Telomerase and Alternative Lengthening of Telomeres coexist in the regenerating zebrafish caudal fins
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Review Commons)
Abstract
Telomeres are essential for chromosome protection and genomic stability, and telomerase function is critical to organ homeostasis. Zebrafish has become a useful vertebrate model for understanding the cellular and molecular mechanisms of regeneration. The regeneration capacity of the caudal fin of wild-type zebrafish is not affected by repetitive amputation, but the behavior of telomeres during this process has not yet been studied. In this study, the regeneration process was characterized in a telomerase deficient zebrafish model. Moreover, the regenerative capacity after repetitive amputations and at different ages was studied. Regenerative efficiency decreases with aging in all genotypes and surprisingly, telomere length is maintained even in telomerase deficient genotypes. Our results suggest that telomere length can be maintained by the regenerating cells through the recombination-mediated Alternative Lengthening of Telomeres (ALT) pathway, which is likely to support high rates of cell proliferation during the tailfin regeneration process. As far as we know, this is the first animal model to study ALT mechanism in regeneration, which opens a wealth of possibilities to study new treatments of ALT dependent processes.
Article activity feed
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The authors Martiěnez-Balsalobre and colleagues found that the regenerative capacity of the zebrafish caudal fin is not limited by the lack of telomerase and showed that the length of telomeres does not decrease substantially after repeated amputations in telomerase-deficient zebrafish. These findings prompt the authors to explore an alternative mechanism that would explain the maintenance of telomere length in this regeneration setting. They produced suggestive evidence for the role of the ALT (Alternative Lengthening of Telomeres) mechanism in the maintenance of telomere length in the …
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The authors Martiěnez-Balsalobre and colleagues found that the regenerative capacity of the zebrafish caudal fin is not limited by the lack of telomerase and showed that the length of telomeres does not decrease substantially after repeated amputations in telomerase-deficient zebrafish. These findings prompt the authors to explore an alternative mechanism that would explain the maintenance of telomere length in this regeneration setting. They produced suggestive evidence for the role of the ALT (Alternative Lengthening of Telomeres) mechanism in the maintenance of telomere length in the absence of telomerase in a regeneration setting.
In my view, several points need to be addressed and clarified.
**There are three major points:**
1.When working with tert mutants, the age at which these fish show a telomere phenotype (namely, loss of body mass and reduced fertility) varies. Therefore, it would be important to state if the fish used in this study were already showing these phenotypic characteristics at each time point studied, namely 4, 8 and 11 months of age
The premature aging phenotype of tert mutant fish has been previously characterized in the paper by Anchelin et al 2013 referenced in the manuscript. We used young fish with no phenotype (4 months old), and aged fish (8 and 11 months old) presenting the already described premature aging phenotypes, such as spinal curvature, loss of fertility, loss of body mass and loss of pigmentation.
The following sentence regarding this has been included in the revised version of the manuscript.
“The fish used showed non-detectable aging phenotype at 4 months old, whereas at 8- and 11-months fish presented the typical tert mutant premature aging phenotypes, i.e. backbone curvature, loss of body mass and hypopigmentation”
2.The knockdown experiments were performed using morpholinos. To confidently use morpholinos it is fundamental to demonstrate first their knockdown efficiency and their specificity. This is lacking in the manuscript.
In this work we have used 3 different morpholinos; tert morpholino has been already used and characterized in the work by Imamura and collaborators in 2008. atr morpholino has been already used and characterized in the paper by Stern at al., 2005.
However, nbs1 morpholino has been designed for this work. A Supplemental Figure (Figure S2) and the following paragraph have been added in the revised version of the manuscript to show the knock-down efficiency of the nbs1 morpholino:
“The knock-down efficiency of the atr morpholino was characterized by Stern and colleagues (Stern et al, 2005). The injection of the *nbs1 *morpholino in zebrafish eggs resulted in the reduction of the expression of nbs1 mRNA at 3dpf (Fig. S2A). Furthermore, PCR using cDNA as a template detected *nbs1 *mRNA species that retained the intron one of the gene as a result of the morpholino effect in blocking the splicing (Fig. S2B).
“The tert morpholino knock-down efficiency has been already showed (Imamura et al, 2008)”
3.The involvement of ALT mechanism in the regeneration process in the absence of telomerase is only suggestive, as the authors show an increase of C-circles and heterogenous telomerase length in telomerase-deficient zebrafish but when trying to establish a functional link the authors resort to the knowndown of genes that may be associated with ATL. Looking at the levels of TERRA and the number of C-circles in the knowndown caudal fins would be essential for their claim.
We have now performed caudal fin regeneration experiments in tert mutant fish microinjected with mo-atr and mo-nbs1 and analyzed the levels of TERRA RNAs and C-circles amount. The results are shown in Supplemental Figure S4. As expected, regeneration capacity decreased in fish microionjected with both morpholinos compared to control fish (FigS4 F). Consistently, TERRA RNAs levels, as well as C-circles amount, increased in the regenerating tissue and this induction was lower when atr and nbs1 gene expression was downregulated by mo-injection (Fig S4 G-J). Taking altogether, these results indicate that ALT mechanism is induced upon amputation and operates in the regenerating tissue of tert deficient fish.
**And several other points:**
4.The regeneration experiments were performed at 32 degrees and this option was never explained nor discussed.
The regeneration experiments in zebrafish typically are performed at 32 °C to accelerate regeneration process. Otherwise, the amount of regenerated blastema at 48 hpa or 72hpa would not be enough to perform any kind of analysis. Furthermore, it could happen that some experimental modifications, for instance the effects of the morpholino injection, do not last if the regeneration process is kept more than 84-96hpa at 28 °C.
This procedure have been used previously by other laboratories (PMID: 8601496, Johnson and Weston,1995; PMID: 12015289 Nechiporuk et al.,2003 and PMID: 16273523 Thumnel et al 2006) to increase the rate of regeneration approximately two fold, a temperature of 33°C was used for the regeneration experiments. In addition, It has been demonstrated normal regeneration at 33°C in wild-type fish
5.When referring to the ALT mechanism, the authors state that "... in about 10% of tumors cells, telomere length is maintained by the Alternative Lengthening of Telomeres (ALT) mechanism ..." and I think it would be more accurate to talk about cancer cells instead of tumor cells.
This has been corrected in the revised version
6.The sentence about C-circles is incorrect. C-circles are mostly single-stranded and not double-stranded as stated.
This has been corrected in the revised version
7.After Figure 2, the authors never mention the age of the fish used.
All the fish used in the amputation experiments after Fig2 are 4 -6 months of age
8.In Figure 1A. The site of amputation does not fit the one described in Mat & Met that states 2 cm from the base of the caudal peduncle. The same stands for Figure 2A.
This is corrected in the new version with a new Figure 1A and 2A
9.In Figure 1B
The Y axis should be named regeneration area instead of rate as the values are a percentage of the area reached after a certain time point after amputation. The same stands for Figure 2B, C. It would be nice to see the real caudal fin images for the relevant time points: before amputation, 0 dpa, when the fins reach 50% of regeneration area and then the last time point.
This has been changed in the new version
The authors should discuss why are the caudal fins reaching more than 100% of regeneration are
This is an intriguing question for which we currently lack an answer. Nonetheless, it does not impact the focus of our ongoing study
10.In Figure 2B. The meaning of ". .. ." on the right side of the graph is not clear. The same stands for Figure 2D.
This has been a mistake when handling the figure folder and has been corrected in the revised version
11.In Figure 2C .Why is the clip 10, 11 and 12 missing from the tert+/- and tert-/- ?
This has been changed in the new version and recalculated the statistical significance. We appreciate the feedback
12.In Figure 2E The proximity of all points at the 12 Clip is indicative of lack of statistical significance, therefore the **** related to which comparisons?
We have modified the data of fig 2E and recalculated the statistical significance
13.In Figure 2D, E
For the measurement of telomere length, the authors state that "Data are average of at least 2 independent experiments." What does this mean exactly? How many animals were used in each experiment?
In the experiments in Fig2, 6 fish total were used per group sampled in at least 2 independent experiments. This has been included in the figure legend and in the Mat&Met section
14.In Figure 3
The authors state that "Data are average of at least 2 independent experiments." What does this mean exactly? How many animals were used in each experiment?
The experiment in Fig 3A was done 3 times with 2 fish per group pooled in each experiment. The telomere length experiment has been done 2 times. This has been added to the figure legend and to the Mat&Met section.
Why were the c-circles evaluated at hpa while the telomere length evaluated at dpa? This should be discussed.
We expect to observe an effect on telomere length after several days of continuous cell proliferation in order to completely regenerate the caudal fin. However, the presence of C-circles in the regenerating tissue is expected to be found as early as 24hpa as a consequence of the action of the ALT mechanism of telomere maintenance, which has to be active from the very beginning. The following sentence has been included in the Discussion section: “ALT activation is expected to happen, and in fact detected, very early in the regeneration process, and eventually results in telomere length heterogenicity several days after amputation, when a lot of cell divisions and telomere recombination have occurred”.
15.In Figure 3A
The meaning of ". .. ." on the top side of the graph is not clear.
t0 should be removed and replaced by 0 hpa and 24hpa and 48hpa for coherence.
This has been a mistake when handling the figure folder and has been corrected in the revised version.
16.In Figure 3B,C 0 hpa replace by 0 dpa
This has been replaced in the new version
17.In Figure 3B
The blue and red stainings in the panels are labelling exactly what? This should be stated in the image and in the legend.
Red staining represents the telomeres and the blue staining are the nuclei. It is shown in the Figure and stated in the figure legend.
18.In Figure 3D
There is a mistake in the legend the should be corrected as follows "Very long telomeres have a higher fluorescence of 200,000 AUF and very short telomeres have a lower fluorescence of 30,000 AUF."
This has been corrected
19.In Figure 4
t0 should be removed and replaced by 0 hpa.
This has been corrected
The meaning of ". .. ." on the top side of the graph is not clear.
This has been a mistake when handling the figure folder and has been corrected in the revised version
The title is an overstatement, as the genes studied are DNA damage genes that may associate with ALT.
The title has been corrected to “The expression of ALT-associated genes is modulated in regenerative tissue of”
20.In Figure 4A, B
The expression of nbs1 and atr in tert-/- increases at 48hpa but the same seems to be true for the tert+/+ and this is never discussed by the authors.
This result would support the idea that both telomerase-dependent and ALT mechanisms operate in the regeneration process in a wild type animal. A sentence in the results and discussion sections has been added to mention and discuss this point:
“These genes were quantified in the regenerated tissue at 24 and 48 hpa. nbs1 and atr mRNA levels increased in telomerase deficient fish at 48 hpa compared to time 0 (0hpa) (Fig. 4A, 4B). The same effect in the expression of these genes was found in wild type fish regenerating fins. Interestingly, atrx and daxx expression decreased (Fig. 4C, 4D) at 24 and 48 hpa, in agreement with published data on ALT in cells (Amorim et al., 2016; Ren et al., 2018; Yost et al., 2019).”
“Curiously we observed an increased expression of ALT activator proteins in both wild type and telomerase deficient zebrafish, and a decrease in ALT inhibitor proteins suggesting that the main players of ALT and their mechanisms are conserved during evolution, and that both mechanisms of telomere maintenance could co-exist in the regeneration process in wild type fish”..
21.In Figure 4C, D
The differences in the expression of atrx and daxx decreases over time in a in tert-/- and this is never discussed by the authors.
As mentioned, and referenced in the manuscript, the proteins are ALT inhibitors, and mutations in these proteins are described to be promoting the activation of ALT mechanisms. Thus, it is expected that in the regenerating fins where ALT is activated, their expression decreases.
22.In Figure 5
An ideal control would be the direct comparison between microinjected+electroporated mo-std in the ventral part of the fin while the dorsal part would be microinjected+electroporated with the mo-gene of interest. This would discard any effect of microinjection+electroporation in the regeneration efficiency.
These experiments are not convincing to show that there is an ALT mechanism is operating here. What this experiment shows if the relevance of these genes for the regenerative capacity of the caudal fin. To show that this is related to the ALT mechanism the authors should investigate the C-circles in these regenerating fins.
We have performed regeneration experiments using WT fish to address this issue. We analyzed the regenerated area of control and morpholino injected fish and then obtained regenerating blastema and analyzed the expression of *tert *and atr. The results are shown in Supplemental Figure S4 (A-E). The regeneration capacity is inhibited in tissues injected with a mix of mo-std+mo-ter, a mix of mo-std+mo-atr, or a mix of mo-tert+mo-atr compared with a control injected with a double dosis of mo-std (std 2x, Fig S4B). In addition, the expression of tert and atr is decreased in the regenerated blastema upon morpholino injection (Fig S4 C and D) indicating that the genetic inhibition of the expression of these genes was efficient. Finally, the levels of TERRA RNAs are increased upon amputation and this induction is reduced when we mo-atr or a combination of mo-atr+tert were microinjected (Fig S4E).
We have also performed caudal fin regeneration experiments in tert mutant fish microinjected with mo-atr and mo-nbs1 and analyzed the levels of TERRA RNAs and C-circles amount. The results are shown in Supplemental Figure S4. As expected, regeneration capacity of the caudal fins of fish microionjected with both morpholinos decreased compared to control fish (Fig S4 F-H). Consistently, TERRA RNAs levels, as well as C-circles amount, increased in the regenerating tissue and this induction was lower when atr and nbs1 gene expression was decreased by mo-injection (Fig S4 I and J). Taking altogether, these results indicate that ALT mechanism is induced upon an injury and operating in the regenerating tissue of both wild type and *tert *deficient fish.
The amputation red lines are not placed in the exact amputation position in some of the panels.
Regeneration rate should be regeneration area.
This has been corrected
23.In Figure 5C, E
Why is the mo-tert more inhibitory of regeneration (Figure 5E - around 30%) than the tert-/- mutant (Figure 5C - around 60%)? This should be discussed.
This point is now discussed: “
24.In Figure 6A
The 2 adult zebrafish shown in the tank with the ATR inhibitor IV should have an amputated caudal fin.
This has been modified
Control is exactly what? Untreated? Treated with vehicle?
The control is fish treated with the same amount of DMSO (vehicle). This is now shown in the panel
Why was the ATR inhibitor IY added immediately after fin amputation while the mo-atr was injected at 48 hpa?
The ATR inhibitor was added immediately after amputation because ALT is then inhibited from the starting of the regeneration process. However, in the case of the *atr *morpholino we need some regenerated tissue to perform the microinjection within and inhibit atr expression specifically in this tissue.
25.Figure 6D, E, F
These panels are a bit out of the focus of this paper. If presented should go to a supplementary figure.
These panels are now moved to the Supplemental Figure S5
26.In Figure S2
The relevant bands should be identified.
We have performed new regeneration experiments in wild type adult fish using ATR inhibitor. The results show that treating fish with ATR inhibitor provokes a clear decrease in the overall phosphorylation status of ATR/ATR substrates within the regenerated tissue (Figure 5B and C). In this case, the intensity of the whole lane was used for quantification.
The gel identifies DMSO, 10uM and 50 uM but the quantification graph identifies Control, 50uM and 100uM.
This has been corrected in the new version
There are no error bars
In the new experiments are now shown.
The authors say that the quantification of various western blot bands was done but how many exactly?
In the new experiments, 3 western blots are quantified
27.In Figure S3
The primers for rps11 are repeated twice.Were these primers design de novo by the authors or did they used previous reported primers, in this case the references should be given.
Tert F2 and R1 should be replaced for F and R for consistency.
This has been corrected and references for the primers used are added in the new Supplemental Figure S6
28.In Figure S4
The sequence of tert mo is missing.
This has been corrected
29.In the methods the genotyping protocol of tert mutants is not described.
A protocol for genotyping the tert deficient zebrafisn has been added in the Mat&Met section.
30.The method to calculate the area of the fin pre- and post-amputation is not described.
The method is already described in the Mat&Met section: “In order to calculate the percentage area of growth between the injected and non-injected part, the values were inserted in the following formula: (Dorsal 48 hpi - Dorsal 0 hpi)/(Ventral 48 hpi -Ventral 0 hpi)*100, where Dorsal is the regenerative area of the MO-treated tissue and Ventral is the regenerative area of the corresponding uninjected half”
Reviewer #1 (Significance (Required)):
The manuscript by Martiěnez-Balsalobre and colleagues deals with a very interesting question on the importance of telomere lengthening during regenerative processes and its relation to ageing. To this end the authors made use of the tert mutant, a telomerase-deficient zebrafish. The authors show a surprising phenotype that telomerase-deficient zebrafish can still regenerate their caudal fins and are able to maintain telomere length during consecutive amputations and I say surprising because it has been shown that telomerase-deficient zebrafish are unable to regenerate their hearts efficiently.
Taking these novel findings, the authors propose that in the zebrafish caudal fin and in the absence of telomerase, telomere length is maintained through the activation of an alternative mechanism called ALT. To my knowledge, the role of ALT as a mechanism of telomere lengthening has never been described in the context of regenerating organs in zebrafish.
We fully appreciate the reviewer´s comments on the significance of the manuscript!
**Referees cross-commenting**
I agree with the comments made by the other reviewers. I would stress the need to tone down the role of ALT during fin regeneration in zebrafish as all the experiments are only indicative of the possible of the involvement of ALT.
We have conducted additional experiments that further support the involvement of ALT. Please read the responses to the other reviewers for more details.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
Using zebrafish as a model for regeneration, the authors find that telomere maintenance by recombination can occur in the absence of telomerase.
Title to Figure 4 perhaps may be too strong, 'ALT mechanism is activated', since only a few features of ALT are assessed. Perhaps, 'ALT features are activated'?
The title to Figure 4 has been changed to “The expression of ALT-related genes is modulated…”
mRNA levels of NBS, ATR are also increased in WT animals (Figure 4A and 4B), but ATRX and DAXX mRNA levels are not decreased in WT animals. Is the increase why the authors in part suggest that ALT is being used in WT animals. If so, what would be the trigger for the use of ALT, as opposed to the trigger to use ALT in tert-/- animals?
Our results indicate the utilization of both telomere maintenance mechanisms to support cell division in regenerative fins among wild-type animals. Consequently, we propose that the signals instigating regeneration are shared between both mechanisms and are present in both wild-type and tert-deficient animals, albeit with varying degrees of contribution.
In Figure 5C, if tert-/- animals are downregulated for nbs1 and atr, would it be expected that the effect on regeneration be more pronounced compared to tert+/+ downregulated for nbs1 and atr than what is observed?
We agree with the reviewer comment, and that is what actually happens. The inhibition of the regeneration in wild type fish is about 40% in mo-nbs1 injection and around 70% in mo-atr injected animals. However, in tert mutants, the decrease in regeneration observed in mo-nbs1 injection is about 56%, whereas is 82% in mo-atr injection.
What are the telomere lengths in tert-/- animals treated with mo-atr or mo-nbs1 or in tert+/+ animals treated with mo-tert and mo-atr compared to singly treated?
The telomere length does not change in mo-atr or mo-nbs1 injected tert mutants compared to mo-std animals.
The telomere length in mo-tert and mo-atr injected wild type animals does not change compared to mo-std injected animals.
This results are now shown in Supplemental Figure S3
Reviewer #2 (Significance (Required)):
Reported findings are novel, timely and model of possible therapeutic value for screening compounds for ALT and/or telomerase inhibitors. Mechanisms of co-existence of ALT and telomerase can also be explored using this model.
We fully appreciate the reviewer´s comments on the significance of the manuscript!
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary:**
Martinez-Balsalobre have examined caudal fin regeneration following surgical transection in WT and telomerase-deficient (tert+/- and tert-/-) zebrafish adults of several ages, and in one experiment, in embryos. They conclude: (1) regeneration efficiency decrease with aging in all genotypes (2) telomere length is maintained, even in a tert-mutant background (3) ALT (alternative lengthening of telomeres) is involved in supporting cell proliferation in tailfin regeneration. The experimental system employs a quantitative area-based measurement as a measure of the degree of regeneration. Functional studies used antisense morpholino gene knockdown and chemical inhibition to implicate ALT involvement.
**Major comments:**
The experimental logic is appropriate, and in general, the data support the conclusions. Strengths of the work include: (1) The quantitative measure of % regeneration appears to be quite objective; (2) the internally controlled experimental design of the morpholino knockdown experiments of Fig 5.
We thank the reviewer for the comment
The Western blot in Fig S2 has some issues. The image is a montage. The experiment appears to have been done only once. The band's identifications by kDa are imprecise (where is the 82 kDa band on the gel? - there are bands smaller and larger than 82 kDa, but none of 82kDa; the 50 kDa band is close to background; the DMSO lane is underloaded relative to the two test lanes (but as the observation is a reduction in the test samples, this does not result in a misinterpretation). What concentration of ATRinhIV were used? The blot has 10 and 50 microM, Fig S1B has 50 and 100 microM, and the text says 1-50 microM).
We have performed new regeneration experiments in wild type adult fish using ATR inhibitor. The results show that treating fish with ATR inhibitor results in a clear decrease in the overall phosphorylation status of ATR/ATR substrates within the regenerated tissue (Figure 5B and C). In this case, the intensity of the whole lane was used for quantification. As mentioned in the text, we used concentrations of 1, 10 and 50 microM, but we do not observe any difference with the 1 microM concentration, thus do not show it. Then we measured the regeneration capacity in both wild type and tert mutant fish using 10microM concentration
The MO-knockdown studies are interpreted as showing synergy of atr and tert knockdown.
There are two problems with them interpretation of synergy: (1) the single result of a greater effect with both MOs does not distinguish between an additive or synergistic effect (and synergistic action is by definition a greater than additive action;
We agree with the reviewer´s comment, and have removed the sentence “Interestingly a synergistic effect was observed when both mechanisms are inhibited” from the Results section.
(2) MO dose is not controlled by a group with an equal total MO doses (mo-std+mo-atr and mo-std+mo-tert). While acknowledging that the issues of using local MO delivery in an adult model are very different from global delivery in an embryonic model, the "synergy" interpretation still requires these experiments/controls be done. These experiments were not accompanied by any molecular evidence that either of the morpholinos targeted expression of the intended gene (which would likely have to be derived from their assessment in another system) - a control that can be challenging, but one that is regarded as essential in the field (https://doi.org/10.1242/dev .001115 ). While this will be difficult to do in the adult setting, it is still appropriate to validate the activity/molecular efficacy of the MO sequence in an experimentally tractable scenario. The specificity of this experiment and interpretation would also be enhanced and corroborated independently by undertaking the atr knockdown in the tert -/- mutant background. Overall, these experiments were preliminary and require further work that could be done withiin 3 months.
We have performed regeneration experiments using WT fish to address this issue. We analyzed the regenerated area of control and morpholino injected fish and then obtained regenerating blastema and analyzed the expression of tert and atr .The results are shown in Supplemental Figure S4 (A-E). The regeneration capacity is inhibited in tissues injected with a mix of mo-std+mo-ter, a mix of mo-std+mo-atr, or a mix of mo-tert+mo-atr compared with a control injected with a double dosis of mo-std (std 2x, Fig S4B). In addition, the expression of tert and atr is decreased in the regenerated blastema upon morpholino injection (Fig S4 C and D) indicating that the genetic inhibition of the expression of these genes was efficient. Finally, the levels of TERRA RNAs are increased upon amputation and this induction is reduced when we mo-atr or a combination of mo-atr+tert were microinjected (Fig S4E).
We have also performed caudal fin regeneration experiments in tert mutant fish microinjected with mo-atr and mo-nbs1, and analyzed the levels of TERRA RNAs and C-circles amount. The results are shown in Supplemental Figure S4. As expected, regeneration capacity of the caudal fins of fish microionjected with both morpholinos decreased compared to control fish (Fig S4 F-H). Consistently, TERRA RNAs levels, as well as C-circles amount, increased in the regenerating tissue and this induction was lower when atr and nbs1 gene expression was decreased by mo-injection (Fig S4 I and J). Taking altogether, these results indicate that ALT mechanism is induced upon an injury and operating in the regenerating tissue of both wild type and tert deficient fish.
Note - the tert MO sequence is missing from the table in Fig S4.
The sequence has been added
The adult experiments have used n=6-10 animals/group. There is no consideration of statistical power (is the analysis of Fig 1C adequately powered?).
The type of statistical test applied in Fig 1C (2-way ANOVA, plus Dunnett´s post-test) compares means of every clip among the 3 genotypes. This is the test that is recommended for this kind of data and experiment.
The degree and nature of replication is not clear in all cases. For example, in Fig 1, were the 6 fish run as one cohort of 6 animals in parallel (which would be just one experiment with 6 animals, each animal being a biological replicate), or were there 6 animals injured at different times (representing multiple independent experiments and represented a greater degree of reproducibility), or something in between. A similar question applies to the other figures.
In the experiments, 6 fish total were used per group sampled in at least 2 independent experiments. This has been included in the figure legend and in the Mat&Met section
For the experiment of Fig 6F, although there are >=100 larvae per group, it is not clear that this experiment has been done more than once.
In the conducted experiments, three independent trials were conducted. The total number of larvae per group utilized in each of the three distinct experiments surpassed 100 larvae per group (approximately 40 larvae in each independent experiment). This data has been incorporated into both the figure legend and the Materials and Methods section."
A few comments about data presentation. "Regeneration rate" and its derivatives are presented as mean +/- SEM. The parameter measured is correctly defined in methods as "Percent fin regeneration", however the graphs where it is plotted have the y-axis labelled as "regeneration rate (%)" (for example. Fig 1B), which is incorrect. The plotted parameter is not a rate - although there is a time dimension (x-axis), what is plotted at each time point is "% regeneration".
This has been corrected and y-axis is now labeled as Regeneration area (% of initial fin area.
Also, in most figures, such as Fig 1B and 1C, mean +/- SD would be more appropriate, as here each of the n=6 data points represents a single observation from one individual in the population, not the mean of 6 small samples of groups of individuals from the population. Furthermore, at these small n-values (6-10 through the report), scatter plots are considered a more appropriate way of displaying the data (some succinct references: DOI: 10.4103/2229-3485.100662 ; from a Nature group journal DOI: 10.1038/s41551-017-0079 ; from a PLOS journal https://doi.org/10.1371/journal.pbio.1002128 ).
This was a mistake in the figure legend, since Fig 1B was already showing mean +/- SD. Fig 1C is now showing mean+/- SD and has been represented with scatter plots.
The use of mean +/- SEM in Fig 4 could be appropriate, but as n is "at least two independent experiments" scatter plots would again be appropriate. Readers would then know which data sets had only two values.
In two instances, the same data are presented in two different ways (Fig 1B, 1C; the column graphs and arrows of Fig 3D).
Fig4 is presented now as scatter plot graphs
How does "data are average of at least 2 independent experiments" apply to Fig 3C?
In the experiments in Fig3C, “Data are average of 2 independent experiments of 3 fish per group pooled”. This has been included in the figure legend.
**Minor comments:**
The paper is written clearly overall. There are multiple minor grammatical/typographical errors, but these did not detract from understanding the manuscript. These were most abundant in the discussion.
A few points:
Discussion p1 - what is meant by "prematurely aged 11-month fish"
This refers to 11 months old tert-/- fish, which has been shown to present accelerated aging features at this age compared to wild type
Discussion p2 - you mean "doubled" rather than duplicated?
Yes; this has been corrected in the new version
tert +/+, tert +/- and tert -/- genotypes for experiments - how were these obtained and genotypically verified? (heterozygous incrosses? WT x homozygous mutant outcrosses?)
All the fish adult fish of the 3 genotypes were obtained from heterozygous incrosses. Then fish were genotyped by PCR. A protocol for genotyping has been added in the Mat&Met section. The wild type larvae used in the tail fin regeneration experiments inhibiting ATR were obtained by wild type cross, whereas the tert-/- were obtained by tert-/- incross
The last paragraph of the discussion makes some valid points, but it seemed out of place and I wondered if it was misplaced.
This paragraph is added to highlight that our work describes new in vivo model to perform drug screening to inhibit ALT mechanism of telomere maintenance, which is of particular importance for the survival of ALT positive tumor cells.
The rps11 primers appear in the Table of Fig S3 twice.
This has been corrected
Reviewer #3 (Significance (Required)):
The authors claim that this is the first in vivo model examining ALT in regeneration.
The paper contributes to the relatively small body of literature using adult zebrafish models (rather than embryonic larval models) in biomedical research. I cannot comment on the telomere/telomerase literature.
This report will be of interest to those working in regenerative medicine, telomere biology, cancer research, and those interested in zebrafish models of disease and physiological processes.
My expertise encompasses zebrafish disease models and functional studies; I do not have special expertise in telomerase or ALT pathways.
We fully appreciate the reviewer´s comments!
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary:
Martinez-Balsalobre have examined caudal fin regeneration following surgical transection in WT and telomerase-deficient (tert+/- and tert-/-) zebrafish adults of several ages, and in one experiment, in embryos. They conclude: (1) regeneration efficiency decrease with aging in all genotypes (2) telomere length is maintained, even in a tert-mutant background (3) ALT (alternative lengthening of telomeres) is involved in supporting cell proliferation in tailfin regeneration. The experimental system employs a quantitative area-based measurement as a measure of the degree of regeneration. Functional studies used antisense …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary:
Martinez-Balsalobre have examined caudal fin regeneration following surgical transection in WT and telomerase-deficient (tert+/- and tert-/-) zebrafish adults of several ages, and in one experiment, in embryos. They conclude: (1) regeneration efficiency decrease with aging in all genotypes (2) telomere length is maintained, even in a tert-mutant background (3) ALT (alternative lengthening of telomeres) is involved in supporting cell proliferation in tailfin regeneration. The experimental system employs a quantitative area-based measurement as a measure of the degree of regeneration. Functional studies used antisense morpholino gene knockdown and chemical inhibition to implicate ALT involvement.
Major comments:
The experimental logic is appropriate, and in general, the data support the conclusions. Strengths of the work include: (1) The quantitative measure of % regeneration appears to be quite objective; (2) the internally controlled experimental design of the morpholino knockdown experiments of Fig 5.
The Western blot in Fig S2 has some issues. The image is a montage. The experiment appears to have been done only once. The band's identifications by kDa are imprecise (where is the 82 kDa band on the gel? - there are bands smaller and larger than 82 kDa, but none of 82kDa; the 50 kDa band is close to background; the DMSO lane is underloaded relative to the two test lanes (but as the observation is a reduction in the test samples, this does not result in a misinterpretation). What concentration of ATRinhIV were used? The blot has 10 and 50 microM, Fig S1B has 50 and 100 microM, and the text says 1-50 microM).
The MO-knockdown studies are interpreted as showing synergy of atr and tert knockdown. There are two problems with them interpretation of synergy: (1) the single result of a greater effect with both MOs does not distinguish between an additive or synergistic effect (and synergistic action is by definition a greater than additive action; (2) MO dose is not controlled by a group with an equal total MO doses (mo-std+mo-atr and mo-std+mo-tert). While acknowledging that the issues of using local MO delivery in an adult model are very different from global delivery in an embryonic model, the "synergy" interpretation still requires these experiments/controls be done. These experiments were not accompanied by any molecular evidence that either of the morpholinos targeted expression of the intended gene (which would likely have to be derived from their assessment in another system) - a control that can be challenging, but one that is regarded as essential in the field (https://doi.org/10.1242/dev.001115). While this will be difficult to do in the adult setting, it is still appropriate to validate the activity/molecular efficacy of the MO sequence in an experimentally tractable scenario. The specificity of this experiment and interpretation would also be enhanced and corroborated independently by undertaking the atr knockdown in the tert -/- mutant background. Overall, these experiments were preliminary and require further work that could be done withiin 3 months. Note - the tert MO sequence is missing from the table in Fig S4.
The adult experiments have used n=6-10 animals/group. There is no consideration of statistical power (is the analysis of Fig 1C adequately powered?).
The degree and nature of replication is not clear in all cases. For example, in Fig 1, were the 6 fish run as one cohort of 6 animals in parallel (which would be just one experiment with 6 animals, each animal being a biological replicate), or were there 6 animals injured at different times (representing multiple independent experiments and represented a greater degree of reproducibility), or something in between. A similar question applies to the other figures. For the experiment of Fig 6F, although there are >=100 larvae per group, it is not clear that this experiment has been done more than once.
A few comments about data presentation. "Regeneration rate" and its derivatives are presented as mean +/- SEM. The parameter measured is correctly defined in methods as "Percent fin regeneration", however the graphs where it is plotted have the y-axis labelled as "regeneration rate (%)" (for example. Fig 1B), which is incorrect. The plotted parameter is not a rate - although there is a time dimension (x-axis), what is plotted at each time point is "% regeneration". Also, in most figures, such as Fig 1B and 1C, mean +/- SD would be more appropriate, as here each of the n=6 data points represents a single observation from one individual in the population, not the mean of 6 small samples of groups of individuals from the population. Furthermore, at these small n-values (6-10 through the report), scatter plots are considered a more appropriate way of displaying the data (some succinct references: DOI: 10.4103/2229-3485.100662 ; from a Nature group journal DOI: 10.1038/s41551-017-0079 ; from a PLOS journal https://doi.org/10.1371/journal.pbio.1002128). The use of mean +/- SEM in Fig 4 could be appropriate, but as n is "at least two independent experiments" scatter plots would again be appropriate. Readers would then know which data sets had only two values. In two instances, the same data are presented in two different ways (Fig 1B, 1C; the column graphs and arrows of Fig 3D). How does "data are average of at least 2 independent experiments" apply to Fig 3C?
Minor comments:
The paper is written clearly overall. There are multiple minor grammatical/typographical errors, but these did not detract from understanding the manuscript. These were most abundant in the discussion. A few points: Discussion p1 - what is meant by "prematurely aged 11-month fish" Discussion p2 - you mean "doubled" rather than duplicated? tert +/+, tert +/- and tert -/- genotypes for experiments - how were these obtained and genotypically verified? (heterozygous incrosses? WT x homozygous mutant outcrosses?) The last paragraph of the discussion makes some valid points, but it seemed out of place and I wondered if it was misplaced. The rps11 primers appear in the Table of Fig S3 twice.
Significance
The authors claim that this is the first in vivo model examining ALT in regeneration.
The paper contributes to the relatively small body of literature using adult zebrafish models (rather than embryonic larval models) in biomedical research. I cannot comment on the telomere/telomerase literature.
This report will be of interest to those working in regenerative medicine, telomere biology, cancer research, and those interested in zebrafish models of disease and physiological processes.
My expertise encompasses zebrafish disease models and functional studies; I do not have special expertise in telomerase or ALT pathways.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Using zebrafish as a model for regeneration, the authors find that telomere maintenance by recombination can occur in the absence of telomerase.
Title to Figure 4 perhaps may be too strong, 'ALT mechanism is activated', since only a few features of ALT are assessed. Perhaps, 'ALT features are activated'?
mRNA levels of NBS, ATR are also increased in WT animals (Figure 4A and 4B), but ATRX and DAXX mRNA levels are not decreased in WT animals. Is the increase why the authors in part suggest that ALT is being used in WT animals. If so, what would be the trigger for the use of ALT, as opposed to the trigger to use ALT in tert-/- animals?
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Using zebrafish as a model for regeneration, the authors find that telomere maintenance by recombination can occur in the absence of telomerase.
Title to Figure 4 perhaps may be too strong, 'ALT mechanism is activated', since only a few features of ALT are assessed. Perhaps, 'ALT features are activated'?
mRNA levels of NBS, ATR are also increased in WT animals (Figure 4A and 4B), but ATRX and DAXX mRNA levels are not decreased in WT animals. Is the increase why the authors in part suggest that ALT is being used in WT animals. If so, what would be the trigger for the use of ALT, as opposed to the trigger to use ALT in tert-/- animals?
In Figure 5C, if tert-/- animals are downregulated for nbs1 and atr, would it be expected that the effect on regeneration be more pronounced compared to tert+/+ downregulated for nbs1 and atr than what is observed?
What are the telomere lengths in tert-/- animals treated with mo-atr or mo-nbs1 or in tert+/+ animals treated with mo-tert and mo-atr compared to singly treated?
Significance
Reported findings are novel, timely and model of possible therapeutic value for screening compounds for ALT and/or telomerase inhibitors. Mechanisms of co-existence of ALT and telomerase can also be explored using this model.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The authors Martínez-Balsalobre and colleagues found that the regenerative capacity of the zebrafish caudal fin is not limited by the lack of telomerase and showed that the length of telomeres does not decrease substantially after repeated amputations in telomerase-deficient zebrafish. These findings prompt the authors to explore an alternative mechanism that would explain the maintenance of telomere length in this regeneration setting. They produced suggestive evidence for the role of the ALT (Alternative Lengthening of Telomeres) mechanism in the maintenance of telomere length in the absence of telomerase in a regeneration …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The authors Martínez-Balsalobre and colleagues found that the regenerative capacity of the zebrafish caudal fin is not limited by the lack of telomerase and showed that the length of telomeres does not decrease substantially after repeated amputations in telomerase-deficient zebrafish. These findings prompt the authors to explore an alternative mechanism that would explain the maintenance of telomere length in this regeneration setting. They produced suggestive evidence for the role of the ALT (Alternative Lengthening of Telomeres) mechanism in the maintenance of telomere length in the absence of telomerase in a regeneration setting. In my view, several points need to be addressed and clarified.
There are three major points:
1.When working with tert mutants, the age at which these fish show a telomere phenotype (namely, loss of body mass and reduced fertility) varies. Therefore, it would be important to state if the fish used in this study were already showing these phenotypic characteristics at each time point studied, namely 4, 8 and 11 months of age.
2.The knockdown experiments were performed using morpholinos. To confidently use morpholinos it is fundamental to demonstrate first their knockdown efficiency and their specificity. This is lacking in the manuscript.
3.The involvement of ALT mechanism in the regeneration process in the absence of telomerase is only suggestive, as the authors show an increase of C-circles and heterogenous telomerase length in telomerase-deficient zebrafish but when trying to establish a functional link the authors resort to the knowndown of genes that may be associated with ATL. Looking at the levels of TERRA and the number of C-circles in the knowndown caudal fins would be essential for their claim.
And several other points:
4.The regeneration experiments were performed at 32 degrees and this option was never explained nor discussed.
5.When referring to the ALT mechanism, the authors state that "... in about 10% of tumors cells, telomere length is maintained by the Alternative Lengthening of Telomeres (ALT) mechanism ..." and I think it would be more accurate to talk about cancer cells instead of tumor cells.
6.The sentence about C-circles is incorrect. C-circles are mostly single-stranded and not double-stranded as stated.
7.After Figure 2, the authors never mention the age of the fish used.
8.In Figure 1A The site of amputation does not fit the one described in Mat & Met that states 2 cm from the base of the caudal peduncle. The same stands for Figure 2A. The experimental procedure refers 1 dpa but this time point is not plotted in the graph in Figure 1B.
9.In Figure 1B The Y axis should be named regeneration area instead of rate as the values are a percentage of the area reached after a certain time point after amputation. The same stands for Figure 2B, C. It would be nice to see the real caudal fin images for the relevant time points: before amputation, 0 dpa, when the fins reach 50% of regeneration area and then the last time point. The authors should discuss why are the caudal fins reaching more than 100% of regeneration area.
10.In Figure 2B The meaning of ". .. ." on the right side of the graph is not clear. The same stands for Figure 2D.
11.In Figure 2C Why is the clip 10, 11 and 12 missing from the tert+/- and tert-/- ?
12.In Figure 2E The proximity of all points at the 12 Clip is indicative of lack of statistical significance, therefore the **** related to which comparisons?
13.In Figure 2D, E For the measurement of telomere length, the authors state that "Data are average of at least 2 independent experiments." What does this mean exactly? How many animals were used in each experiment?
14.In Figure 3 The authors state that "Data are average of at least 2 independent experiments." What does this mean exactly? How many animals were used in each experiment? Why were the c-circles evaluated at hpa while the telomere length evaluated at dpa? This should be discussed.
15.In Figure 3A The meaning of ". .. ." on the top side of the graph is not clear. t0 should be removed and replaced by 0 hpa and 24hpa and 48hpa for coherence.
16.In Figure 3B,C 0 hpa replace by 0 dpa
17.In Figure 3B The blue and red stainings in the panels are labelling exactly what? This should be stated in the image and in the legend.
18.In Figure 3D There is a mistake in the legend the should be corrected as follows "Very long telomeres have a higher fluorescence of 200,000 AUF and very short telomeres have a lower fluorescence of 30,000 AUF."
19.In Figure 4 t0 should be removed and replaced by 0 hpa. The meaning of ". .. ." on the top side of the graph is not clear. The title is an overstatement, as the genes studied are DNA damage genes that may associate with ALT.
20.In Figure 4A, B The expression of nbs1 and atr in tert-/- increases at 48hpa but the same seems to be true for the tert+/+ and this is never discussed by the authors.
21.In Figure 4C, D The differences in the expression of atrx and daxx decreases over time in a in tert-/- and this is never discussed by the authors.
22.In Figure 5 An ideal control would be the direct comparison between microinjected+electroporated mo-std in the ventral part of the fin while the dorsal part would be microinjected+electroporated with the mo-gene of interest. This would discard any effect of microinjection+electroporation in the regeneration efficiency. These experiments are not convincing to show that there is an ALT mechanism is operating here. What this experiment shows if the relevance of these genes for the regenerative capacity of the caudal fin. To show that this is related to the ALT mechanism the authors should investigate the C-circles in these regenerating fins. The amputation red lines are not placed in the exact amputation position in some of the panels. Regeneration rate should be regeneration area.
23.In Figure 5C, E Why is the mo-tert more inhibitory of regeneration (Figure 5E - around 30%) than the tert-/- mutant (Figure 5C - around 60%)? This should be discussed.
24.In Figure 6A The 2 adult zebrafish shown in the tank with the ATR inhibitor IV should have an amputated caudal fin. Control is exactly what? Untreated? Treated with vehicle? Why was the ATR inhibitor IY added immediately after fin amputation while the mo-atr was injected at 48 hpa?
25.Figure 6D, E, F These panels are a bit out of the focus of this paper. If presented should go to a supplementary figure.
26.In Figure S2 The relevant bands should be identified. The gel identifies DMSO, 10uM and 50 uM but the quantification graph identifies Control, 50uM and 100uM. There are no error bars. The authors say that the quantification of various western blot bands was done but how many exactly?
27.In Figure S3 The primers for rps11 are repeated twice. Were these primers design de novo by the authors or did they used previous reported primers, in this case the references should be given. Tert F2 and R1 should be replaced for F and R for consistency.
28.In Figure S4 The sequence of tert mo is missing.
29.In the methods the genotyping protocol of tert mutants is not described.
30.The method to calculate the area of the fin pre- and post-amputation is not described.
Significance
The manuscript by Martínez-Balsalobre and colleagues deals with a very interesting question on the importance of telomere lengthening during regenerative processes and its relation to ageing. To this end the authors made use of the tert mutant, a telomerase-deficient zebrafish. The authors show a surprising phenotype that telomerase-deficient zebrafish can still regenerate their caudal fins and are able to maintain telomere length during consecutive amputations and I say surprising because it has been shown that telomerase-deficient zebrafish are unable to regenerate their hearts efficiently.
Taking these novel findings, the authors propose that in the zebrafish caudal fin and in the absence of telomerase, telomere length is maintained through the activation of an alternative mechanism called ALT. To my knowledge, the role of ALT as a mechanism of telomere lengthening has never been described in the context of regenerating organs in zebrafish.
Referees cross-commenting
I agree with the comments made by the other reviewers. I would stress the need to tone down the role of ALT during fin regeneration in zebrafish as all the experiments are only indicative of the possible of the involvement of ALT.
-
