RESUMO
The laboratory axolotl (Ambystoma mexicanum) is widely used in biological research. Recent advancements in genetic and molecular toolkits are greatly accelerating the work using axolotl, especially in the area of tissue regeneration. At this juncture, there is a critical need to establish gene and transgenic nomenclature to ensure uniformity in axolotl research. Here, we propose guidelines for genetic nomenclature when working with the axolotl.
Assuntos
Ambystoma mexicanum , Cicatrização , Ambystoma mexicanum/genética , Animais , Animais Geneticamente ModificadosRESUMO
Axolotls are uniquely able to resolve spinal cord injuries, but little is known about the mechanisms underlying spinal cord regeneration. We previously found that tail amputation leads to reactivation of a developmental-like program in spinal cord ependymal cells (Rodrigo Albors et al., 2015), characterized by a high-proliferation zone emerging 4 days post-amputation (Rost et al., 2016). What underlies this spatiotemporal pattern of cell proliferation, however, remained unknown. Here, we use modeling, tightly linked to experimental data, to demonstrate that this regenerative response is consistent with a signal that recruits ependymal cells during ~85 hours after amputation within ~830 µm of the injury. We adapted Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) technology to axolotls (AxFUCCI) to visualize cell cycles in vivo. AxFUCCI axolotls confirmed the predicted appearance time and size of the injury-induced recruitment zone and revealed cell cycle synchrony between ependymal cells. Our modeling and imaging move us closer to understanding bona fide spinal cord regeneration.
Assuntos
Proliferação de Células , Análise Espaço-Temporal , Regeneração da Medula Espinal , Ambystoma mexicanum , Animais , Animais Geneticamente Modificados , Ciclo Celular , Biologia Computacional , Epêndima/fisiologia , Traumatismos da Medula Espinal , UbiquitinaçãoRESUMO
In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper.
RESUMO
Genomic manipulation is essential to the use of model organisms to understand development, regeneration and adult physiology. The axolotl (Ambystoma mexicanum), a type of salamander, exhibits an unparalleled regenerative capability in a spectrum of complex tissues and organs, and therefore serves as a powerful animal model for dissecting mechanisms of regeneration. We describe here an optimized stepwise protocol to create genetically modified axolotls using the CRISPR-Cas9 system. The protocol, which takes 7-8 weeks to complete, describes generation of targeted gene knockouts and knock-ins and includes site-specific integration of large targeting constructs. The direct use of purified CAS9-NLS (CAS9 containing a C-terminal nuclear localization signal) protein allows the prompt formation of guide RNA (gRNA)-CAS9-NLS ribonucleoprotein (RNP) complexes, which accelerates the creation of double-strand breaks (DSBs) at targeted genomic loci in single-cell-stage axolotl eggs. With this protocol, a substantial number of F0 individuals harboring a homozygous-type frameshift mutation can be obtained, allowing phenotype analysis in this generation. In the presence of targeting constructs, insertions of exogenous genes into targeted axolotl genomic loci can be achieved at efficiencies of up to 15% in a non-homologous end joining (NHEJ) manner. Our protocol bypasses the long generation time of axolotls and allows direct functional analysis in F0 genetically manipulated axolotls. This protocol can be potentially applied to other animal models, especially to organisms with a well-characterized transcriptome but lacking a well-characterized genome.
Assuntos
Ambystoma mexicanum/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Técnicas de Introdução de Genes/métodos , Técnicas de Inativação de Genes/métodos , Animais , Animais Geneticamente Modificados/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Mutação da Fase de Leitura , Fenótipo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Transgenic animals have been indispensable in elucidating and deciphering mechanisms underlying various biological phenomena. In regeneration, transgenic animals expressing fluorescent protein genes have been crucial for identifying the source cells for regeneration and the mechanism of blastema formation. Animals are usually generated by manipulating their genome using various techniques at/in one cell embryo/fertilized egg stage. Here, we describe the generation of germline transgenic axolotls (Ambystoma mexicanum) using the I-SceI meganuclease and Tol2 transposase.
Assuntos
Ambystoma mexicanum/genética , Técnicas de Transferência de Genes , Animais , DNA/genética , Injeções , Larva/genética , Óvulo/metabolismo , RNA Mensageiro/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transposases/genética , Transposases/metabolismoRESUMO
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/metabolismoRESUMO
BACKGROUND: A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context. RESULTS: We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum) donor embryos into white (d/d) axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl. CONCLUSIONS: Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the "muscle scaffold theory," and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution.