Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
The key discovery of the manuscript is that the authors found that genetically wild type females descended from Khdc3 mutants shows abnormal gene expression relating to hepatic metabolism, which persist over multiple generations and pass through both female and male lineages. They also find dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with Khdc3 mutant ancestry. These data provide solid evidence further support that phenotype can be transmitted to multiple generations without altering DNA sequence, supporting the involvement of epigenetic mechanisms. The authors further performed exploratory studies on the small RNA profiles in the oocytes of Khdc3-null females, and their wild type descendants, suggesting that altered small RNA expression could be a contributor of the observed phenotype transmission, although this has not been functionally validated.
Reviewer #2 (Public review):
Summary:
This manuscript aimed to investigate the non-genetic impact of KHDC3 mutation on the liver metabolism. To do that they analyzed the female liver transcriptome of genetically wild type mice descended from female ancestors with a mutation in the Khdc3 gene. They found that genetically wild type females descended from Khdc3 mutants have hepatic transcriptional dysregulation which persist over multiple generations in the progenies descended from female ancestors with a mutation in the Khdc3 gene. This transcriptomic deregulation was associated with dysregulation of hepatically-metabolized molecules in the blood of these wild type mice with female mutational ancestry. Furthermore, to determine whether small non-coding RNA could be involved in the maternal non-genetic transmission of the hepatic transcriptomic deregulation, they performed small RNA-seq of oocytes from Khdc3-/- mice and genetically wild type female mice descended from female ancestors with a Khdc3 mutation and claimed that oocytes of wild type female offspring from Khdc3-null females has dysregulation of multiple small RNAs.
Finally, they claimed that their data demonstrates that ancestral mutation in Khdc3 can produce transgenerational inherited phenotypes.
However, at this stage and considering the information provided in the paper, I think that these conclusions are too preliminary. Indeed, several controls/experiments need to be added to reach those conclusions.
Additional context you think would help readers interpret or understand the significance of the work
Line 25: this first sentence is very strong and needs to be documented in the introduction.
Line 48: Reference 5 is not appropriate since the paper shows the remodeling of small RNA during post-testicular maturation of mammalian sperm and their sensibility to environment. Please, change it
Line 51: "implies" is too strong and should be replaced by « suggests »
Line 67: reference is missing
Database, the accession numbers are lacking.
References showing the maternal transmission of non-genetically inherited phenotypes in mice via small RNA need to be added
Line 378: All RNA-Seq and small RNA-Seq data are available in the NCBI GEO
We have changed references as requested, and updated portions of the introduction in order to mention specifically genes that seem to regulate an RNA-based genetic nurture effect. We are not aware of any published work that has demonstrated maternal transmission of non-genetic phenotypes via small RNAs; if the reviewer has a specific reference in mind, we would be happy to read it and add it to our manuscript. We did add a few sentences describing why this work has primarily been performed in males/fathers.
Reviewer #1 (Recommendations for the authors):
(1) In addition to the altered hepatic gene expression and metabolites, did the authors notice any overall phenotypes? including body weight, overall growth, eating behavior, etc?
We have added information on more general phenotypes of the mice, including litter size, birth weights, and weights at 3 and 8 weeks of age. We have also performed a metabolic analysis of WT****** mice at 8 months of age. Overall, there are no striking differences in the WT* mice in these broad phenotypic measures, and also no indication that a smaller litter size or larger birthweight are the drivers of our observed hepatic abnormalities.
(2) When analyzing the small RNAs, the authors mentioned that they have mapped the reads aging rRNAs. This should have resulted in the identifications of many rRNA-derived small RNAs (rsRNAs). The authors should also perform analyses on the differential expression of rsRNAs in this context. Both tsRNAs and rsRNAs has been shown to be involved in epigenetic inheritance (at least in sperm) (Nat Cell Biol 2018, PMID: 29695786).
In the oocyte small RNA data, we did not notice many differences in either piRNAs or rRNAs between either the WT and KO oocytes, or the WT and WT** oocytes. The most significant differences by far were in miRNA and tsRNA. We have added that we do not see any differences in rRNAs.
Reviewer #2 (Recommendations for the authors):
To support your conclusion, you should include the following Data/experiments:
(1) In the abstract, you wrote "Our results demonstrate that ancestral mutation in Khdc3 can produce transgenerational inherited phenotypes". The full phenotypic description of the phenotype (weight at birth, 3-weeks, 8weights old, phenotype of the liver...) of each progeny should carefully described/analyzed.
Female KHDC3-deficient mice showed reduced fertility with smaller litter. Given the fact that litter size influences early growth and adult physiology (DOI: 10.1016/j.cmet.2020.07.014), all the metabolic effects observed in the paper could be the result of the litter size. Information about the litter size should be provided. Without this information, it is difficult to evaluate the non-genetic impact of KHDC3 mutation on the metabolism of the progenies.
We have added information on more general phenotypes of the mice, including litter size, birth weights, and weights at 3 and 8 weeks of age (Figure 3). We have also performed a metabolic analysis of WT****** mice at 8 months of age. Overall, there were no striking differences in the WT* mice in these broad phenotypic measures, and also no indication that a smaller litter size or larger birthweight are the drivers of our observed hepatic abnormalities.
We have also added a new figure in order to examine the mechanism of transmission of our observed transcriptional abnormalities (Figure 5). By transferring serum from WT* mice into wild type recipients, we observe alterations to hepatic gene expression, suggesting that serum-based molecules are driving the altered non-genetic factors in the oocyte. This lends further support to the conclusion that the observed changes in WT* mice are from inherited germ cell abnormalities (informed by somatic metabolic abnormalities and communicated via blood), and not a consequence of litter sizes or growth rates.
(2) In addition to the lack of phenotypic information of the progenies, the DEG for the small RNA-seq should be filtered on padj(FDR)<0.05 and not on pvalue<0.05. In Figure 4a, the legend is missing.
We did not alter the filtering on the small RNA-Seq data. We are not focusing on any specific small RNA, rather we are stating that these groups (miRNA, tsRNA) of small RNAs are dysregulated; accordingly we believe that using pval is not inappropriate in this circumstance. The analysis was performed similarly to 4 cell embryo RNA-Seq performed by Harris et al, Cell Reports (PMID 38573852).
