Peer review of: Retention of paternal DNA methylome in the developing zebrafish germline (2020)

Reviewers' comments:  
 
Reviewer #1 (Remarks to the Author):  
 
The study by Skvortsova and colleagues provides the first high resolution analysis of DNA 
methylomes through progression of PGC development. Interestingly and in contrast to mammalian 
PGC development, DNA methylation is retained and specifically exhibits patterns similar to the 
sperm methylome. Accompanying transcriptomes from matched stages revealed the PGC marker 
genes were not subjected to dynamic changes to DNA methylation states. It was fascinating to 
learn that there are a set of genes that are dynamically modified by DNA methylation and that this 
Is shared between somatic and PGC cells. This also coincided nicely with TET1 expression and the 
authors even included analysis of 5hmC at these sites to show it’s enrichment. These data 
collectively reveal that DNA demethylation occurs over time as a result of targeting by Tet. Many 
of the PGC specifically expressed genes are also conserved with mice and humans, indicating the 
quality of the isolated targeted cells is of good quality. Overall, I find this to be a very exciting and 
well-done study combining high resolution epigenomic analysis with the biology of a very 
interesting cell type. The comments below are intended to improve this manuscript.  
 
Minor comments:  
 
1. Figure 1c requires an x-axis label  
2. Figure 1 – d and e are swapped in the figure labelling. In general, I don’t see a need for 1e. The 
correlations could just be stated in the text.  
3. The definition of CpG islands should be clearly explained given the distinction to the classic, yet 
arbitrary, definition used to define them in mammals. The current presentation is only meant for 
those who specialized in understanding non-mammalian vertebrate DNA methylation.  
4. I find the use of the word “argue” overused and unnecessarily confrontational. It is used 
throughout this manuscript.  
5. The section on alternative splicing seems out of place and really doesn’t add much to the story. 
It could be removed or moved to the supplement.  
6. The lines drawn on Figure 5b are odd. It’s a forced line that doesn’t represent the data well. Just 
present the dot plots as is without the line.  
 
 
 
 
Reviewer #2 (Remarks to the Author):  
 
In the manuscript, “Retention of paternal epigenetic memory in the developing teleost germline”, 
Skvortsova et al report whole genome bisulfite sequencing of DNA extracted zebrafish embryos 
and primordial germ cells (PGCs) as four stages of development. Using this data, they address a 
longstanding questing in the field of chromatin biology/germ cell development, which is whether or 
not the methylation state of non-mammalian vertebrate PGCs is reprogrammed in the same way 
as it is in mammals. Importantly, they find that, at least during the first 36 hours of development, 
the methylome of PGCs look like sperm, indicating paternal epigenetic memory. These 
observations are bolstered by transcriptome data identifying genes that are uniquely transcribed in 
PGCs and additional observations noting that many of the genes that are specifically targeted by 
DNA methylation in somatic and germline tissues during development are also misregulated in 
cancer.  
 
Although the work is primarily descriptive in nature, the new data sets on methyl/transcriptomes 
of zebrafish PGCs are likely to be of high value to multiple communities, and the finding of 
paternal epigenetic memory provides an important new insight with relevance to those in the fields 
of germ cell development, DNA methylation, and reprogramming. Major and minor points of 
concern are listed below.  
 
Major:  
1. The authors use transcript levels for key germline markers to show that their PGC samples are 
enriched in this cell type. However, there isn’t any data speaking to the fraction of somatic 

contamination in PGC samples. Can the authors provide some data to address this?  
One approach could be to assess the fraction of cells that are GFP positive after sorting. Analysis of 
transcripts in RNA seq data that should be somatic only could also be useful.  
 
2. The methods section indicates that Repeatmasker was used to mask repeats before 5mC 
analysis was performed. Is this true for figure 1C or only for DMR analysis? While there are 
certainly challenges associated with analysis of repeats, given that the bulk of 5mC resides in 
these sequences, it seems like more attention could be paid to their behavior in this manuscript. 
At the very least, the potential for unreported DMRS to reside in repeats should be addressed in 
the discussion.  
 
3. I was somewhat confused by the section entitled “somatic and germline targets of promoter 
5mC”. It seems like the authors shift their DMR calling strategy in this section on a subset of 
promoters, and suggest that in contrast to previous analysis these sequences are in fact DMRs. 
Why do that just for these sequences? If this reduced stringency is also valid, why not apply it 
genome wide?  
 
 
Minor:  
 
For accuracy, the title should be either:  
Retention of paternal epigenetic memory in a developing teolost germline  
Or Retention of paternal epigenetic memory in the developing zebrafish germline.  
One germline is not enough to definitively speak for all teleosts.  
 
In figure 1 a, the authors refer to embryos as being injected with the kop-egfp transgene at 4 hpf 
ect. Surely, if they were injected it was at the one cell stage? This should be revised to be more 
clear, and if true details about amount of plasmid injected ect should be included in methods. 
However, this description seems to contradict the methods section, where the experiments are 
said to be carried out in embryos carrying the transgene, with reference to a stable transgenic 
line. Clarification is needed.  
 
Figure 1 d and e legends appear transposed.  
 
Figure 3a legend does not explain mir430 data clearly.  
 
Figure 3 b Perhaps label samples in the figure as PGCs for clarity?  
 
Supplemental 1. It would be easier to read these panels if the legend was presented in the same 
order as the data on panels (this would also place both the legend and panels in chronological 
order which would be easier to read).  
 
 
 
Reviewer #3 (Remarks to the Author):  
 
Skvortsova and colleagues characterize the DNA methylome in early zebrafish PGCs using WGBS. 
In the four developmental stages analysed, they do not find evidence of a PGC specific global DNA 
demethylation event – which has been described in mouse and human. In addition, they 
characterise gene-expression changes and find that germ line gene expression appears not to be 
regulated by DNA methylation. Finally, they identify genes that are targeted for DNA methylation 
during early embryogenesis and that are also dysregulated in human cancer.  
 
Overall this is an interesting and well-conducted study, by researchers with significant expertise 
the field. This will undoubtedly be of general interest, particularly to those studying germ line 
development and epigenetic reprogramming. However, there are some issues that need to be 
addressed prior to publication.  
 
1. Developmental stages analysed. There is insufficient discussion/explanation of the stages 

analysed and the relatively limited timescale assessed impacts the conclusions that can be drawn. 
This requires significant attention before the paper is ready for publication. The authors do explain 
their selection strategy to include stages equivalent to the period of epigenetic reprogramming in 
mouse PGCs using RBTS. However, it is not necessarily the case that DNA demethylation occurs at 
the same development stage in zebrafish – especially given that there are significant differences in 
germ line development between mice and zebrafish. I have a number of related 
questions/concerns:  
- is it possible to isolate PGCs any earlier than 3hpf? If so, is it possible that the authors missed an 
early period of DNA demethylation and subsequent remethylation?  
- a figure showing how the timings selected map onto zebrafish PGC development would be 
helpful.  
- Based on the data presented it seems possible that DNA demethylation might occur at a later 
stage? For instance, one another reason to suggest this is the pattern of DNA methylation at germ 
line genes which are methylated throughout the period studied and include relatively late markers 
in the mouse (including ddx4 and dazl). As a rule such genes become expressed and and/or their 
regulatory elements become DNA demethylated around the time of global DNA demethylation in 
mice. When are these genes (and other examples such as Nanos3/Dnd1) demethylated in 
zebrafish PGCs? Can the authors rule out that this is the period which is accompanied by or 
correlated with global DNA demethylation?  
- Page 6: ‘could be potentially linked to sex-determination in juvenile fish’ – the authors do not 
appear to present data to back up this statement?  
In general, the authors should moderate their claims throughout the manuscript to make clear that 
they can only rule out DNA demethylation during the timepoints analysed. Alternatively, they may 
refer to the co-submitted manuscript by Hore and colleagues to support their claims. In addition, 
the term ‘epigenetic reprogramming’ should not be used interchangeably with DNA demethylation 
as it is possible there may be DNA methylation independent epigenetic reprogramming (such as 
changes in histone modifications, histone exchange etc).  
 
2. The quantification of DNA methylation is based solely on WGBS analysis. The merits of this as a 
methodology to assess global DNA methylation levels is disputed. In particular, the presence of 
repetitive elements with unknown copy number is problematic – and they likely represent a 
significant proportion of global DNA methylation. Have the authors validated their findings with an 
orthogonal quantitative method, such as LC-MS?  
 
3. If there is no reprogramming of DNA methylation, is there evidence of more variability in DNA 
methylation patterns between zebrafish individuals (as compared to mice)? Would the authors 
expect differences to emerge in different populations (or across generations), as any sporadic 
accumulation of DNA methylation would not be reset in the germ line? Might this impact the CG 
content in the genome over evolutionary time?  
 
4. Introduction  
- contrary to the authors’ assertion, it has now been established that Tet enzymes are not required 
for active DNA demethylation in either the zygotic or PGCs {Amouroux:2016be, Hill:2018cq}.  
- ‘reacquisition of totipotency during pre-implantation development’. It is not clear what this 
means. Totipotency is present in the zygote, rather than acquired during pre-implantation 
development. Perhaps the authors are referring to the acquisition of pluripotency in the epiblast?  
- ‘lack the initial wave of global DNA demethylation’ Does this refer to active DNA demethylation of 
the paternal genome? Or both this and the subsequent passive DNA demethylation that occurs 
thereafter.  
- ‘phenomenon specific to mammals’. What about plants?  
- ‘Our data demonstrate the absence of genome wide reprogramming events’. This statement is 
too strong and nonspecific. The authors focus on a short timescale and almost entirely on DNA 
methylation. 
 
5. Cancer section: In my view, the study is sufficiently interesting without the human cancer 
section, which could be removed. I’m not sure this builds greatly on current knowledge regarding 
CTAs and reactivation of germ line markers in cancer or really develops the story in a meaningful 
way. In many ways, it’s a distraction.  
 

6. Discussion:  
- Page 16: enhancer demethylation is not really depicted in Figure 2 (?Supp Figure 2C).  
- Page 16: ‘Surprisingly’ – is not a good choice, as this appears to be in keeping with mammals, 
and is rather expected.  
- Page 16: ‘remain methylated… in mature oocytes’. This implies stability of the DNA methylome in 
oocytes – which the authors have not addressed. There could easily be dynamic changes.  
- Page 17. ‘Our data argues against major PGC-specific 5mC remodelling’ – far too strong a 
statement based on the data presented.  
Page 17 – hydroxymethylation. Is there sufficient enrichment to be confident that 5hmC is actually 
leading to significant DNA demethylation? (i.e. how do 5hmC levels equate to 5mC levels?).  
 
7. Other issues:  
- Figure 1. Egg samples. What stage of oogenesis is analysed? It seems surprising that the eggs 
cluster with somatic cells, as they have much lower levels of DNA methylation globally. Also, is 
there an explanation for the difference between the two replicates?  
- Figure 1: legends d and e are swapped  
- Page 9: The statement ‘arguing against a major role for 5mC in PGC fate determination’. Is this 
really a suggested mechanism of fate determination in zebrafish PGCs? (it isn’t thought to be in 
mice). How would this work in the context of germ plasm?  
- Section ‘Somatic and germline targets of promoter 5mC’. It appears that the promoters 
discussed behave differently to the global pattern, and actually increase DNA methylation to be 
more similar to the female pattern. This could perhaps be explained/discussed more clearly, as 
some readers may find this surprising/counterintuitive.