Although, it has been extensively shown that telomeres and telomerase are involved in mammalian aging and cancer promotion and that the zebrafish is now widely used as a good model organism for assessing these complex processes 
, so far there have only been a few incomplete studies about telomeres and telomerase biology in this species. The zebrafish, with a life-span of 3 years, is the only vertebrate system in which telomerase function can be studied in a high-throughput manner. We used this species to perform an exhaustive study of telomerase expression and telomere length among a broad range of tissues throughout its life.
A high level of TERT
gene expression has been reported in almost all zebrafish tissues in contrast to what happens in the corresponding mammalian tissues 
. Our results show that telomerase is expressed in all tissues tested at different stages of life (larvae, juvenile, adult and old fish). However, the expression of TERT mRNA drastically decreased in all tissue examined of old fish (more than two years old), with the exception of the muscle. These results are in line with published data in which continuously proliferating myocytes have also been observed although there was no lipofucsine granule accumulation in the muscle of zebrafish with advancing age 
. These data correlated well with the presence of telomerase activity in all tissues examined of adult fish of different ages and with the significant decrease of telomerase activity in old zebrafish samples, with the exception of muscle and ovary samples.
Despite the observed differences in telomerase expression between mammalian and zebrafish, we detected common transcriptional factor binding sites in both promoters. We observed several Sp1 binding sites, hTERT
has at least nine Sp1 binding sites dispersed along the 4 kb promoter, whereas zfTERT
has 16 Sp1 binding sites. The clustering of Sp1 binding sites is a common event in promoters of TATA-less genes 
such as hTERT
. Another common feature of both promoters is the presence of c-Myc binding sites. Although zfTERT
has only one site located at 1.200 upstream of the start site and hTERT
has several for this transcriptional factor, the induction of zfTERT
promoter in zebrafish embryos by c-Myc is similar to that found with hTERT 
. Similarly, the NF-κB transcription factor also induces the zfTERT
promoter, suggesting that the zebrafish is also a good model for studying the influence of inflammation on TERT expression and cancer development.
In all the analyzed strains, we observed an increased telomere length from larvae to adult fish and a significant telomere shortening in aged fish. Our results are not in agreement with recently published data 
in which, using a TRF assay, the authors conclude that telomeres do not shorten with age. These discrepancies might be explained by the methodology used. It is well-known that the TRF technique is not sensitive enough for detecting changes in telomere length and it is difficult to detect short telomeres, something that is crucial in these aging studies 
. Therefore, we combined this technique with the most accurate Q-FISH technique. Moreover, all our studies have been performed using whole organisms from various strains obtained from different stock centres in order to avoid a particular telomere length associated with a strain and/or to a given stock centre. These results were also confirmed using a specific tissue, such as the caudal fin, where we observed by Flow-FISH that telomere length increased until 18 months old and gradually decreased after this age. Overall, our results showed that the three wild type strains analyzed showed slightly different telomere lengths. TL is the genetic background showing the shortest telomeres and such differences may have an impact on cancer susceptibility and aging. In fact, we have observed that the life-span of the TL background is less than 3.5 years, while the AB and WIK strains have longer life-spans in our zebrafish facility. Further studies should be performed to establish whether these differences are important in aging and cancer processes.
In this study, we have also established that there is a direct relation between the levels of telomerase expression, telomerase activity and telomere length in zebrafish. Haploinsufficiency for TERT leads to premature telomere shortening in human and causes the aging disease known as dyskeratosis congenita 
. This means that telomerase levels control telomere length in human and in zebrafish, and therefore, high TERT expression might prevent telomere erosion and delay senescence in adult animals.
In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures after damage, including heart, spinal cord, retina and fins. This process involves progenitor cells/resident stem cells 
. In mammals, telomerase is expressed in germ cells and in the stem cell compartment of several adult tissues. It has been proposed that telomerase may be important for tissue regeneration after injury. In fact, phenotypes associated with premature loss of tissue regeneration, including the skin (hair loss, hair greying, decreased wound healing) are found in mice deficient for telomerase 
. Our results showed that the zebrafish was able to regenerate the amputated fin at all ages but the fish with the lowest level of telomerase expression; i.e. older fish, had severely impaired fin regeneration. Indeed, the efficiency of regeneration showed a direct correspondence with telomerase expression (3days/young fish versus 12days/old fish). In fact, we have observed an increase in TERT expression in caudal fin, when amputated in young (3 month old) and old fish (24 month old), but only significant in young fish (58% of upregulation). Therefore, young fish responded better than old fish after injury. Importantly, the upregulation of telomerase expression was correlated with telomere length behaviour after reclipping, as the different age groups responded to the injury lengthening their telomeres. Our data were consistent with the idea that telomeres would need to be maintained during increased cell proliferation associated with tissue renewal. Although the strong upregulation of telomerase expression observed in 3 month old fish did not correlate with a strong telomere length increase, this group was the only one able to elongate their telomeres after consecutive amputations. However, fish older than 3 months, although showing an increase in telomere length after a second clip, are not able to maintain this elongation after a prolonged injury (clip 3). This behaviour might be related to the inefficient activation of telomerase expression in fish older than 3 months. Curiously, whereas the oldest fish had a similar telomeric response to adult fish, they showed incomplete and deficient fin regeneration. However, regeneration is a complex process in which many genes/factor are involved. Therefore, further studies are required to clarify the role of telomerase and telomere length in regeneration. Further gain- and loss-of-function experiments for two telomerase components will shed light on the role of telomerase in regeneration.
Another important insight of our study is the declined telomerase expression, telomere length and regeneration capacity as biomarkers for aging in zebrafish. Until now, SA B-Gal activity, melatonin deficiency and cognitive function were all used as aging biomarkers 
. All these biomarkers, together with the ones described in this study, can discriminate between two age groups; that is, those younger than 18 months old and older than 18 months old (). These results indicate that telomere length, telomerase expression and regeneration capacity are highly dependent on zebrafish age and, therefore, they are useful for evaluating the aging process of zebrafish.
Potential aging biomarkers in the zebrafish.
Further studies are necessary to establish if all the cells express telomerase or only a specific population. A transgenic zebrafish expressing a reporter driven by TERT promoter might be useful for identifying in vivo cells with high/low telomerase activity, i.e. progenitor- stem cells/aging cells. Although there are obvious differences between human and zebrafish, such as the high expression of TERT alongside its life-span, both species show declined telomere expression and telomere length with aging. Their promoters are up-regulated with the same intensity and by similar key transcription factors. We therefore propose that the zebrafish can be used to identify genes and drugs that affect the ability to restore aging phenotypes using telomere length or telomerase expression, which have been identified as good aging biomarkers in this study.