29 research outputs found
The Regenerative Capacity of the Zebrafish Caudal Fin Is Not Affected by Repeated Amputations
Background: The zebrafish has the capacity to regenerate many tissues and organs. The caudal fin is one of the most convenient tissues to approach experimentally due to its accessibility, simple structure and fast regeneration. In this work we investigate how the regenerative capacity is affected by recurrent fin amputations and by experimental manipulations that block regeneration. Methodology/Principal Findings: We show that consecutive repeated amputations of zebrafish caudal fin do not reduce its regeneration capacity and do not compromise any of the successive regeneration steps: wound healing, blastema formation and regenerative outgrowth. Interfering with Wnt/Ă-catenin signalling using heat-shock-mediated overexpression of Dickkopf1 completely blocks fin regeneration. Notably, if these fins were re-amputated at the non-inhibitory temperature, the regenerated caudal fin reached the original length, even after several rounds of consecutive Wnt/Ă-catenin signalling inhibition and re-amputation. Conclusions/Significance: We show that the caudal fin has an almost unlimited capacity to regenerate. Even after inhibition of regeneration caused by the loss of Wnt/Ă-catenin signalling, a new amputation resets the regeneration capacity within the caudal fin, suggesting that blastema formation does not depend on a pool of stem/progenitor cells that require Wnt/Ăcateni
Behaviour of telomere and telomerase during aging and regeneration in zebrafish.
Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration
Immunolocalization of the telomeraseâ1 component in cells of the regenerating tail, testis, and intestine of lizards
Premature aging in telomerase-deficient zebrafish
SUMMARY
The study of telomere biology is crucial to the understanding of aging and cancer. In the pursuit of greater knowledge in the field of human telomere biology, the mouse has been used extensively as a model. However, there are fundamental differences between mouse and human cells. Therefore, additional models are required. In light of this, we have characterized telomerase-deficient zebrafish (Danio rerio) as the second vertebrate model for human telomerase-driven diseases. We found that telomerase-deficient zebrafish show p53-dependent premature aging and reduced lifespan in the first generation, as occurs in humans but not in mice, probably reflecting the similar telomere length in fish and humans. Among these aging symptoms, spinal curvature, liver and retina degeneration, and infertility were the most remarkable. Although the second-generation embryos died in early developmental stages, restoration of telomerase activity rescued telomere length and survival, indicating that telomerase dosage is crucial. Importantly, this model also reproduces the disease anticipation observed in humans with dyskeratosis congenita (DC). Thus, telomerase haploinsufficiency leads to anticipation phenomenon in longevity, which is related to telomere shortening and, specifically, with the proportion of short telomeres. Furthermore, p53 was induced by telomere attrition, leading to growth arrest and apoptosis. Importantly, genetic inhibition of p53 rescued the adverse effects of telomere loss, indicating that the molecular mechanisms induced by telomere shortening are conserved from fish to mammals. The partial rescue of telomere length and longevity by restoration of telomerase activity, together with the feasibility of the zebrafish for high-throughput chemical screening, both point to the usefulness of this model for the discovery of new drugs able to reactivate telomerase in individuals with DC
Very old fish have telomerase activity.
<p>Telomerase activity was measured quantitatively and qualitatively in whole zebrafish embryos (3 days post-fertilization, dpf, nâ=â100) and in several organs from adults with different ages (6, 12 and 30 months old, nâ=â4). <b>A,</b> Q-TRAP assay using 1 ”g of protein extract. Results are expressed as the mean value ± S.E. from triplicate samples relative to telomerase-positive cells. Different letters denote statistically significant differences between different ages of each sample according to a Tukey test. <b>B,</b> TRAP assay using protein extract from whole zebrafish embryos. A ladder of bands indicates the presence of telomerase activity. The lowest band (56 pb) is the internal control (IC). Lanes Câ and C+ correspond to telomerase- negative and positive controls, respectively. In all cases, to confirm the specificity of the assay, a negative control is included for each sample, treated with 1 ”g of RNAse at 37°C for 20 min. The Q-TRAP assay was also performed using 0.1 ”g of protein extract and the same relative results were obtained (data not shown).</p
Dynamic of telomere length assayed by Q-FISH.
<p><b>A</b>, Graph showing the mean telomeric fluorescence values. Data are mean values ± S.E. and statistical significance was assessed using the Tukey test (<i>p<0.05</i>). <b>B</b>, Histograms showing telomere fluorescence frequencies. The red lines demarcate both the shortest (<1000 auf) and the longest telomere percentage (>3000 auf) clearly illustrating telomere lengthening throughout the life cycle to adulthood and shortening in old age.</p
Behaviour of telomere length during fin regeneration by Flow-FISH assay.
<p><b>A</b>, Experimental design of the Flow-FISH assay. Clip 1 (1<sup>st</sup> fin excision), clip 2 (2<sup>nd</sup> excision) and clip 3 (3<sup>rd</sup> excision). <b>B</b>, Representation of the zebrafish caudal fin cells distribution (clip 1, clip2 and clip3) according to their telomere length. MFI is indicated for each age, (nâ=â5). The same trend was observed in the three independent experiments <b>C</b>, Graphic representation of the percentage of cells with long telomere (LTC), medium telomere (MTC), and short telomere (STC), delimited by dotted red lines, from clip1, clip 2 and clip 3, at different ages.</p