Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response

  1. Rebecca A. Jones
  2. Fay Cooper
  3. Gavin Kelly
  4. David Barry
  5. Matthew J. Renshaw
  6. Gopal Sapkota
  7. James C. Smith

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The FAM83 ( Fam ily with sequence similarity 83 ) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When over-expressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation of fam83f knock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the hatching gland of developing zebrafish embryos, and that fam83f KO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate that fam83f KO embryos are more sensitive to ionizing radiation than WT embryos—an unexpected finding, bearing in mind the previously-reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss of fam83f leads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when over-expressed in HEK293T cells, and that this localization is dependent upon a C’ terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo.

Article activity feed

  1. Review Commons

    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)):____ ____ __ In this manuscript, Jones et al. report on a potential role for fam83fa in zebrafish hatching, radiation response and autophagy. The authors are commended for generating multiple KO lines and maternal-zygotic embryos for analysis. However, important controls are lacking and the data is circumstantial throughout with very little mechanistic insight into the precise roles, if any, of fam83f in these processes.

    We thank the reviewer for recognizing the strengths of our manuscript, and highlighting areas we might improve. Please see the specific comments below addressing the points raised. In respect of mechanistic insight, while we agree that our manuscript does not provide this, it was not intended to. Rather, we aim to communicate our descriptive findings on the role of Fam83fa in vivo, providing data for follow-up studies by other researchers into the mechanistic role of Fam83fa.

    Validation of the KO phenotypes (hatching, IR sensitivity) requires rescue with WT fam83fa WT mRNA, but not 1-500 or fam83fb mRNA.

    We thank the reviewer for raising the issue of rescue experiments. Such experiments are frequently used in knock-down experiments, where non-specificity may be a problem, but they are used more rarely in genetic knock-outs, where the gene defect is well defined. In the case of Fam83fa, a particular difficulty is that overexpression of fam83fa itself causes a p53-mediated DNA damage response (DDR) (Salama et al., 2019). Moreover, we have shown by both qRT-PCR and western blotting that injection of fam83fa mRNA into zebrafish embryos (the traditional technique by which rescue experiments are performed) induces a p53-mediated DDR. As a result, it would be very difficult to interpret the results of any rescue experiment, because one would have to be absolutely certain that levels of fam83fa re-expression recapitulate and do not exceed endogenous levels. As a tool for specificity, we therefore used more than one fam83fa-/- mutant line, carrying a different genomic mutation, and validated that the same phenotype was present in both. We are happy to provide the qRT-PCR and western blot data confirming the results of *fam83fa *mRNA injection, if required. We have included an additional section into the manuscript detailing this issue.

    While the hatching phenotype (Fig 3) is convincing, there is no data on HG development in the null embryos. Does the HG develop normally in the absence of fam83fb? If so, this would support the authors conclusions that the role of fam83fb is functional rather than developmental (indirect effect). In situs as in Fig.1 might be helpful here.

    Thank you to the reviewer for this helpful suggestion. We agree that we did not investigate whether the hatching gland develops normally in the MZ-fam83fa-/- mutant embryos. No gross morphological differences were observed that led us to investigate this, although we agree it is an interesting question for a future project. In terms of functional vs developmental effects, we are confident that MZ-fam83fa-/- mutant embryos develop at a normal temporal rate, as evidenced by the machine learning based classifier used to assess temporal developmental trajectory (Figure S3 and Jones et al., 2022, 2024). This strongly suggests that the effect of fam83fa KO is functional rather than indirect and caused by (for example) developmental delay.

    While the IR sensitivity phenotype (Fig S4) is convincing, IR-induced cell death/apoptosis was not analyzed. There is a large literature describing straightforward assays for cell death/apoptosis detection in zebrafish with assays such as acridine orange or TUNEL labeling, or active casp3 whole-mount IF. Is IR-induced cell death enhanced in fam83fa KOs?

    We thank the reviewer for their positive comments and agree that investigating the nature of the cell death occurring following IR would be very interesting. We did make use of both acridine orange and TUNEL labeling following injection of fam83fa mRNA (see 1 above), and whilst the assays themselves were relatively straightforward, due to technical issues the quantification of fluorescence intensity was not. Similarly, we suspect that a significant degree of necrosis is also occurring, which further complicates the issue of data interpretation from both these approaches. We do, however, think this is an important avenue of questioning, and hope that other researchers will explore the mechanism of IR induced cell death in the MZ-fam83fa-/- mutants in the future,

    Similarly, there are multiple tools to assay autophagy in zebrafish (e.g., Moss et al., Histochem Cell Biol 2020, PMC7609422; Mathai et al., Cells 2017, PMC5617967). Is autophagy affected in the KOs, with or without IR? These experiments might directly implicate fam83fa in autophagy.

    We agree that there are exciting tools with which to assay autophagy in zebrafish, and although we considered some of these, including caudal fin regeneration, we deemed these experiments to be beyond the descriptive scope of this paper, given the time and resources available to us. We hope that other researchers will use our data as a basis for investigating the role of Fam83fa in autophagy further, using assays such as these suggested by the reviewer.

    Figure 4: Isn't there a slight reduction in p53 induction at 10 hours?

    Although the western blot in Figure 4A gives this impression, this is probably due to loading variability (see the anti-β-actin loading control band). Moreover, over three independent experiments (Figure 4B), this apparent difference is not statistically significant. Taken together with other evidence that the p53-mediated DNA damage response is not affected in MZ-fam83fa-/- mutants, we are confident there is no detectable change in the level of stabilized p53 in the MZ-fam83fa-/- mutants compared to WT.

    Given the widely documented, dominant role of p53 in zebrafish IR-sensitivity, the authors should test if the IR sensitivity of fam83fa KO animals is p53-dependent, ideally via a cross into p53 null, but at least via injection of p53 morpholinos.

    We agree that p53 is widely documented as playing an essential role in the IR induced DNA damage response in zebrafish. All our experiments suggest there is no difference between the levels of p53 (protein or mRNA) or any of the p53-induced downstream effectors (that we tested) in MZ-fam83fa-/- mutants compared to WT embryos. This was true whether or not the embryos were subjected to genotoxic stressors, including IR treatment. We therefore conclude that the increased sensitivity phenotype we observe as a result of loss of Fam83fa is not caused by a change in p53 activity, at least not as part of the DNA damage response.

    Do autophagy inhibitors phenocopy the hatching and IR-sensitivity defects of fam83fa embryos? Do the inhibitors exacerbate the mutant phenotypes or synergize with M or Z mutant phenotypes? (I may have missed this but do M and Z fam83fa null embryos have any phenotype? Or do the phenotypes only manifest in MZ embryos?)

    This is an excellent question, and indeed one we attempted to address. We tried to optimize several autophagy inhibitors including bafilomycin A1, chloroquine and wortmannin, as well as the proteasomal inhibitor MG132. In addition, we tried to optimize the autophagy promoters Torin1 and rapamycin. Unfortunately, we regularly saw global effects in zebrafish embryos that were difficult to characterize and control by dosage. At the same time, we were also working to confirm the specific effects of these drugs on autophagy using p62 and LC3-I and LC3-II western blots, which themselves were difficult to optimize. We attempted to optimize these experiments for 6 months before the COVID lockdown occurred, at which point they were abandoned. We would be delighted for future researchers to continue these experiments, as we are now unable to pursue this further due to closure of the Smith lab, but we agree that these are very pertinent questions. We hope the descriptive data provided in our paper will prompt other researchers in the autophagy field to further explore the role of Fam83fa in autophagy. In response to the zygotic phenotype question, this was something we did not investigate. As there was no immediately apparent phenotype in the zygotic generation, for ease of screening larger numbers of embryos we proceeded immediately to the maternal-zygotic (MZ) generation.

    Reviewer #1 (Significance (Required)):

    The role of Fam83f is not known. This study in zebrafish might be the first to clarify the function of this protein in vivo.

    We thank the reviewer for this positive insight, and we agree that our work is the first do so in vivo.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Fam83f is one of the proteins about which little is known. The authors Jones et al., tried to shed light on Fam83f function by knocking out the gene in zebrafish. Here they found that fam83 is expressed in the hatching gland and that larvae without Fam83f hatch significantly earlier than wild-type animals. The authors furthermore investigated the response of fam83f knock-out animals to DNA damage and found increased sensitivity to ionizing radiation and MMS. In order to find out more about Fam83f function in the DNA damage response, the authors performed RNA-seq after employing DNA damage and here they saw upregulation of several autophagy/lysosome-associated proteins and downregulation of some phosphatidylinositol-3-phosphate binding proteins, among others. Finally, the authors found that Fam83f is targeted to the lysosome. The manuscript is overall well written and clear in its general statement.

    We thank the reviewer for their encouraging comments.

    In the manuscript, the authors describe the investigation of several aspects of Fam83f function and particularly the role in hatching seems to be important for Fam83f as the gene is strongly expressed in the hatching gland and its absence leads to a clear and considerable earlier hatching. Unfortunately, all aspects of Fam83f function that are described in the manuscript are investigated very superficially, the conclusions are not supported by data and important controls are lacking. As such, the RNA-seq results are not confirmed by qRT-PCR, the role of the Fam83f LIR domain is not confirmed by co-IPs and it has not been investigated whether the presence of Fam83f in lysosomes is due to its degradation or whether it has a function in this cellular compartment.

    We thank the reviewer for their input and will address each point raised below: -

    All aspects of Fam83f function are investigated superficially.

    We agree that we have not provided an in-depth analysis of the mechanistic role of Fam83fa. It was because there were so many roles that we decided to make this paper rather descriptive in nature, hoping that the observations will prove useful to other researchers who may wish to define the mechanistic roles of Fam83fa more deeply. Even without in-depth investigation, our findings are previously unreported and the phenotypes we report are clear. We have amended our manuscript to make it apparent that this paper is intended to be descriptive in nature, and we hope this addresses this issue.

    Important controls are lacking - RNA-seq results are not confirmed by qRT-PCR

    We thank the reviewer for their comment. We did not include qRT-PCR data as a control for the RNA-seq data because 1) each RNA-seq experiment was repeated on three biological replicates across three independent experiments and 2) we conducted RNA-seq on two different MZ-fam83fa-/- mutant lines and only considered genes that were mis-regulated in both mutants. Taken together, we considered this to be sufficient validation for the manuscript. However, we also performed confirmatory qRT-PCR for several of the differentially expressed genes identified, including the three main PI(3)P binding genes. We have now included these data in the supplementary information as an additional control - see Figure S6G which is now also referred to in the main text, and additional primer sequences have been added to Table S1.

    The role of the Fam83f LIR domain is not confirmed by co-Ips

    We agree with the reviewer that this is an important experiment, and we worked closely with Dr Brian Ludwig and Dr Karen Vousden (The Francis Crick Institute) to test this. We tried to express zebrafish Atg8 and Gabarap (the two main ATG8 proteins that bind to LIR domains) but were unable to express sufficient levels of protein to perform the co-Ips. The text in the manuscript has now been amended to reflect that this experiment is required to confirm the role of the putative LIR domain in Fam83fa.

    *it has not been investigated whether the presence of Fam83f in lysosomes is due to its degradation or whether it has a function in this cellular compartment

    Whilst we agree with the reviewer that this is an important question, we did not intend this paper to expand beyond a descriptive role of the observations we made following the loss of Fam83fa in vivo. These are important questions to follow up on to determine the mechanism of action of Fam83fa, and we hope that other researchers will pursue these avenues of investigation following the publication of our observations.

    Also, there is no leading concept in the manuscript. Starting from a role in hatching, the authors go to the DNA damage response and finally to the presence of Fam83f in lysosomes. How are these different aspects linked? Is the presence of Fam83f in lysosomes important for the suppression of hatching and how does Fam83f delays this process? (One would have wished that the authors would not have been that broad and were more focused on a particular aspect which then could have been investigated in depth.)

    We agree with the reviewer that the paper gives a broad overview of our observations and does not examine the underlying mechanisms in detail. However, we believe that descriptive papers such as this, where observations following genetic perturbation are reported, are equally important, providing as they do important foundational data for other researchers to take forward. We do postulate on the links between the hatching, DNA damage and lysosomal phenotypes we observe in the discussion section, and we have expanded on this following the reviewers' comments, to make our hypothesized link between these phenomena clearer.

    Specific comments:

    • All materials should be described in material and methods including the antibodies that have been used

    The antibodies used together with concentrations and catalog numbers are now in Materials and Methods

    • Abbreviations should be explained

    The manuscript has been revised to ensure all abbreviations are explained. We thank the reviewer for bringing this oversight to our attention.

    • Figure 4A: Levels of p53 should also be shown for untreated fam83f -/-KO1 and KO2 animals

    The authors thank the reviewers for raising this point. Extracts from untreated MZ-fam83fa-/- KO1 and KO2 embryos were not included on this particular blot, as p53 was observed to be undetectable in all embryos, across all our experiments (WT and both mutants) unless genotoxic stress was applied. No quantification could therefore be performed as the expression level was essentially zero. However, we have now included an example p53 western blot in Supplemental Figure 5A, which shows WT, MZ-fam83fa-/- KO1 and MZ-fam83fa-/- KO2 untreated blots for p53 (all undetectable) alongside treated embryos (detected).

    • Some references are missing (e.g. page 17, lane 320/321: As this group of cells arises....)

    This citation and reference have now been added; thank you to the reviewer for highlighting this omission.

    • Lane 369: The authors write about 4 KO lines but only two are shown in the figure.

    We thank the reviewer for this observation. In Figure 2B only KO1 and KO2 schematic diagrams are shown for simplicity (as these are the lines taken forward for further investigation). We have now amended the manuscript text to make this clear.

    • Lane 374/375: The NMD is not proven

    Absolutely - we have now revised the text to change this sentence accordingly and thank the reviewer for noting this.

    • Lane 380: how can RNA levels of fam83fa be upregulated when the gene has been knocked out? Why are these genes only upregulated in KO1? How relevant is this?

    This was a typographical error, and we are very grateful to the reviewer for picking up on this. It should have read 'fam83fb'. As nonsense-mediated decay and associated transcriptional adaptation have been previously reported in zebrafish, this finding may be of considerable interest to the community. It is a side observation, and not necessarily directly related to the role of Fam83fa in vivo, but we felt it important to include. Indeed, as a result of this observation we have recently shared our MZ-fam83fa-/- lines with another group who are planning to investigate precisely this question - why are fam83fb and fam83g only upregulated in KO1?

    • Figure 3C is not mentioned in the text and lacks any labelling

    Figure 3C is now clearly referred to in the text and a label added to the figure.

    • Lane 434/435: all relevant data should be shown (can be done as supplementary figure)

    We have now amended this to include an additional supplemental figure (Figure S5A).

    • Lane 434: The reference to the figure seems to be incorrect (5A4A)

    Amended accordingly - thank you for pointing out this mistake.

    • Figure 4C and 4D: what is the difference?

    Thank you to the reviewer for noticing this omission. These data are from t1 (+2hrs) and t2 (+10hrs) and have now been labelled accordingly.

    • S5C and S5D: why are there 3 clusters?

    We thank the reviewer for raising this as it has provided us with an opportunity to present our data more clearly. There are 3 clusters that represent the combination of the two first principal components, which are time and treatment. Therefore, the clusters represent i) untreated at t1, ii) treated at t1 and iii) treated at t2. However, having two plots with different color schemes made this confusing/misleading. We have now replaced the two PCA plots with one that is colored and labelled accordingly with the 3 aforementioned clusters.

    • Lane 495 to 505: What does this mean that the GO analysis shows upregulation and downregulation of endopeptidases and why "in contrast"?

    We thank the reviewer for this comment, and we agree that this paragraph was misleading/confusing. This has now been rewritten in the main text, clarifying that endopeptidases were consistently upregulated at both timepoints.

    Reviewer #2 (Significance (Required)):

    The strength of the manuscript is certainly that it provides inside into Fam83f function as there is not much known about Fam83f.

    We thank the reviewer for the positive comment, and we agree that very little is known about this highly conserved protein.

    These study is probably most interesting for people in the zebrafish and related fields as the authors convincingly show the expression of Fam83f in the hatching gland and also the earlier hatching in the absence of the protein is very clear.

    Thank you for the positive feedback.

    The weakness of the study is clearly that it does not provide an in-depth analysis. As such, it shows that Fam83f is involved in hatching and can delay the process but it remains elusive how this is achieved. (Likwise, also the investigation into the DNA damage response remains very superficial and does not prove a specific role for Fam83f in the DNA damage response or whether the increased sensitivity is more unspecifically caused by the absence of a gene or eventually even connected to the earlier hatching.

    Please refer to responses above (and changes made to the manuscript) clarifying that this study is intended to be descriptive, and provides important foundational data for further in-depth mechanistic studies by other researchers interested in the role of Fam83fa in vivo.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):____ ____ __ In their manuscript "Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response", Jones et al. provide an interesting exploration for the function of a poorly studied protein, Fam83f in embryonic development. Using the zebrafish as a model organism, the study combines loss-of-function genetics, phenotypic analysis and RNA-sequencing to characterize and explore the result of Fam83f loss. Upon critical review of the manuscript and the results we offer suggestions to improve the manuscript (see 'minor technical issues'). Additionally, we would like to highlight a weakness of the study in making the connection between Fam83f to the observed phenotype (increased sensitivity to DNA damage), see 'major issues'.

    Major issues:

    Most of our concern stems from relatively incomplete connection of the loss of fam83f to increased sensitivity to DNA-damage and lysosome function.

    Please refer to comments above and changes made to the manuscript to clarify this is a descriptive paper that is not intended to provide in-depth mechanistic insight into the role of Fam83fa.

    Is the increased sensitivity in fam83f KO embryos a direct effect to fam83f loss? A rescue experiment (by introduction of Fam83fa mRNA into their KO2 fish line) in the presence of ionizing radiation would help us understand the functional role of this protein in this process. Furthermore, can overexpression of any of the down-regulated genes involved in lysosome function restore the early hatching phenotype or the sensitivity to DNA damage? Fam83fa rescue experiments would be very difficult to interpret - please see comments above and the corresponding changes to our manuscript.

    In terms of over-expressing some of the downregulated genes identified in the RNA-seq and qRT-PCR to see if the phenotype can be rescued, we feel these are excellent suggestions and we hope other researchers in future will attempt such experiments.

    Minor technical issues:

    -Methods line 203, clarify how many embryos were used per sample for RNA-seq (this was only described as 15 embryos in the main body results text).

    Text has been amended to clarify this. We thank the reviewer for noticing this oversight.

    -Comment about the expansion of fam83f orthologs in mammals (8) as opposed to only 2 in zebrafish

    We apologize for any confusion: mammals do not have 8 fam83f orthologs. Mammals and zebrafish have 8 FAM83 genes (FAM83A-FAM83H). Zebrafish, unlike mammals, have genome duplication and although mammals have only one FAM83F gene, zebrafish have two: Fam83fa and Fam83fb. We trust this clarifies this issue and believe this to be clear in our main text. However, we are happy to make any suggested amendments should the reviewer consider our wording confusing.

    -Supplementary figure 1C: please include representative images of secondary axis formation in fam83fa overexpressed Xenopus embryos.

    We have not included any images as these are already published in our related paper on FAM83F (Dunbar et al., 2020) which we refer to in the figure legend text. No additional images were captured specifically for this publication.

    -Provide more information about the mis-regulated genes in the RNA-seq analysis, how many are up or down regulated? Perhaps a better plot than a Venn diagram can be an MA-plot with the Venn diagram moved to a supplementary figure.

    The Venn diagrams in Figure 5A-C are to illustrate the number of differentially expressed genes that are shared between KO1 and KO2 (whether up or down regulated), and only those that are common to both lines are taken forward. Following the reviewer's comments, we have now displayed the behavior of the common genes across all replicates in one heatmap, with the data normalized to the WT untreated samples, and the normalized variance stabilized count indicates whether a gene is up or down regulated across each of the replicates and conditions. We believe this addresses the reviewer's comment as these data are now displayed in a more direct way and the genes that are consistently up or downregulated across all replicates (and indeed those that are not) can be clearly seen. We thank the reviewer for raising this and improving our data representation.

    -A better comparison of mis-regulated genes in the fam83f knockouts would be a comparison of KO2 and perhaps KO3, as the compensatory effects in KO1 can lead to additional indirect effect on the transcriptome. We understand the time and cost involved in this experiment and suggest that the differential gene expression analysis be performed individually on up or down regulated genes from KO2, or a comparison of such analysis will be provided with the differential gene expression analysis that was performed on shared mis-regulated genes between KO1 and KO2.

    The reviewer raises an excellent point. At the time of experimental design, we were concerned that omitting KO1 in favor of another line (e.g. KO3) would bias our results by excluding potentially important data. Similarly, as transcriptional adaptation occurs in a sequence specific manner, and the phenotype was present in KO1 regardless, we didn't want to exclude these data. However, with hindsight, we agree that it may have been prudent to exclude KO1 on this basis, and we may have seen an increased concordance of differentially expressed genes (DEGs) between KO2 and KO3. However, this is not possible to repeat now due to the Smith lab closing, and our documented findings are valid and important regardless. We acknowledge however that, with hindsight, what the reviewer suggests may have been better experimental design.

    -Can you confirm with the RNA-seq analysis that fam83g is upregulated in KO1 as opposed to KO2? (i.e. can the compensatory analysis you have observed with qRT-PCR be confirmed with the RNA-seq data?)

    This is an excellent question, and we thank the reviewer for raising this. fam83fb passed our threshold for significance to be deemed as differentially expressed (upregulated) in KO1 only, in accordance with our qRT-PCR data. fam83g did not pass the significance threshold, but perhaps this is not surprising as both fam83fb and fam83g are expressed at particularly low levels to start with and would probably require much greater sequencing depth to be detected.

    Reviewer #3 (Significance (Required)):

    There is fundamental value in clarifying the in vivo function of poorly characterized protein-coding genes. This study fills a gap in the literature, but the broader conceptual impact is limited. The authors do a thorough job at generating and characterizing CRISPR/Cas9 mediated knock-out zebrafish animals. It is further commended that the authors do a meticulous job in a quantitative description of the resulting phenotype. This is a thorough study, with the only major concern being the lack of rescue experiments that would be needed to substantiate the the role of fam83f in sensitivity to DNA damage and lysosome function.

    We thank the reviewer for their comments and trust we have addressed the issues concerned with the changes described above.

  2. Review Commons

    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

    In their manuscript "Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response", Jones et al. provide an interesting exploration for the function of a poorly studied protein, Fam83f in embryonic development. Using the zebrafish as a model organism, the study combines loss-of-function genetics, phenotypic analysis and RNA-sequencing to characterize and explore the result of Fam83f loss. Upon critical review of the manuscript and the results we offer suggestions to improve the manuscript (see 'minor technical issues'). Additionally, we would like to highlight a weakness of the study in making the connection between Fam83f to the observed phenotype (increased sensitivity to DNA damage), see 'major issues'.

    Major issues:

    Most of our concern stems from relatively incomplete connection of the loss of fam83f to increased sensitivity to DNA-damage and lysosome function.

    Is the increased sensitivity in fam83f KO embryos a direct effect to fam83f loss? A rescue experiment (by introduction of Fam83fa mRNA into their KO2 fish line) in the presence of ionizing radiation would help us understand the functional role of this protein in this process. Furthermore, can overexpression of any of the down-regulated genes involved in lysosome function restore the early hatching phenotype or the sensitivity to DNA damage?

    Minor technical issues:

    • Methods line 203, clarify how many embryos were used per sample for RNA-seq (this was only described as 15 embryos in the main body results text).
    • Comment about the expansion of fam83f orthologs in mammals (8) as opposed to only 2 in zebrafish
    • Supplementary figure 1C: please include representative images of secondary axis formation in fam83fa overexpressed Xenopus embryos.
    • Provide more information about the mis-regulated genes in the RNA-seq analysis, how many are up or down regulated? Perhaps a better plot than a Venn diagram can be an MA-plot with the Venn diagram moved to a supplementary figure.
    • A better comparison of mis-regulated genes in the fam83f knockouts would be a comparison of KO2 and perhaps KO3, as the compensatory effects in KO1 can lead to additional indirect effect on the transcriptome. We understand the time and cost involved in this experiment and suggest that the differential gene expression analysis be performed individually on up or down regulated genes from KO2, or a comparison of such analysis will be provided with the differential gene expression analysis that was performed on shared mis-regulated genes between KO1 and KO2.
    • Can you confirm with the RNA-seq analysis that fam83g is upregulated in KO1 as opposed to KO2? (i.e. can the compensatory analysis you have observed with qRT-PCR be confirmed with the RNA-seq data?)

    Significance

    There is fundamental value in clarifying the in vivo function of poorly characterized protein-coding genes. This study fills a gap in the literature, but the broader conceptual impact is limited. The authors do a thorough job at generating and characterizing CRISPR/Cas9 mediated knock-out zebrafish animals. It is further commended that the authors do a meticulous job in a quantitative description of the resulting phenotype. This is a thorough study, with the only major concern being the lack of rescue experiments that would be needed to substantiate the the role of fam83f in sensitivity to DNA damage and lysosome function.

  3. Review Commons

    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

    Fam83f is one of the proteins about which little is known. The authors Jones et al., tried to shed light on Fam83f function by knocking out the gene in zebrafish. Here they found that fam83 is expressed in the hatching gland and that larvae without Fam83f hatch significantly earlier than wild-type animals. The authors furthermore investigated the response of fam83f knock-out animals to DNA damage and found increased sensitivity to ionizing radiation and MMS. In order to find out more about Fam83f function in the DNA damage response, the authors performed RNA-seq after employing DNA damage and here they saw upregulation of several autophagy/lysosome-associated proteins and downregulation of some phosphatidylinositol-3-phosphate binding proteins, among others. Finally, the authors found that Fam83f is targeted to the lysosome. The manuscript is overall well written and clear in its general statement. In the manuscript, the authors describe the investigation of several aspects of Fam83f function and particularly the role in hatching seems to be important for Fam83f as the gene is strongly expressed in the hatching gland and its absence leads to a clear and considerable earlier hatching. Unfortunately, all aspects of Fam83f function that are described in the manuscript are investigated very superficially, the conclusions are not supported by data and important controls are lacking. As such, the RNA-seq results are not confirmed by qRT-PCR, the role of the Fam83f LIR domain is not confirmed by co-IPs and it has not been investigated whether the presence of Fam83f in lysosomes is due to its degradation or whether it has a function in this cellular compartment. Also, there is no leading concept in the manuscript. Starting from a role in hatching, the authors go to the DNA damage response and finally to the presence of Fam83f in lysosomes. How are these different aspects linked? Is the presence of Fam83f in lysosomes important for the suppression of hatching and how does Fam83f delays this process? (One would have wished that the authors would not have been that broad and were more focused on a particular aspect which then could have been investigated in depth.)

    Specific comments:

    • All materials should be described in material and methods including the antibodies that have been used
    • Abbreviations should be explained
    • Figure 4A: Levels of p53 should also be shown for untreated fam83f -/-KO1 and KO2 animals
    • Some references are missing (e.g. page 17, lane 320/321: As this group of cells arises....)
    • Lane 369: The authors write about 4 KO lines but only two are shown in the figure.
    • Lane 374/375: The NMD is not proven
    • Lane 380: how can RNA levels of fam83fa be upregulated when the gene has been knocked out? Why are these genes only upregulated in KO1? How relevant is this?
    • Figure 3C is not mentioned in the text and lacks any labelling
    • Lane 434/435: all relevant data should be shown (can be done as supplementary figure)
    • Lane 434: The reference to the figure seems to be incorrect (5A<->4A)
    • Figure 4C and 4D: what is the difference?
    • S5C and S5D: why are there 3 clusters?
    • Lane 495 to 505: What does this mean that the GO analysis shows upregulation and downregulation of endopeptidases and why "in contrast"?

    Significance

    The strength of the manuscript is certainly that it provides inside into Fam83f function as there is not much known about Fam83f.

    These study is probably most interesting for people in the zebrafish and related fields as the authors convincingly show the expression of Fam83f in the hatching gland and also the earlier hatching in the absence of the protein is very clear.

    The weakness of the study is clearly that it does not provide an in-depth analysis. As such, it shows that Fam83f is involved in hatching and can delay the process but it remains elusive how this is achieved. (Likwise, also the investigation into the DNA damage response remains very superficial and does not prove a specific role for Fam83f in the DNA damage response or whether the increased sensitivity is more unspecifically caused by the absence of a gene or eventually even connected to the earlier hatching.

  4. Review Commons

    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

    In this manuscript, Jones et al. report on a potential role for fam83fa in zebrafish hatching, radiation response and autophagy. The authors are commended for generating multiple KO lines and maternal-zygotic embryos for analysis. However, important controls are lacking and the data is circumstantial throughout with very little mechanistic insight into the precise roles, if any, of fam83f in these processes.

    1. Validation of the KO phenotypes (hatching, IR sensitivity) requires rescue with WT fam83fa WT mRNA, but not 1-500 or fam83fb mRNA.
    2. While the hatching phenotype (Fig 3) is convincing, there is no data on HG development in the null embryos. Does the HG develop normally in the absence of fam83fb? If so, this would support the authors conclusions that the role of fam83fb is functional rather than developmental (indirect effect). In situs as in Fig.1 might be helpful here.
    3. While the IR sensitivity phenotype (Fig S4) is convincing, IR-induced cell death/apoptosis was not analyzed. There is a large literature describing straightforward assays for cell death/apoptosis detection in zebrafish with assays such as acridine orange or TUNEL labeling, or active casp3 whole-mount IF. Is IR-induced cell death enhanced in fam83fa KOs?
    4. Similarly, there are multiple tools to assay autophagy in zebrafish (e.g., Moss et al., Histochem Cell Biol 2020, PMC7609422; Mathai et al., Cells 2017, PMC5617967). Is autophagy affected in the KOs, with or without IR? These experiments might directly implicate fam83fa in autophagy.
    5. Figure 4: Isn't there a slight reduction in p53 induction at 10 hours?
    6. Given the widely documented, dominant role of p53 in zebrafish IR-sensitivity, the authors should test if the IR sensitivity of fam83fa KO animals is p53-dependent, ideally via a cross into p53 null, but at least via injection of p53 morpholinos.
    7. Do autophagy inhibitors phenocopy the hatching and IR-sensitivity defects of fam83fa embryos? Do the inhibitors exacerbate the mutant phenotypes or synergize with M or Z mutant phenotypes? (I may have missed this but do M and Z fam83fa null embryos have any phenotype? Or do the phenotypes only manifest in MZ embryos?)

    Significance

    The role of Fam83f is not known. This study in zebrafish might be the first to clarify the function of this protein in vivo.

  5. Version published to 10.1101/2024.02.10.579757v1 on bioRxiv