Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract

Thank you for choosing to send your work entitled “Ovulation in Drosophila requires reproductive tract secretions controlled by the nuclear hormone receptor Hr39” for consideration at eLife. Your article has now been peer reviewed and we regret to inform you that your work will not be considered further for publication at this time. Your submission has been evaluated by 3 reviewers, one of whom is a member of our Board of Reviewing Editors, and the decision has been discussed further with one of eLife's Senior editors. The Reviewing editor and the outside reviewers discussed their comments before we reached this decision.

Each reviewer has acknowledged the importance of the work, but all three are unanimous in suggesting that the work presented is not complete and ready for publication. Reviewer 1 points to the lack of a detailed developmental analysis or identification of gene function, aspects that echo the comments of Reviewer 3. Similarly, Reviewer 2 feels that the nature of the secreted product, indeed the nature of secretion itself, is not fully examined, a view shared by Reviewer 1. Finally, Reviewer 3, an expert in the specific field, raises many technical questions and also would like to be further convinced that some of the points raised in different parts of the manuscript have indeed been settled by the results that are presented. The detailed comments of each reviewer are included below.

Reviewer #1:

The paper “Ovulation in Drosophila requires reproductive track secretions controlled by nuclear hormone receptor Hr39” explores an interesting link between secretions from female reproductive tract glands (Spermathecae and Parovaria) with ovulation in Drosophila. The authors show that a developmental defect in the specification of secretory organs in the female reproductive tract (through mutations in genes such as lozenge and Hr39) causes significant ovulation defects. These defects are neither due to mating deficiencies, nor caused by defects in neurons that innervate the oviduct muscles.

Based on the stereotypical division patterns seen in cells of the secretory unit precursor (SUP) cells and the expression of Notch signaling reporters at high levels, the authors test and establish that Notch signaling is required for the specification of secretory cells. Similarly, the authors establish a role for the transcription factor Hindsight, which is also expressed in these cells. Finally, using a Janelia Gal4 driver line expressed specifically in developing but not in mature secretory cells, the authors establish that secretory cells are needed in sufficient numbers to allow normal ovulation and sperm storage. Previous studies have shown that secretory cells in the female reproductive glands release their secretions into a structure called the end apparatus, which is in close apposition to the microvilli of the secretory cells. The authors test the role of secretions from these cells using a specific Gal4 and block exocytosis from these cells by over-expressing a dominant negative version of Dynamin (shi[ts]). This causes a defect in sperm storage but not in ovulation. The authors conclude that either the secretory factors from SC that regulate ovulation are independent of exocytosis or they are needed at very low levels, not disrupted in the chosen experimental design.

This is largely an observational paper, with few broader insights to report. That the secretory cells control ovulation, and that this process is Notch dependent is an interesting finding, described and proven with well-planned experiments and originality in the paper. But the manuscript does not explore much beyond this basic premise.

The data presented are of high standard but the paper lacks a cohesive narrative that moves the field forward. For example, the paper does not explore in detail the developmental aspects of the SC cells, and uses an assortment of mutations (in Hr39, lz, Hindsight, Notch) only as ways to eliminate the function of these cells rather than explore their interdependencies, or developmental relevance. Similarly, no attempt is made on the functional side to determine the nature of the secretions that have such profound effects on ovulation. In the end, the negative result with shibire-ts leaves the question wide open for further exploration. This work represents an exploratory project that has a lot of future potential but is not yet ready to move the field forward.

Reviewer #2:

The findings of Spradling and colleagues are potentially interesting. The paper clearly shows the requirement of secretory cells in the events leading to attraction and storage of the sperm. However, the data on the nature of the secretory components is not satisfactory. The authors report the involvement of Shibre (Dynamin); however, shibre is known to affect endocytosis in the neurons: the significance of this mutation on exocytosis is, therefore, unclear. It is important to know if the defect is due to secretion of extracellular matrix components or some growth factors or cytokines. I do not expect the authors to reveal the identity of the essential secreted component(s); they must, however, provide the clear involvement of the secretory process. Does it involve the regulated or the constitutive secretory pathway? The authors should test the effect of mutations in the genes that block constitutive (ER-Golgi-plasma membrane) and the regulated (Golgi-secretory storage granules-PM) in sperm attraction and attachment to support their proposal.

Reviewer #3:

There are a number of interesting conclusions in this paper; however, the title suggests a link between Hr39 and secretory cell function in the spermathecae, but the only clear role suggested by the experiments for Hr39 is a role in the formation of the spermathecae and the parovaria, not in the functioning of fully formed secretory cells in these tissues. In fact, using Hr39 RNAi with the dpr5-Gal4 driver showed what appears to be a small drop in ovulation after 6 hrs (from 64.5 to 45.8 although the RNAi control is missing) – however, no drop in egg laying after 48 hours was seen. In addition, no significant drop in sperm storage was seen. Based on these findings, it is difficult to see why the authors used Hr39 in the title of this paper. In addition it seems that the interesting conclusions are not highlighted in the Abstract or Introduction. There needs to be a clear differentiation in the writing of the paper between conclusions based on the absence of the entire tissue, and those effecting cell number on adult function. Overall, the paper should be rewritten to clearly highlight the more interesting conclusions regarding the role of the secretory cells of the spermathecae (i.e. ovulation defects and sperm storage defects), and the conclusions based on hr39 and lz should be more clearly stated for what they are, a study of the loss of the tissues they help form.

Specific comments:

* The authors should state in the introduction the previously established link between the secretory cells of the spermathecae and sperm storage/ovulation – although Schnakenberg et al., (2011) is mentioned in the Discussion, it should also be mentioned up front in the Introduction.

* The authors need to clearly state that lzGal4 expression has previously been shown to recapitulate actual lz expression. When does the lzGal4 line come on in development?

* The authors state: “lz is expressed in the subset of these neurons near the oviduct-uterus junction, but the number of fru⁺ppk⁺ neurons was not affected by knocking down cycA or hr39 using the lzGal4 driver.” How do the authors know that knocking down cycA or hr39 should change neuronal numbers? Do they know if lz is required for their proliferation? What if it has another function in these neurons?

* Why is hr39 knockdown using RNAi tested in lz cells, but not lz-RNAi itself? Has hr39 previously been shown to be expressed in these lz neurons? ppk-Gal4 with hr39-RNAi does not effect egg laying – why don't the authors test lz?

* The result in Figure 2E is not that mSP expression in fru⁺ neurons induces egg laying, an already published finding (Yang et al., 2009), but that mSP expression in fru⁺ neurons can not bypass the egg-laying defect in hr39^-/- mutants. Therefore, the SP behavioral switch can happen in the absence of Hr39 (and therefore no spermathecae or parovaria are needed); however, the loss of egg-laying in hr39^-/- cannot be overcome by the SP behavioral switch.

* It appears from the methods that the shi[ts] flies are shifted just prior to mixing males and females. Is one possible explanation for the shi[ts] phenotype that all the important secretions for ovulation/egg-laying are already in place prior to mating?

* There appears to be no mention of statistical analysis: the types of analysis should be clearly stated along with every figure/table where they have been used and statistically significant differences/similarities should always be highlighted.

* fru⁺/ppk⁺ neurons have recently be shown to also be dsx-positive (Rezával et al., 2012).

* There are many different Gal4 lines used in this study: the authors should make it clear throughout the paper exactly where the lines have been shown to be expressed (either previously published or by the authors themselves). For example, the syt12-Gal4 line is introduced near the end of the paper but its overall expression pattern and reasons for use are far from clear.

* The first paragraph of the Discussion is confusing (for example, using double-negatives): this section should be re-worked to clearly state what was previously shown, and then clearly state what the current study adds to the story. The authors have shown a clear ovulation defect that was previously not shown; this is an interesting result that currently gets lost in the detailed explanation of the previous study.

Thank you for choosing to send your work entitled “Ovulation in Drosophila requires reproductive tract secretions controlled by the nuclear hormone receptor Hr39” for consideration at eLife, and for telephonic discussions with Detlef Weigel and K. VijayRaghavan on December 21, 2012, which were useful. This has been followed up with discussions amongst the editors and we suggest the following points for a revision of the manuscript.

1)We strongly encourage you to change the title because it has been a source of confusion about what the paper is about. The current title can easily be read as implying that the paper will reveal the identity of the Hr39-dependent secretions. The title should instead reflect that this paper shows for the first time an unexpected way by which ovulation is controlled by the female reproductive tract.

2)The clear responses given to Referee 1 could be included in the text so that the paper better documents the assertion that this is indeed a foundational paper.

3)Next, experiments documenting the adult role of Hr39 should be included. Experiments that report the effects of disrupting at least one key gene in the ER/Golgi/PM secretory pathway(s) on ovulation would also greatly strengthen the manuscript. Your appeal letter indicates that both types of experiments are readily feasible.

4)Finally, a measured and brief analysis of your views on the Schnakenburg et al. paper, in the context of the current work, will be useful for readers to compare the two studies and to reach their own conclusions about the similarities and differences between them.