Vilya, a component of the recombination nodule, is required for meiotic double-strand break formation in Drosophila

Questions related to the role of Vilya for DSB formation:

1) It is not entirely clear if one can conclude that no DSB are formed in the absence of Vilya. In the subsection “vilya is required for programmed DSB formation in early pachytene”, the authors state: "Immunofluorescence analysis of early pachytene oocytes reveals a complete failure to initiate programmed DSBs". It would be better to use more nuanced language: This experiment suggests a failure or a strong reduction, and the resolution of the picture does not allow for the conclusion "complete" failure. What is the estimated limit of sensitivity of the gH2AV staining? The data of the recombination map is clear but if Vilya is required for CO, then some remaining DSB could be repaired to NCO in the mutant.

We now use more nuanced language throughout the manuscript, suggesting that there is a strong reduction rather than asserting that there is a complete elimination of DSB formation during pachytene. In support of our assertion that there is a very strong reduction in crossing over, we have also added a recombination analysis for the entire 3rd chromosome to complement the X chromosome recombination data already included (see Figure 2—figure supplement 2). These new data show that the frequency of recombination on the 3rd chromosome in vilya⁸²⁶ is reduced over 50-fold compared to wild type. In addition, we have now included DSB numbers for the transheterozygote (Df/vilya⁸²⁶). Together with the data already included in the manuscript, these experiments show that there is a very strong reduction in both the initiation of DSBs and in crossing over.

2) Is Vilya required for DSB formation in nurse cells? This is quite important and would allow to distinguish between a direct role for DSB formation and a regulatory role as observed for C(3)G.

We have included data regarding the lack of induction of DSBs in nurse cells in the vilya mutant in a new figure (Figure 1—figure supplement 4). In this figure we show the pattern of DSB induction within all nuclei in region 2A for both wild type and c (3)G (which shows a pattern similar to wild type). However, we show that mei-W68, mei-P22, vilya⁸²⁶ and Df/vilya⁸²⁶ all exhibit a strong reduction in the initiation of DSBs in both nurse cells and oocyte nuclei. These images further demonstrate that Vilya has a general role in DSB formation, similar to Mei-W68 and Mei-P22, rather than an oocyte-specific regulatory role in DSB formation, as has been reported for C(3)G (Mehrotra and McKim, 2006).

3) Also, an alternative interpretation, may be not likely, but that should be mentioned is that the mutant phenotypes could be explained by assuming a fast repair of DSB to NCO (or sister) in Vilya mutant and not requiring Rad54.

We have added this as a possibility in the subsection “Meiosis in Drosophila”.

4) The interplay between Vilya and C(3)G should be better documented: Is Vilya localization dependent on C(3)G? If yes, how could one explain the DSB activity detected in C(3)G mutant?

We have now included data to show that the localization of Vilya to discrete foci is not dependent on C(3)G. However no linear element Vilya^3XHA staining was observed in the c (3)G mutant. Our data show that 75% of the Vilya^3XHA foci observed in region 2A oocytes colocalize/associate with the γH2AV marks (see new Figure 7). However, very few region 2B oocyte nuclei retained any Vilya^3XHA foci. We speculate that the inability to retain Vilya^3XHA at discrete foci is the result of the failure to convert the DSBs that do form into crossovers – a process known to require the SC. We have added these observations to the subsection “The formation of discrete Vilya^3XHA foci is dependent on programmed DSB formation but not the SC” in the Results section.

What is the DSB level in Vilya C(3)G double mutant?

Previous correspondence with the editor led to an agreement that the analysis of the vilya; c (3)G double mutant, while interesting, was beyond the scope of this study.

5) An important test for the deficiency of DSB formation is the suppression of okra mutant phenotypes. One missing piece of information is the detection of gH2AV in vilya okra double mutant (region 2A and region 3).

Again, previous discussions with the editor regarding measuring the DSB level in the vilya; okra double mutant led to the agreement that such experiments could be deferred to a future paper.

6) The interpretation of the ring mutations on the interaction with MeiP-22 is not convincing as the mutant protein may have an improper folding or conformation, this alternative should be taken into account in the Discussion. Western blots should be provided as well as two hybrid data for the RING domain mutants.

We agree that the failed interaction between Mei-P22 and the Vilya RING domain point mutants may be the result of improper protein folding or confirmation. We have now added this possibility to the Results section. We have also provided the yeast two-hybrid data and the Western blot as a new figure (Figure 9—figure supplement 1). In addition, we have added a new Materials and methods section regarding this experiment in the yeast two-hybrid section, as well as a figure legend for the new figure.

7) The colocalization of Vilya with gH2Av is not convincing. If Vilya foci mark CO, one does not expect a high colocalization frequency between gH2AV and Vilya (may be only 30% or less). In addition the protocol used for image analysis and evaluating colocalization is poorly described. What does closely associated mean? What does adjacent mean? How is this different from expected by chance? Randomization controls should be performed to evaluate the degree of overlap, e.g., by flipping one of the fluorescence channels by 180° and re-evaluating the degree of fortuitous overlap. In the third paragraph of the subsection “The formation of discrete Vilya^3XHA foci is dependent on programmed DSB formation”: The difference in Vilya-gH2AV association does not appear to be significant (Fisher's exact test gives p = 0.175). Please clarify.

We have added more details regarding the process of image analysis to the Materials and methods. Our definition of adjacent simply means no apparent gap between the foci. In addition, as suggested, we performed randomization controls (see subsection “The formation of discrete Vilya^3XHA foci is dependent on programmed DSB formation but not the SC” of Results and subsection “Microscopy and image analysis “of Materials and methods) and have now included them in the Results and Methods sections. Since we wanted to be able to perform the randomization controls on all oocytes scored for colocalization, we used only images that contained one oocyte – many region 2A oocytes cannot be individually and completely cropped due to their proximity to another SC-containing nucleus, even though you can clearly distinguish between the two nuclei in 3D. This is why the numbers in the Results section regarding this data have changed in the revised version. The overall association of γH2AV and Vilya^3XHA foci changed only slightly from 58% to 60.5% in scoring these 11 nuclei.

We have also attempted to better explain the uncertainties that surround the quantitative relationship between the numbers of Vilya and γH2AV foci in terms of laying the groundwork for the model presented in the Discussion of the paper. As Vilya is required for DSB formation (a dynamic process) and is also likely marking CO sites which no longer bear the γH2AV mark, it is more difficult to predict the frequency of association that would be predicted in region 2A when DSBs are being induced. We do know that the number of Vilya^3XHA foci in region 2B is consistent with that of RNs/COs.

8) In terms of Vilya foci quantification, it is not clear what the parameters to identify a focus are, in particular taking into account that their intensity varies and that there is signal in the central region. For instance in Figure 6A, there seems to be more than 12 Vilya foci in the nucleus.

We understand how the reviewer came to the conclusion that there were more than 12 foci in Figure 6A. In this particular image, a few Vilya foci are not in the main nucleus shown. This is because it is sometimes difficult to crop out a region 2A nucleus in such a manner as to be free of sections from neighboring nuclei. In region 2A, up to four neighboring nuclei contain full-length SC and enter meiosis. Although you can clearly see the boundary in 3D, upon projection of the entire stack, these nuclei looked connected, when in fact they are not. In retrospect we should have chosen a better image that was able to be cropped and displayed as information from a single nucleus. We have now changed that image and also added in the Materials and methods section more information on foci quantification (for both Vilya and DSBs).

Questions related to the role of Vilya for Crossover control:

9) To firm up the speculation about a possible late role for Vilya and to reconcile the cytology and the phenotypic analysis, do X-rays rescue Vilya foci in a mei-P22 or mei-W68 mutant? This experiment would help clarify whether Vilya has functions downstream of DSB formation, as speculated.

We were able to include the experiment suggested by the reviews in analyzing whether DSBs created by X-ray can recruit Vilya to them as discrete foci. The short answer is that “yes” X-ray-induced breaks can induce Vilya to form foci. We have included that new data as a new figure (Figure 8).

10) In the first paragraph of the subsection “Vilya's role in DSB formation and crossing over” and Figure 8: The authors' model is not convincing as spelled out. If Mei-W68 catalyzes DSBs at Mei-P22 sites, and if Vilya is then recruited to a subset of DSB sites, one should expect to see more Mei-P22 foci than Vilya foci. This data is missing in the manuscript and is important to support the model in Figure 8. Furthermore, in Liu et al., 2002, the average number of Mei-P22 foci per SC containing cell in region 2A was calculated at 8.7, which is not significantly higher than the average number of Vilya3XHA foci in early pachytene region 2A calculated at 8.0 in the present study (Figure 3B). Moreover, the poor colocalization (13,5%) observed between γH2AV and MEI-P22 foci in Mehrotra & Mckim, 2006 doesn't fit with MEI-P22 being a mark of future DSB sites. The manuscript would be significantly strengthened if these issues could be addressed. Why not integrate trem in the model?

As we pointed out in our response to reviewer concern number 6, we were not able to obtain additional information regarding the localization of Mei-P22 during the revision process. Therefore, we have chosen to produce a minimalistic model with the information we currently have. As we only ever see a fraction of the DSBs made in region 2A using the antibody to γH2AV (the only marker we have in flies), we cannot predict what the expected number of Mei-P22^3XHA or Vilya^3XHA foci should be in this region (see above) compared to the number of γH2AV foci. We can only report on the number of foci per stage of meiosis and their overall trend throughout the germarium. We have also removed the suggestion that Mei-P22 is marking future DSB sites.

Although we would like to eventually incorporate Trem into the model, the null allele of trem doesn't allow us to look at localization of Vilya in this mutant. This is because the P-element insertion in trem is a pBac[WH] element that contains a UAS in the 5' UTR of the trem gene. Therefore, expression of Vilya using the GAL4-UAS system in this trem null allele also expresses trem. In the future we will need to make either a tagged germline vilya expression construct for this analysis or use CRISPR to tag the enodogenous vilya gene, as we have been completely unsuccessful at making a Vilya antibody after numerous attempts (three additional attempts even during this revision process). Therefore, until we are able to fully analyze their relationship or get preliminary data, we have chosen to leave Trem out of the model for now.

11) Figure 3: As Vilya3XHA shows two distinct types of staining (linear and foci), the specificity of the anti-HA antibody should be shown in a control experiment (e.g. anti-HA on a WT oocyte).

We have added a control image of the rat anti-HA antibody as part of Figure 3—figure supplement 1. In Figure 3—figure supplement 1B we show a region 2A nucleus that has both types of staining (where the foci predominate) and a later stage (stage 4) showing the specificity to the linear staining. (Stage 4 is a stage between the region 3 and the stage 6 already shown in Figure 3—figure supplement 1A.) In both cases the controls show insignificant staining levels. The remaining panels in this figure have been moved from B and C to C and D. We have also added details regarding how the control images were collected in the Materials and methods and have pointed to this location in the figure legend.

12) Information about the level of expression of the transgene (relative to endogene) expressing tagged Vilya is needed. Possible artefacts due to overexpression should be discussed for interpreting the localization of Vilya.

We mention in the manuscript that we are using an overexpression system that is highly expressed throughout the ovariole. We also have been very aware to use throughout the manuscript the 3XHA reference when discussing the localization of Vilya, as to remind the reader this is a tagged overexpressed construct. We have included in the manuscript caveats to using an overexpression construct for certain experiments as well.

We have not included levels of expression of the transgene for the following reasons: 1) We know and mention that in using the nos-GAL4-UAS system, we are highly expressing Vilya at all stages of oogenesis. 2) The nurse cells undergo many rounds of endoreduplication leading to hundreds of copies of the genome per each of the 15 nurse cells. These nurse cells synthesize both protein and RNAs, which are dumped into the oocyte. We would be unable to determine whether a certain level of increase in mRNA resulted in that level of protein expression, as well due to the inability to isolate germarium, we are unable to look at expression levels specifically in the area of the ovary we are studying in this manuscript.