagem da Célula
19.
Dev Dyn ; 242(7): 847-60, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23592243

RESUMO

BACKGROUND: The mammalian central nervous system is incapable of substantial axon regeneration after injury partially due to the presence of myelin-associated inhibitory molecules including Nogo-A and myelin associated glycoprotein (MAG). In contrast, axolotl salamanders are capable of considerable axon regrowth during spinal cord regeneration. RESULTS: Here, we show that Nogo-A and MAG, and their receptor, Nogo receptor (NgR), are present in the axolotl genome and are broadly expressed in the central nervous system (CNS) during development, adulthood, and importantly, during regeneration. Furthermore, we show that Nogo-A and NgR are co-expressed in Sox2 positive neural progenitor cells. CONCLUSIONS: These expression patterns suggest myelin-associated proteins are permissive for neural development and regeneration in axolotls.


Assuntos
Ambystoma mexicanum/metabolismo , Proteínas de Anfíbios/metabolismo , Proteínas da Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Regeneração da Medula Espinal/fisiologia , Proteínas de Anfíbios/genética , Animais , Proteínas da Mielina/genética , Glicoproteína Associada a Mielina/genética , Proteínas Nogo , Traumatismos da Medula Espinal/genética , Regeneração da Medula Espinal/genética
20.
J Mech Behav Biomed Mater ; 150: 106341, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-38160643

RESUMO

BACKGROUND: The extracellular mechanical environment plays an important role in the skeletal development process. Characterization of the material properties of regenerating tissues that recapitulate development, provides insights into the mechanical environment experienced by the cells and the maturation of the matrix. In this study, we estimated the viscoelastic material properties of regenerating forelimbs in the axolotl (Ambystoma mexicanum) at three different regeneration stages: 27 days post-amputation (mid-late bud) and 41 days post-amputation (palette stage), and fully-grown time points. A stress-relaxation indentation test followed by two-term Prony series viscoelastic inverse finite element analysis was used to obtain material parameters. Glycosaminoglycan (GAG) content was estimated using a 1,9- dimethyl methylene blue assay. RESULTS: The instantaneous and equilibrium shear moduli significantly increased with regeneration while the short-term stress relaxation time significantly decreased with limb regeneration. The long-term stress relaxation time in the fully-grown time point was significantly lower than 27 and 41 DPA groups. The GAG content was not significantly different between 27 and 41 DPA but the GAG content of cartilage in the fully-grown group was significantly greater than in 27 and 41 DPA. CONCLUSIONS: The mechanical environment of the proliferating cells changes drastically during limb regeneration. Understanding how the tissue's mechanical properties change during limb regeneration is critical for linking molecular-level matrix production of the cells to tissue-level behavior and mechanical signals.


Assuntos
Ambystoma mexicanum , Regeneração , Animais , Análise de Elementos Finitos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA