Transgenerational inheritance of ethanol preference is caused by maternal NPF repression

Essential revisions:

1) An important distinction between preference in exposed flies vs. their progeny is the requirement of memory. The experiment in Figure 1C suggests some degree of dissociation between long term memory of preference and transgenerational inheritance of preference, as long-term memory impairment partially but not completely impairs the transgenerational inheritance. Is this partial transgenerational deficit because the parent can't hang onto the memory, a situation akin to "brood 2" (Figure 1—figure supplement 2) where the parental memory is gone, or does the lack of Orb2 in the mutant impair the inheritance of the offspring (not an unreasonable idea given the role of Orb2 in development)? To distinguish these two models, the authors should do one of several things: impair memory by expressing Orb2 RNAi in the adult mushroom body and assessing the inheritance, or use another memory mutant, or use some other acute MB manipulation.

The reviewers’ point is well taken. To address this possibility we have added two additional experiments (Figure 1—figure supplement 1). First, the experiment was repeated with the memory mutant amn¹, thereby addressing the concerns about an Orb2 specific effect. Secondly, we used a conditional knockdown of Orb2 in the mushroom body; thus generating F₁ progeny that did not have expression of the RNAi transgene.

2) The authors need to clean the paper up and provide the necessary info in figure legends for the reader to understand what they have done.

We have made particular changes to address the specific comments.

Specific points:

1) In the Introduction, “wasp” should be “wasps”.

We have corrected this typo in the text.

2) There are many grammatical errors throughout the paper e.g. “treated to” ethanol.

We apologize for the grammatical errors in the previous draft. We have taken additional steps to proof read and correct these errors.

3) Do flies exposed to wasps prefer to consume EtOH as well? Are they more likely to be addicted? Just curious.

The reviewer raises a very interesting question. Although we have previously shown that adult flies continue to eat following wasp exposure, we do not have any direct data to conclusively show that they preferentially consume ethanol. Unfortunately, to investigate this question one would need a very different experimental design and specialized apparatus. While this is an area of interest for us, we feel that it is better suited to a future investigation and outside of the scope of the current study.

4) Figure 1C is not completely clear. The flies assayed are ALL F₁? Perhaps label this better?

We apologize for the confusion. In Figure 1C data is shown for legacy (F₁) flies, collected during or post wasp exposure. We have clarified this in the figure itself as well as in the figure legend.

5) Are the dcp1 and drice knockdowns in ovary only? What is the driver? This information should be added to the legend and text.

The driver line used in these experiments is specific to the female germline. We have added the transgene information into the text in hopes of clarifying this point: “Nevertheless, maternal germline knockdown of the effector caspases Dcp-1 and drice (maternal-αtubulin-Gal4 > UAS-Dcp-1[RNAi], and maternal-αtubulin-Gal4 > UAS-Drice[RNAi] respectively) produce offspring without an ethanol preference, regardless of parental treatment (Figure 2C).”

We have also added this information into the figure legend: “Genetic knockdown of the germline effector caspases, Drice or Dcp-1, was achieved by expressing a RNA hairpin in the female germline (driven by the maternal-αtubulinGal4).”

6) Figure 3.

A)Transgene controls (UAS-NPF and UAS-NPRRNAi) are missing.

We have added in these data as a supplemental figure (Figure 3—figure supplement 1). We did not include them in the primary graph because the experiments were conducted at different times and we felt that this might invite inappropriate statistical comparisons. Importantly, both transgene lines behaved similarly to wild type.

B) There is no label for what the bars are in Figure 3B/C.

We apologize for the oversight; we have added labeling for the violin plots in Figure 3.

C) Why are only some of the pairwise comparisons shown? Are all others N.S.? If so, this should be stated.

The results of all of our statistical tests are shown in the figure. Statistical tests that yielded a non-significant result are indicated with “N.S” in the figure itself. The chosen statistical comparisons were selected to test specific hypotheses: First, comparisons between treatment group of the same genotype were performed to assay for changes due to wasp exposure. Secondly, we explored the role of NPF signaling, manipulated through genetic means without wasp exposure. Therefore, the unexposed NPF-manipulation group was compared to the unexposed background line. We restricted our statistical tests so that they were hypothesis driven, and although significance for all pairwise comparisons could be calculated we feel that all pairwise comparisons would not be informative and may be statistically inappropriate. For instance, conducting all pairwise tests would necessitate the comparison between samples differing both in treatment and genotype, resulting in a meaningless significance value (in other words, comparisons that are not considering the carefully paired control group for each experimental treatment and/or genotype).

D) It’s not completely clear in the text if the legacy RNAi flies also have both the driver and RNAi? I would guess not (were they mated to WT males? other males?) but you should state this explicitly in the Results.

The reviewers are correct, the legacy RNAi flies are not carrying both transgenes, as this would have greatly complicated the interpretation of our results. We have stated this more clearly in the Results section: “For these experiments, it was critical to ensure that F₁ flies did not share the maternal genotype, and a crossing scheme was devised to avoid progeny with transgene expression (see Materials and methods section).”

E) Where is NPFR knocked down? Brain? Ovary? Is there NPF in ovary/reproductive tract?

The driver line used in the NPF-R experiments was a pan-neuronal driver (ElavGal4). We have added this to the figure legend: “Knockdown of the NPF-receptor in neurons (using the pan neuronal driver Elav-Gal4) leads to increased germline apoptosis”.

F) Where is NPF overexpressed? Brain? Ovary?

The genetic NPF experiments (overexpression and knockdown) used the NPF-Gal4 line. We have added this detail to the figure legend: “NPF overexpression (OE) or knockdown (KD) was achieved in the NPF-expression pattern (NPF-Gal4).”

7) In general, the figure legends are not well written. They lack a huge amount of detail that the reader needs to evaluate the data, e.g. N, the type of statistical tests used, labels, genotypes etc.

We have reworked the figure legends to include greater experimental and statistical detail. We have added information regarding statistical measures, genotype, and sample size (sample size for the ethanol preference was not added, as the n of 10 was standard across experiments).

8) Figure 4. What was the ROI for the quantification? Cell body? Neuropil? Maybe show on the image?

We thank the reviewers for this suggestion. We have generated a supplemental figure that addresses the regions of quantification (Figure 4—figure supplement 2).

9) Figure 5. Blind? How? It actually matters – many 'visual' mutations are pleiotropic. A genotype/condition must be given in either text or legend.

The flies used were ninaB¹ mutants. We have added clarification both in the figure itself as well as the figure legend.

10) Results, subsection “Maternal Chromosomal Inheritance of Ethanol Preference Behavior”: there actually are paternally-inherited regulator RNAs, at least in mice, so this is not completely clear cut.

We absolutely agree with the reviewers, and we did not mean to suggest that cytoplasmic factors (or small RNAs) can only be passed on maternally. Rather, we were trying to disentangle the ‘chromatin mark’ mechanism from the ‘cytoplasmic’ mechanism; which could be either maternal or paternal, however in this case we have shown that males do not participate in the initial transmission of the epigenetic program. We have removed the reference to small RNAs in an attempt to clarify our thinking in the text.

11) Figure 3E heading says overexpression and knockdown but the data shows only knock down.

We thank the reviewers for finding this error; the figure has been corrected.

12) Transcriptomic data: clarify whether changes are relative to unexposed flies of the same generations or relative to exposed flies of different generations. For example, are the F₁ and F₂ changes relative to F₀ exposed flies or F₁/F₂ unexposed flies? At what point do transcriptomes change?

Each generation had a paired exposed and unexposed group. The fold change data was normalized within the generation (F₁ exposed legacy normalized to F₁ unexposed legacy). We have added text to the Results section to make this clearer: “Heads from the exposed legacy F₁ and F₂ generation were collected and normalized to their respective unexposed legacy group.”

13) Figure 1—figure supplement 3A suggests female genotype is not important for transgenerational inheritance, while Figure 5 deals with the necessity of female genotype. This needs better clarification.

Figure 1—figure supplement 3A deals specifically with the F₁ males being able to pass on the ethanol preference to the F₂ generation. Alternatively, Figure 5 explores the initial transmission from the F₀ generation (exposed group) to the legacy flies. We believe these data point to differences between the initial transmission (following wasp exposure) and the transmission of the behavior between legacy flies (i.e. activation vs. maintenance). We have attempted to clarify this in several ways. First, we have more clearly labeled the graphs in Figure 1—figure supplement 3A. We have also added text to the Results section to better highlight this difference: “Interestingly, F₁ offspring from an exclusively maternal wasp exposure inherit ethanol preference, while F₁ offspring from an exclusively paternal exposure did not (Figure 5A). Importantly, this demonstrates a key difference between the F₀ males and the F₁ males, and may point to an ‘activation phase’ and ‘maintenance phase’ of the epigenetic program.”