Germline protein, Cup, non-cell autonomously limits migratory cell fate in Drosophila oogenesis

  1. Banhisikha Saha
  2. Sayan Acharjee
  3. Gaurab Ghosh
  4. Purbasa Dasgupta
  5. Mohit Prasad

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Abstract

Specification of migratory cell fate from a stationary population is complex and indispensable both for metazoan development as well for the progression of the pathological condition like tumor metastasis. Though this cell fate transformation is widely prevalent, the molecular understanding of this phenomenon remains largely elusive. We have employed the model of border cells (BC) in Drosophila oogenesis and identified germline activity of an RNA binding protein, Cup that limits acquisition of migratory cell fate from the neighbouring follicle epithelial cells. As activation of JAK-STAT in the follicle cells is critical for BC specification, our data suggest that Cup, non-cell autonomously restricts the domain of JAK-STAT by activating Notch in the follicle cells. Employing genetics and Delta endocytosis assay, we demonstrate that Cup regulates Delta recycling in the nurse cells through Rab11GTPase thus facilitating Notch activation in the adjacent follicle cells. Since Notch and JAK-STAT are antagonistic, we propose that germline Cup functions through Notch and JAK-STAT to modulate BC fate specification from their static epithelial progenitors.

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  1. Version published to 10.1371/journal.pgen.1010631
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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary:

    • Saha et al. characterize Drosophila egg chambers that are mutant for cup and identify an increase in the number of a specialized type of follicle cells, the border cells. They demonstrate that this increase correlates with an expanded domain of STAT activity and reduced Notch signaling in anterior follicle cells. Determining that cup is required in the germline cells, the authors postulate and provide some evidence that cup mutants prevent germline Delta from properly signaling to follicle cells. In line with this, they also show that blocking endocytosis phenocopies some aspects of cup mutants, particularly border cell numbers and Delta levels, which they monitor cytoplasmically and at the cell surface. Lastly, they demonstrate that activation of Rab11 can rescue Delta levels and border cell number in cup mutants. They conclude that a key function of Cup in the germline is to traffic Delta to signal to follicle cells, and that the endocytic processing of Delta is required for its function.*

    Major comments:

    • The findings of this study are interesting and novel. The authors have completed a lot of experiments and analyzed the results carefully and in great detail. Experimental design is described adequately and statistical analysis is sufficient. While the main results are largely convincing and support the conclusions, there are some weaknesses that need to be addressed.*

    Response: We thank the reviewer for appreciating our work and we have tried to address concerns of the reviewers to the maximal possible extent with the hope to strengthen our claims further.

    One major concern is that the vast majority of the experiments were conducted with a single homozygous allele for cup. The authors claim this was necessary because other alleles arrest oogenesis, which is understandable, but it leaves the potential problem that the allele, a P-element insertion, may affect other genes, or there may be other unidentified mutations on the mutant chromosome. The authors are able to partially rescue the border cell phenotype with overexpression of Cup and can also mimic the outcome with RNAi in the germline, which helps alleviate some of this concern, but this was only done for one set of experiments (those in figure 1). Similar experiments need to be included to demonstrate the same outcomes when cut is disrupted by other alleles/methods for at least some of the Notch/Delta analyses since this is key to the paper's conclusions.

    Response____: We acknowledge the concern raised by reviewer and to address it, we evaluated different allelic combination of Cup to rule out issues with background mutation. We evaluated the Delta count, NICD and border cell numbers in a different allelic background of cup8/ cup01355. Satisfyingly we observed similar results like that observed for cup01355/ cup01355 homozygotes. This result is included as (Fig S1E-G)

    In addition, we have specifically downregulated Cup function in the germline employing the RNAi approach and validated the non-cell autonomous effect of Cup function in border cell fate specification. This result is included in (Fig 1M-O)

    A second concern is that some evidence is circumstantial or indirect. Specifically, the authors argue that the effect of Cut is due to trafficking of Delta, but do not consider the possibility that Delta could be more directly regulated or that other factors may be relevant. Border cell specification is rescued by increasing recycling in cup mutants, but this could be due to recycling of more factors besides Delta. To address this more directly, the authors should overexpress Delta in the germline of cut mutants. It is possible the disruption of Delta in cut mutants is due to changes in Delta protein stability/levels, so the experiment may also clarify this issue. If this is the case, it may be that hypomorphic Delta mutants would have a defect on border cell number, which could be examined separately. If Delta levels are low, endocytosis and recycling increases may also rescue cut mutants indirectly, but the conclusion about what Cut regulates may differ.

    Response: As per the suggestion of the reviewer, we did attempt to over express Delta in the germline of cup mutants egg chambers. Unfortunately, we couldn’t record any Delta overexpression as the available vector (UASt- Delta) can drive stable expression only in the somatic cells but not in the germline cells. However, to check out the possibility if Delta was being directly regulated by Cup, we compared the levels of proteins between wild type and Cup mutant egg chambers (Figure 4E-G). Unlike our expectation we didn’t observe any significant differences in the levels of Delta in Cup compared to the control. This kind of supports our belief that Cup may not be directly regulating the levels of Delta in the germline.

    Another concern is that Cup's main role is a confusing since it regulates many things, including cytoskeleton and cytoskeleton is necessary for general health and vesicle trafficking in the egg chamber - how do the authors think Rab11 upregulation is overcoming these defects?

    Response: We appreciate the reviewer for raising this concern as it kind of intrigued us to examine if the overexpression Rab11CA was rescuing the cytoskeleton too. Interestingly, we observed that Rab11CA overexpression restored the actin filament in Cup mutant germline(figure S6H-K). This result is in line with report that Rab11 effector Nuf can modulate actin polymerization (Jian Cao et al.,2008).

    Rab11CA rescues Delta levels almost completely in cut mutants but only partially rescues Notch activation, suggesting there are other problems in these egg chambers that could contribute to the defects. While exploring possible other factors is beyond the scope of this work, the authors may want to acknowledge this issue.

    Response: We do agree with the reviewer that we only observe partial rescue of the NRE GFP with Rab11CA, it suggests that Cup can affect different aspect of egg chamber development independent of Rab11 function.

    Minor comments:

    It would help the presentation of the paper to introduce Notch/Delta signaling during oogenesis in the introduction. More introduction and clarity about the number of polar cells at early stages and their role in the border cell cluster may also be useful to the reader.

    Response: We have modified the introduction to highlight the role of Notch/ Delta signaling in early oogenesis.

    It is notable that the primary phenotype of a change in border cell numbers is quite subtle, often only affecting 1-2 cells, and the variation in different genotypes and experiments is sometimes also that large. The authors do a good job of being careful to count the cells at a specific developmental time and do appropriate statistical tests within an experiments. Still, it difficult to be sure that the effects are due to the gene being manipulated specifically or the genetic background. Related to this, a few issues should be addressed. Notably, at earlier stages, Notch signaling impacts cell division, so some of the phenotypes might be explained by there being more total cells in the domain instead of more signaling. The authors show Cut is in the same domain and pH3 is similar, but they didn’t seem assess overall numbers.

    Response: As per the suggestion of the reviewer, we assessed the total number of follicle cell nuclei in stage 8 egg chambers. This analysis was done each confocal z slide of the egg chamber taking care that each nuclei (DAPI) was counted only once. Satisfyingly we didn’t observe any significant difference in the number of follicle cell nuclei between wild type and cup mutant egg chambers supporting our earlier claims with pH3 and Cut antibody that cell proliferation is not responsible for the excessive border cell fate in Cup mutants. This result in included in (Fig S2O-Q)

    Secondly, for the stat suppression of cut (figure 2L), the authors need to show the stat-/+ control for comparison to make a conclusion about suppression versus additive effects.

    Response: As per the suggestion of the reviewer, we have included the data for statp1681/+ control in figure 2L.

    In addition, prior work (Wang et al 2007) expressed DN Kuz in border cells and did not see a change in specification, unlike what is claimed here. In the experiment in question, the control has lower than normal numbers of border cells and the DN Kuz has a number more typical of the controls in other experiments- so this is a concern that there is something else in the genetic background influencing the numbers. Other controls could help make this case, but ultimately this result is probably not necessary for the main argument. Thus the authors might consider leaving it out the Kuz analysis or perhaps can comment on the discrepancy with prior published results.

    Response: We have removed the data on Kuzbanian and have added data that suggests that Notch activation in the follicle cells downstream of Cup facilitates specification of appropriate number of migratory border cells (Fig 3K-N).

    Can the authors comment on why the volume of the border cell cluster increases more dramatically (>2x) than the number of cells (30% more)?

    • Does the increase in border cell number change the migratory capacity? That is, do the clusters in cut mutant egg chambers migrate normally while the egg chamber looks okay?*

    Response: We believe that dramatic increase in the volume of the border cell cluster I (>2x) than the number of cells (30% more) is due the loose arrangement of the cells in the border cell cluster. Interestingly, the cup mutant border cell clusters do exhibit migration defect that we are examine as part of separate study.

    Several of the figure legend titles state conclusions that are over interpretations of the data shown:

    - Figure 3 legend is overstated- these experiments do not assay STAT activity, only border cell number, so the title can be simplified to say that.

    Response: We have modified the Figure legend in line with the data presented.

    - For figure 4, both cytoskeleton and Delta are shown to be disrupted in cup mutants, but they are not directly linked, eg, the experiments do not show a change in Delta in cytoskeletal mutants alone. While it is interesting that cup mutants have disrupted cytoskeleton, ultimately this result is not well connected to the main issue of Notch/Delta signaling; in fact, it becomes confusing how anything can be trafficked to the cell surface if there is poor cytoskeletal organization. Since the authors favor the hypothesis that the cytoskeleton is not the key to the border cell specification difference, they may want to move this result out of figure 4.

    __Response: __We have included the data that suggests that cytoskeleton organization is critical for Delta trafficking. Specifically we demonstrate that treatment of egg chambers with Cytochalasin D exhibits accumulation of Delta in the nurse cell cytoplasm (Fig S5D-F).

    - The Figure 5 legend is also overstated- these experiments show that Delta is higher in cup mutants and endocytosis mutants AND that endocytosis (of something) is required in the germline for border cell number- but these results are not linked in this figure. More evidence for this connection does come later in figure 6.

    • Some figure legends are quite brief and could benefit from a little more detail on what is being shown*.

    __Response: __We have modified the title of the Figure legends with respect to data presented.

    Figure layout could be improved by keeping images consistent sizes and making sure graph text is large enough to read easily. Figures in general could be streamlined by having negative results and less pertinent results in supplemental data.

    __ Response:__ We have reorganized the figures and worked on the graph text for easy read.

    Not all papers cited in the text are in the reference list.

    Responses: We have modified the title of the figure legends and cross checked our reference list with the papers mentioned in the main text.

    CROSS-CONSULTATION COMMENTS

    I generally agree with the other reviewers that there are concerns with the precise function of cup in this context, and that some revision is needed, including editing of the writing. In response to reviewer 2, prior published studies only detected Cup in germline, but it is possible that it is expressed in follicle cells at a low level. The mutant clonal experiment in follicle cells that the authors did had no effect on border cells, so that provides some evidence the role is non-autonomous. I agree with reviewer 2's concern that the authors overstate the connection between cup and Delta and border cells based on their data and need a few more experiments to tie things together. I understand reviewer 3's concerns that the experimental effects on border cell numbers are very small and variable- I listed this as a minor concern, though, since this number is mainly being used as a read-out for STAT signaling levels and the data were extensively quantified and statistically tested.

    Reviewer #1 (Significance (Required)):

    My expertise is in cell migration, developmental biology, and Drosophila genetics. This paper will be of broad interest in these fields as it incorporates aspects of each in its characterization of a new regulatory mechanism to induce a motile cell population non-cell-autonomously, which is an exciting finding. Specifically, the work increases our understanding of the intersection between Notch and Jak/STAT signaling, which many researchers study - these were both known to be involved in border cell specification. The study provides more detailed characterization of the signaling and specification process in general, and makes significant advances in understanding how Delta signals are produced and presented from germline cells to receiving cells in the soma. Cut has not been previously implicated in these signaling pathways, so that is also novel, although its precise mechanistic role here is still somewhat unclear.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, Saha et al. made a detailed description of the role of the mRNA binding protein Cup in specifying the number of Border Cells (BC) during Drosophila melanogaster oogenesis. First of all, they show that females homozygote for a hypomorph allele of cup have higher number of BCs compared to Wild Type (WT) females. They present a series of experiments that points towards the phenotype being due to a specific role of cup in the nurse cells that non-cell autonomously regulates BC specification. Also, they show that this phenotype is the result of an increase in the levels of JAK/STAT signalling in the BC, a major determinant of BC

    fate. In addition, they show that cup mutant egg chambers exhibit a downregulation of

    the Notch (N) pathway function in the BCs and that over-activating Notch results in the rescue of the number of BCs. Moreover, the authors present data on the effect of cup in Delta (Dl) trafficking in the nurse cells: They found that cup mutant egg chambers show increased number of Dl puncta within the cytoplasm of the nurse cells, but reduced numbers in the nurse cell-Anterior Follicle Cell (AFC) boundary as a result of defective Dl endocytosis. Finally, they were able to rescue the Dl trafficking phenotype, as well as the number of BC by overexpressing an active form of Rab11.

    Mayor points:

    In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:

    o The authors show that cup01355 mutant egg chambers have a phenotype in Dl trafficking. Although they analysed in detail the effects on Dl in the nurse cells, their images show that there might be a defect in Dl levels/trafficking in the Follicle Cells (FCs) as well (Fig5A-B). It has been shown that Dl mut FCs have reduced levels of Notch activity due to reduced lateral inhibition (Poulton et al., 2011), so there is a possibility that the reduced levels of Notch activity in the cup01355 egg chambers might be due, partially, to defects in Dl trafficking/levels in the FCs, rather than in the nurse cells. o The authors tested the role of the Notch pathway in the cup mutant phenotypes by measuring the number of NICD puncta in the signal receiving cells as proxy for Notch activity (Fig4). Although I understand the rationale, I am not convinced that they can completely rule out that the changes in NICD puncta number in FCs is not due to some effect of cup LOF on Notch trafficking in these cells.

    o In figure 6, the authors show that expression of a constitutively active form of Rab11 specifically in the nurse cells restores the BC number to that of the WT. However, the levels of Dl particles and, especially the levels of NRE-GFP expression, remains slightly lower than in the WT conditions.

    Response: We do agree with the reviewer that we only observe partial rescue of the NRE GFP with Rab11CA, it suggests that Cup can affect different aspect of egg chamber development independent of Rab11 function. This has been acknowledged in the main text and it now reads as “We did note that irrespective of partial rescue in the levels of NRE-GFP and Delta puncta count, a complete reversion to wild type border cell numbers was observed when Rab11CA was overexpressed in the cup mutant germline. This may suggest either that border cell fate specification is quite robust beyond a certain base level of signaling or Cup may affect other aspects of egg chamber development independent of Rab11 function.”

    One of the main conclusions of this study is that cup regulates BC specification through a non-cell autonomous mechanism that involves communication between nurse cells and AFCs. For that reason, I think in order to conclusively say that, the authors need to try to remove the function of cup specifically in the nurse cells. They mentioned they have tried different ways of doing this unsuccessfully, but do not specify how they have tried. I suggest using the cup-RNAi line combined with a nurse cell specific Gal4 and a ubiquitous gal80ts line (tub-Gal80ts), if they have not try this. I do not expect the authors to repeat all the experiments with this condition, but at least they should test the main findings i.e. number of BCs, JAK/STAT overactivation and Notch attenuation.

    Response: To further support the non-autonomous role of Cup in border cell fate specification, we down regulated Cup function in germline nurse cells employing Mat-alpha GAL4 and Cup RNAi. Since Mat-alpha GAL4 driver has weak expression in the nurse cells of early stage chambers, it enabled us to evaluate Cup function during mid oogenesis. Consistent with our expectation, we observed higher number number of border cells in the migratory cluster compared to the control supporting our conclusion that germline Cup modulates the number of adjacent anterior follicle cells that acquire migratory border cell fate. The above results are included in (Fig 1M-O). In addition over expression if Cup cDNA in the anterior follicle cells failed to the rescue the excessive border cells observed in the Cup mutant egg chambers supporting the germline role of Cup further. This result in included in (Fig S1L-O).

    • The authors have shown in Figure 3 that there is a decrease in Notch signalling in the AFCs in cup01355 egg chambers. In order to test that the BC number phenotype observe in this condition is due to that effect on Notch signalling they have done a rescue experiment using the antimorphic Notch allele Nax-16. Since in this condition all cells (nurse cells and FCs) have increased levels of Notch, they cannot conclusively say that the increase in Notch function in the FCs rescues the cup

    phenotype. If they want to show that the function of Notch is specifically needed in the FCs, they should over-activate Notch exclusively in the AFCs. For instance, they could express a constitutively active form of Notch, such as UAS-NICD (Go et al., 1998) or UAS-NDECD (Fortini et al., 1993), specifically in the AFCs. Otherwise, they should re-write the text since they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype.

    Response: Following the suggestion of the reviewer, we attempted over expression of NICD in the follicle using driver slbo-GAL4 in the cup mutant background. Gratifyingly, we observed rescue in the border cell fate of Cup mutant egg chambers. However, we didn’t observe any rescue in the morphology of nurse cell nuclei of Cup mutants. This supports our conclusion that increase in Notch function in the FCs rescues the cup phenotype with respect to the border cell fate only. (Fig 3K-N).

    • The authors had made a great effort to prove that proper Delta endocytosis in the nurse cells is essential for adequate Notch signalling in the AFCs and right number of BCs recruitment. Specifically:

    o They checked the consequences on Dl trafficking of down-regulation of rab5 or auxilin, but they did not test the effect in BC numbers

    • o They show that downregulating the function of shi affects the number of BCs, but did not show the effect of this condition in Dl trafficking.*
    • Consequently, they cannot conclusively say that effects on trafficking of Dl affect number of BCs, since they haven't really tested both effects on the same background. I think that for simplification, they should test both, effects on Dl trafficking and number of BCs in one of those genetic backgrounds and leave the other two for supplementary material. Alternatively, they should re-write their conclusion for this section.*

    Response: As Rab11GTPase over expression rescued the excessive border cell fate in the cup mutants, to test the specificity we downregulated Rab11 function in the germline itself to check Delta trafficking and border cell fate specification. We employed a late expressing GAL4 driver in the germline and observed that down regulation of Rab11 function resulted in more number of follicle cell acquiring border cell fate and decrease in the number of Delta puncta at the interface of Anterior follicle cells and nurse cells. This phenotype is reminiscent of the Cup mutants suggesting that perturbing the recycling component of endocytosis perse affects border cell fate and Delta trafficking. This result in included in (Fig 6D-I)

    • Their results clearly show that Dl accumulates in puncta, suggesting that there might be a defect in Dl trafficking, and although their rescue experiments point towards an scenario where Rab11-dependent Dl recycling is being affected, I think there are some weak points on their arguments. The fact that Rab11-KD does not generally affect Notch signalling in the FCs, as shown in (Windler & Bilder, 2010) argues against their conclusion that the effect of cup in nurse cells on Rab11 function is responsible for the defects in Dl trafficking and, subsequently, on Notch activity in AFCs. An alternative explanation is that Rab11 overactivation in the Cup mutant background compensates for a different defect on Dl trafficking, for example, Rab4-dependent recycling pathway. Another possibility is that AFCs could be specially sensitive to changes in Rab11-dependent Dl trafficking defects in the nurse cells. To distinguish between these two possibilities, they should perform some of the following experiments:
    • o First of all, there are a number of endosome markers that can be used to check in which step of the endocytic route Dl is being accumulated, including (but not limited to) anti-Rab11 antibody, anti-Rab5, anti-Rab7, tub-Rab4-mcherry. They should do co-localization experiments with Dl and endosomal markers.*
    • o Also, they could check what happens to the number of BCs and Dl trafficking when Rab11 function is blocked in the nurse cells, in a similar way to what they did with Auxillin, Rab5 and Shi. They could use some of the tools described in (Satoh et al., 2005)*

    Response: We have perturbed Rab11 function during mid oogenesis which is quite distant from early stage egg chambers examined by Windler & Bilder. We observed that down regulation of Rab11 activity in germline affects both border cell fate in the AFCs and Delta trafficking in the germline itself. Protein Trap analysis of Rab11 in wild type and Cup mutant background suggests Rab11 is enriched in the trans-golgi network where the activity of Rab11 is modulated through nucleotide exchange. Over all our results suggest that Rab11 activity is diminished in the cup01355 egg chambers and thus stimulating the recycling endocytosis restores Notch signalling in the AFCs, limiting JAK-STAT activation and restricting BC cell fate specification.

    • The authors final model is one in which cup in the nurse cells regulates Rab11 function to ultimately control JAK/STAT signalling in the AFCs. However, they have not looked at the status of JAK/STAT signalling in their Rab11-CA rescue experiments. I think this experiment will really round-up their work.* Response: The border cell fate is linked to activation of JAK-STAT signaling in the anterior follicle cells. As we have already exhausted the STAT antibody, it will difficult to access the levels of STAT perse.

    Minor points:

    • The authors tested if the extra BC phenotype observed in the cup mutant egg chambers is due to defects in FCs endoreplication. I have two questions related to this section.*

    • o First of all, I do not understand the rationale behind this idea that defects in FCs endoreplication would result in extra BCs. Please explain and add any relevant references.*

    • o Secondly, they say that they used Cut and Phospho-Histone3 as endoreplication markers. I believe that what they mean is that the absent of these two markers indicates that FCs have exit the cell cycle and enter the endocycle (Sun & Deng, 2005), however they are not markers of endoreplication. Please, re-write to make this clear.*

    Response: The follicle cell exhibits a switch from mitotic to endocycle phase at a particular stage of oogenesis (Sun & Deng’ 2005). Our premise is that incase this switch is delayed, will the extra proliferation can account for the excessive border cell fate? In this context we have modified the text to render clarity to this section.

    The authors tested whether the levels of Notch activity were altered in the cup mutant egg chambers. For that, they used an NRE-GFP construct that shows a clear reduction in the levels of Notch activity in the AFCs. They also used the number of NICD and NECD puncta in signal receiving and sending cells respectively, as proxy of Notch activity. Although I understand the rationale, there are other explanations for this phenotype as discussed above. Thus, if they want to have an alternative way of showing the dampening of Notch signalling, they could use the levels of expression of well characterised targets of Notch in the FCs, such us hnt and E(spl)mb-CD2 or E(spl)m7. Response: We believe that our new set of data with NICD over expression (in the AFCs) rescuing border cell fate in Cup mutants coupled with NRE-GFP, NICD, NECD data now lends stronger support to our claim that Notch signaling in the follicle cells is indeed downstream of Cup function in developing egg chambers.

    • In M&M the authors explain that NRE-GFP levels were expressed in Fold change.* However, in figure 3C the units of the graph are Fluorescence Intensity in a.u. Please,

    check this small inconsistency

    Response: We have modified this as per reviewer’s suggestion.

    In figure 4, they show the quantification of tubulin fibres within the nurse cells, however they are missing a similar analysis of Phalloidin (Pha) fibres/levels. I think this experiment and figure will be more complete if the authors added such a quantification of the effects of cup LOF in Pha distribution. Also, the authors do not show the single Pha channel in Fig4C, which would greatly helped to appreciate the differences between the WT and Cup LOF nurse cells. I suggest modifying the figure to better show the changes in Pha distribution. Response: We have modified the figure and included quantitation of actin fibre length in Supplementary figure 6H- K.

    In figure 4F-G the authors are showing the general effect of cup LOF in Delta distribution. They indicate with yellow arrowheads the cytoplasmic Dl puncta accumulation in the nurse cells, however it is almost impossible to see such puncta with that level of magnification/resolution. I suggest removing the arrowheads, since the figure 4H-I shows the same puncta more clearly. Response: We have modified the figure to render clarity

    In the Dl trafficking experiments (Fig4 H-I,K,L and Fig5A-C), the authors measured the number of puncta in the anterior nurse cell-follicle cell junction. In order to do those types of quantifications they need to be able to tell the cell boundaries that separate FCs from the nurse cells. Please, clarify the criteria for determining if the puncta are within the FCs or the underlying nurse cells. __Response: Delta, NICD, NECD proteins marks the apical surface of the follicle cells. We used this as a reference to segregate nurse cell puncta with respect to follicle cells. This has been elaborated in the Material & Method section. __

    In figure 6C-D the authors show example images of egg chambers expressing Rab11-CA-YFP using the germline specific nos-Gal4. However, in the images it looks like the YFP signal is coming from the surrounding stretched FCs. Please check that these are the right images or explain the inconsistency.

    Response: We have crosschecked the images and the YFP signaling is from nurse cell periphery which gives the wrong impression that it is from stretched follicle cells.

    • In figures 1R, 2L, 3Q, 6I, 6M, the authors should show the results of the statistical analysis between all the conditions tested. I think that this is crucial to be able to tell whether some of the rescues are complete or only partial. *Responses: To avoid cramming the Figures, we have including some of the p values in the Figure legends. *

    • Line 174: should say "mutant egg chambers".*
    • Line 281: There is a reference that is missing from reference list: Liu et al., 2010;*
    • Line 292: The reference for the NRE-GFP construct is not the correct one, since that references to a review article. Please, add the correct reference.*
    • In line 462 of the manuscript you have a reference that is missing from your reference list.*
    • In line 394 the authors say: "protein, it's enrichment in the cytoplasmic fraction of the cup mutant egg chambers", but I think that they meant mutant nurse cells.*

    Response: We have modified the text as per the all the suggestions above Reviewer #2 (Significance (Required)):

    The BC migration is an excellent model to study collective cell migration and how epithelial cells can acquire migratory behaviours. After years of study, there is good understanding of the signals and genetic circuits that regulate BCs specification and migration (Montell et al., 2012), but there are not many studies, to my knowledge, that describe a role of nurse cells in specifying or guiding the migration of these cells. Thus, this study by Saha and colleagues is one of the first studies that show a role for nurse cells in specifying the number of BCs.

    My field of expertise is in cell-cell communication through different pathways, including Notch and Integrin signalling. I have studied the role of endocytosis in regulating Notch signalling in various contexts, including follicular epithelium in Drosophila ovaries.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    This manuscript describes an investigation into the signaling that induces the differentiation of follicle cells into border cells in the Drosophila ovary. Previous studies have established the border cells as an informative model for studying how epithelial cells delaminate and undergo collective cell migration, and have identified the JAK-STAT and Notch pathways as important regulators of the process. Here, the authors performed a forward genetic screen and identified cup as another gene that is involved in the regulation of border cell differentiation. Their findings are consistent with a model in which cup is required in germ cells for the endocytosis of the Notch ligand, Delta. In cup mutants, impaired trafficking of Delta leads to decreased Notch signaling in follicle cells, which allows for increased JAK-STAT expression in follicle cells and an increase in the number of follicle cells that differentiate into border cells. Overall, the approach is thorough and the phenotypes are clear and well-described. The quantification of phenotype penetrance and of aspects of the images, such as pixel intensities and the number of particles in a region is a strength of the paper. The use of multiple independent methods to test key points is another strength. However, there are several concerns that should be addressed before the paper is considered for publication:

      1. The central phenotype that this paper is based on is a difference in the number of border cells per cluster in wildtype and mutant genotypes. However, this phenotype is fairly subtle in some cases (e.g. in Fig. 2L, it varies by only about 10% between control and mutant) and it is somewhat variable. For example, the number of cells in border cell clusters of the controls range from 4.49 in Fig. 3M to 6.41 in Fig. 1F. Considering that the mutant values fall within this range in some cases (e.g. 5.98 in Fig 3M) and the difference between the means from control and mutant genotypes is often less than two, the significance of this phenotype is unclear. How does this compare to other mutants that have been described to affect border cell specification? Are there any consequences for the differentiation of the follicle or the function of the egg caused by this defect?*

    Response: We are using the border cell number as readout for the output of JAK-STAT signaling. Though the difference in numbers may appear to be subtle, we believe our data clearly demonstrates that Cup non cell autonomously regulates border cell fate by modulating Notch signaling in the follicle cells*. *

    • Wang, et al. (PMID 17010965) have described previously that Notch signaling, and*

    Kuzbanian specifically, is required for border cell migration. The authors should cite this paper and discuss their findings in light of this study. For example, if Notch signaling is impaired in cup mutants, is border cell migration also impaired? Likewise, the citation of the Assa-Kunik, 2007 study as evidence that Notch and JAK-STAT signaling act antagonistically (Line 286) is a bit of an oversimplification. While that study does show that Notch and JAK-STAT act antagonistically at earlier stages of follicle development, Fig. 6 of that paper shows that a Notch reporter and a JAK-STAT reporter are both expressed concomitantly in border cells of a Stage 10 follicle and in the anterior follicle cells of what looks like a Stage ~8 follicle. The authors should discuss the apparent contradiction between their findings and this study.

    Response: We provide genetic evidence to support our claims that Cup in the germline modulates Notch activation in the anterior follicle cells thus limiting border cell fate specification to a few. The overlap in the expression of Notch reporter m7-lacz and STAT in the follicle cells and border cells is interesting and will need further investigation in real time to decipher any comparison between the two studies.

    • Lastly, the manuscript contains many grammatical errors, incomplete sentences, improper punctuation and spacing, and informal writing, such as the use of contractions. It should be thoroughly edited for content and clarity.*

    Response: We have tried to edit the manuscript with the aim to improve on the language, grammar and punctuations.

    Reviewer #3 (Significance (Required)):

    Although the identification of cup as a contributor to the regulation of border cell differentiation is novel, the other main regulators investigated in this study, including Notch and JAK-STAT signaling, have been identified previously. The role of cup in this context seems to be to fine tune Notch signaling and it seems to play a relatively minor role in the process of border cell specification. In addition, the conclusions of this paper are not well-integrated into the existing literature on Notch and JAK-STAT signaling in border cells, and the discussion about the broader implications of this study for the understanding of Notch signaling was not well-developed. However, the careful documentation and quantification of the phenotypes reported in this study adds rigor and allows for firm conclusions. For these reasons, this study may have a lasting but perhaps somewhat incremental impact on the study of border cell migration in the Drosophila ovary.

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    Referee #3

    Evidence, reproducibility and clarity

    This manuscript describes an investigation into the signaling that induces the differentiation of follicle cells into border cells in the Drosophila ovary. Previous studies have established the border cells as an informative model for studying how epithelial cells delaminate and undergo collective cell migration, and have identified the JAK-STAT and Notch pathways as important regulators of the process. Here, the authors performed a forward genetic screen and identified cup as another gene that is involved in the regulation of border cell differentiation. Their findings are consistent with a model in which cup is required in germ cells for the endocytosis of the Notch ligand, Delta. In cup mutants, impaired trafficking of Delta leads to decreased Notch signaling in follicle cells, which allows for increased JAK-STAT expression in follicle cells and an increase in the number of follicle cells that differentiate into border cells. Overall, the approach is thorough and the phenotypes are clear and well-described. The quantification of phenotype penetrance and of aspects of the images, such as pixel intensities and the number of particles in a region is a strength of the paper. The use of multiple independent methods to test key points is another strength. However, there are several concerns that should be addressed before the paper is considered for publication:

    1. The central phenotype that this paper is based on is a difference in the number of border cells per cluster in wildtype and mutant genotypes. However, this phenotype is fairly subtle in some cases (e.g. in Fig. 2L, it varies by only about 10% between control and mutant) and it is somewhat variable. For example, the number of cells in border cell clusters of the controls range from 4.49 in Fig. 3M to 6.41 in Fig. 1F. Considering that the mutant values fall within this range in some cases (e.g. 5.98 in Fig 3M) and the difference between the means from control and mutant genotypes is often less than two, the significance of this phenotype is unclear. How does this compare to other mutants that have been described to affect border cell specification? Are there any consequences for the differentiation of the follicle or the function of the egg caused by this defect?
    2. Wang, et al. (PMID 17010965) have described previously that Notch signaling, and Kuzbanian specifically, is required for border cell migration. The authors should cite this paper and discuss their findings in light of this study. For example, if Notch signaling is impaired in cup mutants, is border cell migration also impaired? Likewise, the citation of the Assa-Kunik, 2007 study as evidence that Notch and JAK-STAT signaling act antagonistically (Line 286) is a bit of an oversimplification. While that study does show that Notch and JAK-STAT act antagonistically at earlier stages of follicle development, Fig. 6 of that paper shows that a Notch reporter and a JAK-STAT reporter are both expressed concomitantly in border cells of a Stage 10 follicle and in the anterior follicle cells of what looks like a Stage ~8 follicle. The authors should discuss the apparent contradiction between their findings and this study.
    3. Lastly, the manuscript contains many grammatical errors, incomplete sentences, improper punctuation and spacing, and informal writing, such as the use of contractions. It should be thoroughly edited for content and clarity.

    Significance

    Although the identification of cup as a contributor to the regulation of border cell differentiation is novel, the other main regulators investigated in this study, including Notch and JAK-STAT signaling, have been identified previously. The role of cup in this context seems to be to fine tune Notch signaling and it seems to play a relatively minor role in the process of border cell specification. In addition, the conclusions of this paper are not well-integrated into the existing literature on Notch and JAK-STAT signaling in border cells, and the discussion about the broader implications of this study for the understanding of Notch signaling was not well-developed. However, the careful documentation and quantification of the phenotypes reported in this study adds rigor and allows for firm conclusions. For these reasons, this study may have a lasting but perhaps somewhat incremental impact on the study of border cell migration in the Drosophila ovary.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Saha et al. made a detailed description of the role of the mRNA binding protein Cup in specifying the number of Border Cells (BC) during Drosophila melanogaster oogenesis. First of all, they show that females homozygote for a hypomorph allele of cup have higher number of BCs compared to Wild Type (WT) females. They present a series of experiments that points towards the phenotype being due to a specific role of cup in the nurse cells that non-cell autonomously regulates BC specification. Also, they show that this phenotype is the result of an increase in the levels of JAK/STAT signalling in the BC, a major determinant of BC fate. In addition, they show that cup mutant egg chambers exhibit a downregulation of the Notch (N) pathway function in the BCs and that over-activating Notch results in the rescue of the number of BCs. Moreover, the authors present data on the effect of cup in Delta (Dl) trafficking in the nurse cells: They found that cup mutant egg chambers show increased number of Dl puncta within the cytoplasm of the nurse cells, but reduced numbers in the nurse cell-Anterior Follicle Cell (AFC) boundary as a result of defective Dl endocytosis. Finally, they were able to rescue the Dl trafficking phenotype, as well as the number of BC by overexpressing an active form of Rab11.

    Major points:

    • In this study, the authors employed an hypomorph allele of Cup to generate egg chambers where both germline and somatic cells are mutant for Cup. They did a series of experiments to try to demonstrate that the Border Cell (BC) specification phenotype they observe is non-cell autonomous and that is due to the Loss of Function (LOF) of Cup exclusively in the nurse cells. Although I appreciate the difficulties of eliminating or reducing the levels of Cup specifically in the nurse cells only during mid-oogenesis, I feel like this is key to be able to claim that this effect of Cup in BC specification is really non-cell autonomous. The reasons why I still have some doubts that there might be some cell autonomous effects in the FCs are the following:
      • The authors show that cup01355 mutant egg chambers have a phenotype in Dl trafficking. Although they analysed in detail the effects on Dl in the nurse cells, their images show that there might be a defect in Dl levels/trafficking in the Follicle Cells (FCs) as well (Fig5A-B). It has been shown that Dlmut FCs have reduced levels of Notch activity due to reduced lateral inhibition (Poulton et al., 2011), so there is a possibility that the reduced levels of Notch activity in the cup01355 egg chambers might be due, partially, to defects in Dl trafficking/levels in the FCs, rather than in the nurse cells.
      • The authors tested the role of the Notch pathway in the cup mutant phenotypes by measuring the number of NICD puncta in the signal receiving cells as proxy for Notch activity (Fig4). Although I understand the rationale, I am not convinced that they can completely rule out that the changes in NICD puncta number in FCs is not due to some effect of cup LOF on Notch trafficking in these cells.
      • In figure 6, the authors show that expression of a constitutively active form of Rab11 specifically in the nurse cells restores the BC number to that of the WT. However, the levels of Dl particles and, especially the levels of NRE-GFP expression, remains slightly lower than in the WT conditions.

    One of the main conclusions of this study is that cup regulates BC specification through a non-cell autonomous mechanism that involves communication between nurse cells and AFCs. For that reason, I think in order to conclusively say that, the authors need to try to remove the function of cup specifically in the nurse cells. They mentioned they have tried different ways of doing this unsuccessfully, but do not specify how they have tried. I suggest using the cup-RNAi line combined with a nurse cell specific Gal4 and a ubiquitous gal80ts line (tub-Gal80ts), if they have not try this. I do not expect the authors to repeat all the experiments with this condition, but at least they should test the main findings i.e. number of BCs, JAK/STAT overactivation and Notch attenuation.

    • The authors have shown in Figure 3 that there is a decrease in Notch signalling in the AFCs in cup01355 egg chambers. In order to test that the BC number phenotype observe in this condition is due to that effect on Notch signalling they have done a rescue experiment using the antimorphic Notch allele Nax-16. Since in this condition all cells (nurse cells and FCs) have increased levels of Notch, they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype. If they want to show that the function of Notch is specifically needed in the FCs, they should over-activate Notch exclusively in the AFCs. For instance, they could express a constitutively active form of Notch, such as UAS-NICD (Go et al., 1998) or UAS-NECD (Fortini et al., 1993), specifically in the AFCs. Otherwise, they should re-write the text since they cannot conclusively say that the increase in Notch function in the FCs rescues the cup phenotype.
    • The authors had made a great effort to prove that proper Delta endocytosis in the nurse cells is essential for adequate Notch signalling in the AFCs and right number of BCs recruitment. Specifically:
      • They checked the consequences on Dl trafficking of down-regulation of rab5 or auxilin, but they did not test the effect in BC numbers
      • They show that downregulating the function of shi affects the number of BCs, but did not show the effect of this condition in Dl trafficking. Consequently, they cannot conclusively say that effects on trafficking of Dl affect number of BCs, since they haven't really tested both effects on the same background. I think that for simplification, they should test both, effects on Dl trafficking and number of BCs in one of those genetic backgrounds and leave the other two for supplementary material. Alternatively, they should re-write their conclusion for this section.
    • Their results clearly show that Dl accumulates in puncta, suggesting that there might be a defect in Dl trafficking, and although their rescue experiments point towards an scenario where Rab11-dependent Dl recycling is being affected, I think there are some weak points on their arguments. The fact that Rab11-KD does not generally affect Notch signalling in the FCs, as shown in (Windler & Bilder, 2010) argues against their conclusion that the effect of cup in nurse cells on Rab11 function is responsible for the defects in Dl trafficking and, subsequently, on Notch activity in AFCs. An alternative explanation is that Rab11 overactivation in the Cup mutant background compensates for a different defect on Dl trafficking, for example, Rab4-dependent recycling pathway. Another possibility is that AFCs could be specially sensitive to changes in Rab11-dependent Dl trafficking defects in the nurse cells. To distinguish between these two possibilities, they should perform some of the following experiments:
      • First of all, there are a number of endosome markers that can be used to check in which step of the endocytic route Dl is being accumulated, including (but not limited to) anti-Rab11 antibody, anti-Rab5, anti-Rab7, tub-Rab4-mcherry. They should do co-localization experiments with Dl and endosomal markers.
      • Also, they could check what happens to the number of BCs and Dl trafficking when Rab11 function is blocked in the nurse cells, in a similar way to what they did with Auxillin, Rab5 and Shi. They could use some of the tools described in (Satoh et al., 2005)
    • The authors final model is one in which cup in the nurse cells regulates Rab11 function to ultimately control JAK/STAT signalling in the AFCs. However, they have not looked at the status of JAK/STAT signalling in their Rab11-CA rescue experiments. I think this experiment will really round-up their work.

    Minor points:

    • The authors tested if the extra BC phenotype observed in the cup mutant egg chambers is due to defects in FCs endoreplication. I have two questions related to this section.
      • First of all, I do not understand the rationale behind this idea that defects in FCs endoreplication would result in extra BCs. Please explain and add any relevant references.
      • Secondly, they say that they used Cut and Phospho-Histone3 as endoreplication markers. I believe that what they mean is that the absent of these two markers indicates that FCs have exit the cell cycle and enter the endocycle (Sun & Deng, 2005), however they are not markers of endoreplication. Please, re-write to make this clear.
    • The authors tested whether the levels of Notch activity were altered in the cup mutant egg chambers. For that, they used an NRE-GFP construct that shows a clear reduction in the levels of Notch activity in the AFCs. They also used the number of NICD and NECD puncta in signal receiving and sending cells respectively, as proxy of Notch activity. Although I understand the rationale, there are other explanations for this phenotype as discussed above. Thus, if they want to have an alternative way of showing the dampening of Notch signalling, they could use the levels of expression of well characterised targets of Notch in the FCs, such us hnt and E(spl)m-CD2 or E(spl)m7.
    • In M&M the authors explain that NRE-GFP levels were expressed in Fold change. However, in figure 3C the units of the graph are Fluorescence Intensity in a.u. Please, check this small inconsistency
    • In figure 4, they show the quantification of tubulin fibres within the nurse cells, however they are missing a similar analysis of Phalloidin (Pha) fibres/levels. I think this experiment and figure will be more complete if the authors added such a quantification of the effects of cup LOF in Pha distribution. Also, the authors do not show the single Pha channel in Fig4C, which would greatly helped to appreciate the differences between the WT and Cup LOF nurse cells. I suggest modifying the figure to better show the changes in Pha distribution.
    • In figure 4F-G the authors are showing the general effect of cup LOF in Delta distribution. They indicate with yellow arrowheads the cytoplasmic Dl puncta accumulation in the nurse cells, however it is almost impossible to see such puncta with that level of magnification/resolution. I suggest removing the arrowheads, since the figure 4H-I shows the same puncta more clearly.
    • In the Dl trafficking experiments (Fig4 H-I,K,L and Fig5A-C), the authors measured the number of puncta in the anterior nurse cell-follicle cell junction. In order to do those types of quantifications they need to be able to tell the cell boundaries that separate FCs from the nurse cells. Please, clarify the criteria for determining if the puncta are within the FCs or the underlying nurse cells.
    • In figure 6C-D the authors show example images of egg chambers expressing Rab11-CA-YFP using the germline specific nos-Gal4. However, in the images it looks like the YFP signal is coming from the surrounding stretched FCs. Please check that these are the right images or explain the inconsistency.
    • In figures 1R, 2L, 3Q, 6I, 6M, the authors should show the results of the statistical analysis between all the conditions tested. I think that this is crucial to be able to tell whether some of the rescues are complete or only partial.
    • Line 174: should say "mutant egg chambers".
    • Line 281: There is a reference that is missing from reference list: Liu et al., 2010;
    • Line 292: The reference for the NRE-GFP construct is not the correct one, since that references to a review article. Please, add the correct reference.
    • In line 462 of the manuscript you have a reference that is missing from your reference list.
    • In line 394 the authors say: "protein, it's enrichment in the cytoplasmic fraction of the cup mutant egg chambers", but I think that they meant mutant nurse cells.

    Significance

    The BC migration is an excellent model to study collective cell migration and how epithelial cells can acquire migratory behaviours. After years of study, there is good understanding of the signals and genetic circuits that regulate BCs specification and migration (Montell et al., 2012), but there are not many studies, to my knowledge, that describe a role of nurse cells in specifying or guiding the migration of these cells. Thus, this study by Saha and colleagues is one of the first studies that show a role for nurse cells in specifying the number of BCs.

    My field of expertise is in cell-cell communication through different pathways, including Notch and Integrin signalling. I have studied the role of endocytosis in regulating Notch signalling in various contexts, including follicular epithelium in Drosophila ovaries.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Saha et al. characterize Drosophila egg chambers that are mutant for cup and identify an increase in the number of a specialized type of follicle cells, the border cells. They demonstrate that this increase correlates with an expanded domain of STAT activity and reduced Notch signaling in anterior follicle cells. Determining that cup is required in the germline cells, the authors postulate and provide some evidence that cup mutants prevent germline Delta from properly signaling to follicle cells. In line with this, they also show that blocking endocytosis phenocopies some aspects of cup mutants, particularly border cell numbers and Delta levels, which they monitor cytoplasmically and at the cell surface. Lastly, they demonstrate that activation of Rab11 can rescue Delta levels and border cell number in cup mutants. They conclude that a key function of Cup in the germline is to traffic Delta to signal to follicle cells, and that the endocytic processing of Delta is required for its function.

    Major comments:

    The findings of this study are interesting and novel. The authors have completed a lot of experiments and analyzed the results carefully and in great detail. Experimental design is described adequately and statistical analysis is sufficient. While the main results are largely convincing and support the conclusions, there are some weaknesses that need to be addressed. One major concern is that the vast majority of the experiments were conducted with a single homozygous allele for cup. The authors claim this was necessary because other alleles arrest oogenesis, which is understandable, but it leaves the potential problem that the allele, a P-element insertion, may affect other genes, or there may be other unidentified mutations on the mutant chromosome. The authors are able to partially rescue the border cell phenotype with overexpression of Cup and can also mimic the outcome with RNAi in the germline, which helps alleviate some of this concern, but this was only done for one set of experiments (those in figure 1). Similar experiments need to be included to demonstrate the same outcomes when cut is disrupted by other alleles/methods for at least some of the Notch/Delta analyses since this is key to the paper's conclusions.

    A second concern is that some evidence is circumstantial or indirect. Specifically, the authors argue that the effect of Cut is due to trafficking of Delta, but do not consider the possibility that Delta could be more directly regulated or that other factors may be relevant. Border cell specification is rescued by increasing recycling in cup mutants, but this could be due to recycling of more factors besides Delta. To address this more directly, the authors should overexpress Delta in the germline of cut mutants. It is possible the disruption of Delta in cut mutants is due to changes in Delta protein stability/levels, so the experiment may also clarify this issue. If this is the case, it may be that hypomorphic Delta mutants would have a defect on border cell number, which could be examined separately. If Delta levels are low, endocytosis and recycling increases may also rescue cut mutants indirectly, but the conclusion about what Cut regulates may differ.

    Another concern is that Cup's main role is a confusing since it regulates many things, including cytoskeleton and cytoskeleton is necessary for general health and vesicle trafficking in the egg chamber - how do the authors think Rab11 upregulation is overcoming these defects? Rab11CA rescues Delta levels almost completely in cut mutants but only partially rescues Notch activation, suggesting there are other problems in these egg chambers that could contribute to the defects. While exploring possible other factors is beyond the scope of this work, the authors may want to acknowledge this issue.

    Minor comments:

    It would help the presentation of the paper to introduce Notch/Delta signaling during oogenesis in the introduction. More introduction and clarity about the number of polar cells at early stages and their role in the border cell cluster may also be useful to the reader.

    It is notable that the primary phenotype of a change in border cell numbers is quite subtle, often only affecting 1-2 cells, and the variation in different genotypes and experiments is sometimes also that large. The authors do a good job of being careful to count the cells at a specific developmental time and do appropriate statistical tests within an experiments. Still, it difficult to be sure that the effects are due to the gene being manipulated specifically or the genetic background. Related to this, a few issues should be addressed. Notably, at earlier stages, Notch signaling impacts cell division, so some of the phenotypes might be explained by there being more total cells in the domain instead of more signaling. The authors show Cut is in the same domain and pH3 is similar, but they didn't seem assess overall numbers. Secondly, for the stat suppression of cut (figure 2L), the authors need to show the stat-/+ control for comparison to make a conclusion about suppression versus additive effects. In addition, prior work (Wang et al 2007) expressed DN Kuz in border cells and did not see a change in specification, unlike what is claimed here. In the experiment in question, the control has lower than normal numbers of border cells and the DN Kuz has a number more typical of the controls in other experiments- so this is a concern that there is something else in the genetic background influencing the numbers. Other controls could help make this case, but ultimately this result is probably not necessary for the main argument. Thus the authors might consider leaving it out the Kuz analysis or perhaps can comment on the discrepancy with prior published results.

    Can the authors comment on why the volume of the border cell cluster increases more dramatically (>2x) than the number of cells (30% more)?

    Does the increase in border cell number change the migratory capacity? That is, do the clusters in cut mutant egg chambers migrate normally while the egg chamber looks okay?

    Several of the figure legend titles state conclusions that are over interpretations of the data shown:

    • Figure 3 legend is overstated- these experiments do not assay STAT activity, only border cell number, so the title can be simplified to say that.
    • For figure 4, both cytoskeleton and Delta are shown to be disrupted in cup mutants, but they are not directly linked, eg, the experiments do not show a change in Delta in cytoskeletal mutants alone. While it is interesting that cup mutants have disrupted cytoskeleton, ultimately this result is not well connected to the main issue of Notch/Delta signaling; in fact, it becomes confusing how anything can be trafficked to the cell surface if there is poor cytoskeletal organization. Since the authors favor the hypothesis that the cytoskeleton is not the key to the border cell specification difference, they may want to move this result out of figure 4.
    • The Figure 5 legend is also overstated- these experiments show that Delta is higher in cup mutants and endocytosis mutants AND that endocytosis (of something) is required in the germline for border cell number- but these results are not linked in this figure. More evidence for this connection does come later in figure 6. Some figure legends are quite brief and could benefit from a little more detail on what is being shown.

    Figure layout could be improved by keeping images consistent sizes and making sure graph text is large enough to read easily. Figures in general could be streamlined by having negative results and less pertinent results in supplemental data.

    Not all papers cited in the text are in the reference list.

    Referees cross-commenting

    I generally agree with the other reviewers that there are concerns with the precise function of cup in this context, and that some revision is needed, including editing of the writing. In response to reviewer 2, prior published studies only detected Cup in germline, but it is possible that it is expressed in follicle cells at a low level. The mutant clonal experiment in follicle cells that the authors did had no effect on border cells, so that provides some evidence the role is non-autonomous. I agree with reviewer 2's concern that the authors overstate the connection between cup and Delta and border cells based on their data and need a few more experiments to tie things together. I understand reviewer 3's concerns that the experimental effects on border cell numbers are very small and variable- I listed this as a minor concern, though, since this number is mainly being used as a read-out for STAT signaling levels and the data were extensively quantified and statistically tested.

    Significance

    My expertise is in cell migration, developmental biology, and Drosophila genetics. This paper will be of broad interest in these fields as it incorporates aspects of each in its characterization of a new regulatory mechanism to induce a motile cell population non-cell-autonomously, which is an exciting finding. Specifically, the work increases our understanding of the intersection between Notch and Jak/STAT signaling, which many researchers study - these were both known to be involved in border cell specification. The study provides more detailed characterization of the signaling and specification process in general, and makes significant advances in understanding how Delta signals are produced and presented from germline cells to receiving cells in the soma. Cut has not been previously implicated in these signaling pathways, so that is also novel, although its precise mechanistic role here is still somewhat unclear.

  10. Version published to 10.1101/2022.07.11.499544v1 on bioRxiv