Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte

  1. Ana Milas
  2. Jorge de-Carvalho
  3. Ivo A. Telley

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Abstract

In Drosophila melanogaster the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the posterior end of the oocyte while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. This mutual exclusion leads to a polarized microtubule network and accumulation of posterior determinant oskar later in oogenesis. Reciprocal biochemical interactions between Par proteins can explain their cortical exclusion and domain formation – for example, Par-1 excludes Par-3 by phosphorylation. However, past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, despite modern screening approaches and genetic manipulation, neither the molecular identity nor the nature of the signal is known. Here, we provide the first evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. We show that Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. This occurs before Par-1 is removed suggesting that phosphorylation of Bazooka by Par-1 is not sufficient to maintain Bazooka exclusion in the absence of PFC contact. Furthermore, we provide evidence that PFC contact maintains Par-1 and oskar localization and microtubule cytoskeleton polarity in the oocyte. Our observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.

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    Referee #3

    Evidence, reproducibility and clarity

    The Drosophila oocyte is a classical model to study establishment of cell polarity, and it is known for decades how Bicoid and Oskar define the anterior-posterior axis of the embryo. However, Bicoid and Oskar are not conserved so that these findings cannot be generalized. The situation is different for the Par proteins, which have been identified in C. elegans. They are not only conserved but also their mode of of action seems to be preserved. 20 years ago it was a very surprising finding that the Par proteins contribute to establishment of polarity in the Drosophila oocyte. A fascinating and simple mutual inhibition model emerged over the years, in which the same molecular mechanisms establish cell polarity in the C. elegans one-cell embryo and in the Drosophila oocyte: Anteriorly localised Par-3/Bazooka recruits aPKC kinase, which excludes Par-1 by phosphorylation, whereas posteriorly localised Par-1 kinase excludes Par-3/Bazooka by phosphorylation. The manuscript by Milas et al. challenges this model by closely analysing Par localisation in living Drosophila oocytes. The authors provide strong evidence that the kinetics of Par-1 and Bazooka localisation are not consistent with the model.

    Milas et al. first describe a morphological difference between the anterior-lateral and posterior cortex of the oocyte by showing that only the posterior cortex is tightly connected to the overlaying epithelium. This morphological difference correlates with the localisation of Par-1, which is restricted to the posterior, while Bazooka localises only to those regions of the cortex, where there is a gap between the oocyte and the epithelium. This gap expands towards the posterior cortex during stage 10A and encloses it at stage 11. Unexpectedly, Par-1 and Bazooka localisations overlap at the posterior cortex when the gap expands, which contradicts the mutual inhibition model. The authors hypothesised that the close contact of epithelium with the oocyte might influence Par-1/Bazooka localisation. To test this they mechanically detached the epithelium from the oocyte and also ablated groups of epithelial cells. These manipulations resulted in posterior spreading of Bazooka protein within 30-60 minutes. Interestingly, the authors found that in those regions of the posterior cortex, where cells have been ablated, Par-1 and Bazooka colocalise for 30 minutes, which is difficult to reconcile with a model in which Par-1 excludes Bazooka by phosphorylation. The authors also show that Par-1 finally disappeared form the regions where epithelial cells have been ablated. However, aPKC, the kinase that is supposed the exclude Par-1 by phosphorylation, appeared only after Par-1, which argues against the idea that aPKC prevents Par-1 localisation. In summary, the described localisation kinetics are in conflict with the current model, in which direct phosphorylation activities of Par-1 and aPKC orchestrate the mutual exclusive Par domains in the Drosophila oocyte. The data suggest that the mechanisms underlying mutual inhibition are more complex than thought and involve contact with posterior epithelial cells.

    The microscopy used by the authors is state of the art, the data are of high quality and the quantitative analysis is convincing. The results are surprising but conclusive since the experiments were performed and presented in a professional way. This combination makes the manuscript very interesting.

    Major points:

    1. The finding that the posterior cortex is in close contact to the epithelium, while there is a gap between the remaining oocyte cortex and the epithelium is very interesting, and should be quantified and characterised more precisely. When does the gap form and how exactly does it spread posteriorly? Is it possible to distinguish the gap from the attachment zone by using markers for the ECM (e.g. viking-GFP) or adhesion proteins (e.g. Integrin)?
    2. The authors suggest that direct contact between the epithelium and the oocyte is required to exclude Bazooka from the posterior oocyte cortex. The polar cells of the follicular epithelium have almost no contact to the oocyte. One would expect that if only the polar cells are ablated, this would not lead to posterior spreading of Bazooka. Such a control experiment could support the author´s model.

    Minor points:

    1. There are repeatedly double negations which make the text difficult to understand (e.g. "Bazooka exclusion was lost...." (line 104) or "Par-1 does not delocalise from the posterior pole prior to accumulation of Bazooka" (line 163). I see that this follows the logic of the published molecular mechanisms but for the sake of comprehensibility, the authors should try to formulate the results in a positive way (at least in a repeating sentence).
    2. Based on the kinetics of Par-1 localisation the authors the conclude that Par-1 binds to diffusible binding sites at the oocyte cortex, which are modulated by posterior epithelial cells. This is one possible explanation for their results but other interpretations are equally possible. Since the authors provide no further evidence for the existence of Par-1 binding sites their interpretation should be formulated more carefully.
    3. The authors should mention that they use the Par-1 isoform (N1S) which fully rescues the par-1 mutant phenotype (see Doerflinger et. al, Curr Biol, 2006). What is known about the rescuing activity of the Bazooka transgenes that were used in the manuscript?
    4. In principle it is possible that the posterior spreading of Bazooka (after follicle cell detachment or ablation) is caused by premature ooplasmic steaming. However, the movies show that this is not the case. This should be stated in the text.

    Significance

    The Drosophila oocyte is a classical model to address the fundamental biological question of how cell polarity is established. The current model of mutual Par protein inhibition is a critical part of our understanding of cell polarisation, and was proposed to be conserved between flies and worms. In the case of Drosophila this model mainly relies on a combination of genetic and biochemical data. Milas et al. tested this model by using in vivo imaging, and found that the kinetics of Par localisation do not correspond to the existing model. This suggests that central aspects of the proposed mechanisms controlling mutual Par inhibition in the Drosophila oocyte are not conserved or not fully understood. The work makes therefore a surprising and important contribution to the understanding of cell polarity.

    I work for many years on Drosophila oogenesis and my main interest switched from cell polarity to membrane trafficking.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript examines an important and unsolved question concerning the establishment of the polarity axis in the Drosophila oocyte, namely how the follicle cells located at the posterior of the egg chamber trigger a signal to the oocyte for its subsequent polarization. To address this question the authors, center their studies on the localization of PAR proteins which are distributed along the antero-posterior axis. They more specifically focus on the mutual exclusion of Par1 and Bazooka/Par3 (Baz)at the posterior of the oocyte. The signaling event from the posterior follicle cells toward the oocyte is an essential process however it remains unsolved despite numerous screening and genetic manipulation approaches, leaving open the possibility that classical signaling involving a diffusible ligand emitted by follicular cells with its receptor located at the plasma membrane of the oocyte, would not be applied here.

    Here the authors are using original biophysical approaches to address whether signaling between the follicular cells and the oocyte would involve mechanical features.

    The authors focus at the dual exclusion between Baz and Par1 between the stages 10 and 11. To specifically follow these two proteins in the oocyte without being disrupted by their expression in the follicle cells, they used the Gal4/UASp system to express Baz and Par1. They found that Baz accumulate again at the posterior of the oocyte at stage 10B following the loss of contact between the posterior follicle cells (PFCs) and the oocyte whereas Par1 is gradually lost at that position. By using a glass micropipette to aspirate and pull on the PFCs they observed a premature Baz accumulation at the oocyte posterior. Then, to spatially improve the targeted area in the PFCs, the authors use a pulsed UV lazer, and show that PFCs are required to locally maintain posterior exclusion of Baz. Using a similar setup, they show that similarly Par1 is eliminated at the oocyte cell cortex region that had been in contact with ablated PFCs. However, Par1, with a kinetic slower to the one of Baz, is never disappearing before Baz appearance. Although difficult to distinguish, the authors report that the disappearance of Par1 is locally connected by an increase in microtubules (see major points). Finally, upon PFCs ablation, the authors show that the posterior reappearance of Baz is followed by the appearance of aPKC. However, the reappearance of PKC is slower than the removal of Par1, suggesting that in this case Par1 is not removed by PKC.

    The particularly interesting results of this work show that cellular contacts between PFCs and the oocyte are necessary to maintain Baz exclusion and Par1 localization. Furthermore, the ablation results suggest that individual PFCs are required to maintain local posterior exclusion of Baz. Overall it is an interesting observation, and most of the data are presented in a clean organized manner.

    Major comments

    1. The authors concentrate their studies on the distribution of Par3 and Par1 at the posterior part of the oocyte, mainly at stage 10 according to the images in the figures and movies. The involvement of Par3 and Par1 on polarized transport to the posterior pole of the oocyte has been well characterized previously between stages 7 and 9. The results of the authors are very interesting but they do not show that beyond the return of Baz and the disappearance of Par1 at the developmental stage they are looking at, the antero-posterior polarization and more particularly the localization of oskar in the posterior is affected. This is an important point as the authors propose that follicle cell contact maintains main body axis polarity. This would be possible by monitoring the impact of PFC ablation on the maintenance of oskar localization by tracking osk RNA with the MCP-MS2 system, or also by visualizing the staufen protein with a stau-GFP transgene.
    2. The authors use the Jupiter protein fused to the cherry protein to track MTs. This is perfectly fine to highlight the cytoplasm in the oocyte and to outline the cell-cell contacts between the PFCs and the oocyte. However, with Jupiter-cherry the microtubules are not clearly detected in the oocyte in the data presented.This is a problem because the authors want to make an important point with the potential reappearance of microtubules in the oocyte while Par1 has disappeared in the vicinity of the destroyed PFCs. (Fig5). The authors should use another microtubule reporter that allows better detection of microtubules in the oocyte, Jupiter-GFP, EB1-GFP, Ensconsin MT binding domain (EMTB)-RFP.

    Minor comments:

    1. The stage of the oocyte is not always indicated, this is particularly the case with the Fig2 with the pulling experiment with a glass micropipette.
    2. With the Fig 3E, to highlight the fact that the intensity of Baz increases very quickly after the removal of PFCs (1 mn) the authors should include an insert with a shorter time scale. The authors could also comment on the difference in velocity in baz reappearance when the ablation of PFCs includes or not polar cells.
    3. In the discussion line 240, this is not myosin II but myosin V which anchored oskar mRNA at the posterior.
    4. For the suppl figure 5, the n is not mentioned in the legend

    Significance

    Nature and significance of the advance and work in the context of existing literature

    This manuscript examines an important and unsolved question concerning the establishment of the polarity axis in the Drosophila oocyte, namely how the follicle cells located at the posterior of the egg chamber trigger a signal to the oocyte for its subsequent polarization (Gonzalez-Reyes et al ; Nature 1995 ;doi: 10.1038/375654a0) and (Roth et al; 1995; Cell; doi: 10.1016/0092-8674(95)90016-0). To address this question the authors, center their studies on the localization of PAR proteins which are distributed along the antero-posterior axis. They more specifically focus on the mutual exclusion of Par1 and Bazooka/Par3 (Baz) at the posterior of the oocyte. The signaling event from the posterior follicle cells toward the oocyte is an essential process however it remains unsolved despite numerous screening and genetic manipulation approaches, leaving open the possibility that classical signaling involving a diffusible ligand emitted by follicular cells with its receptor located at the plasma membrane of the oocyte, would not be applied here. We still know little about the modalities of this signaling between the follicular cells and the oocyte necessary for the polarization of the latter. We know that the first sign of anteroposterior polarization in the oocyte is posteriorly the recruitment of Par1 and subsequently the elimination of Baz. However, we do not know the nature of this signaling. Furthermore, we do not know whether this signaling must be maintained in order to maintain the polarization of the oocyte and more particularly to maintain the localization of oskar RNA, the posterior determinant of the oocyte, Here the authors are using original biophysical approaches to address whether signaling between the follicular cells and the oocyte would involve mechanical features. Important results of this work show that cell contacts between PFCs and the oocyte are necessary to maintain baz exclusion and Par1 localisation. Furthermore, the ablation results suggest that individual PFCs are required to maintain local posterior exclusion of Bazooka.

    Audience: These results will be of interest to those interested in the relationship between cell signaling and polarization in particular in a developmental context.

    Reviewer's area of expertise: Cell polarity, microtubule-associated transport, oocyte development in Drosophila.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The formation of mutually exclusive domains of Partition defective (Par) proteins works as a foundation for establishment of cell polarity in a variety of cells. The Drosophila oocyte is a well-known model system to study mechanisms of the asymmetric distribution Par proteins. At stage 6/7 of oogenesis, an unknown signal the posterior follicle cells (PFC) induces the recruitment of the Par-1 kinase to the posterior cortex of the oocyte and the concomitant exclusion of aPKC/Par-6 from this region. By contrast, Bazooka (Par-3) remains at the posterior with Par-1 and only disappears from the posterior at early stage 9. Millas et al. investigate the nature of the PFC signal and whether PFC continue to play a role in keeping Bazooka away from the posterior after the original signal is received by the oocyte. They do so by following the distribution of Bazooka and other Par proteins in living oocytes after pulling away or ablating the PFC at various stages of oogenesis.

    Major comments

    1. Quality of live imaging Judging from the appearance of the polar follicle cells and the size of the follicle cells, the authors constantly have an issue with maintaining a steady focal plane during live imaging in most movies (Figure 2 and video1; Figure 3 and video 2; Figure 4 and video 4; Figure 5 and video 5, FigureS5 and video 6). The conclusions of the paper are based on measuring changes in fluorescence intensity at the oocyte posterior over time, and this will be undermined by a varying focal plane. Considering the bullet shape of the oocyte, imaging the posterior at different focal planes could also cause artefacts. Supplementary Fig 3D-E and video 3 (a control experiment) are examples where the focal plane did not drift.
    2. Mechanical contact of PFC with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity By physically pulling PFC away from the oocyte at stage 10b (Figures 1-2) the authors observed that in some oocytes Bazooka re-localises to posterior and concluded that it is a mechanical contact between PFC and the oocyte cortex that keeps Bazooka away from the posterior. Although this is an interesting observation per se, this is after the polarity of the oocyte has been defined (stages 6-9) and the posterior determinant, oskar mRNA has been localised. Could the authors do the same experiment at stages 6-9 to directly address whether the distance between the PFC and the oocyte cortex actually matters, considering that Bazooka remains at the posterior up to early 9 when the PFC and the oocyte are still at close contact?

    The conclusion that the signal between PFC and the oocyte could be mechanical is only one of potential interpretations of the experiment. It still could be a short range/ non-diffusible biochemical signal that is sensitive to the distance between the PFC and the oocyte membrane. The authors do not provide any evidence for or against either interpretation.

    1. Figure 5B is supposed to demonstrate that local loss of Par-1 at the posterior causes the re-growth of microtubules from this region. However, the data provided are not convincing. The accumulation of red vesicles at the posterior cortex 150 min post ablation does not look like a specific signal for Jupiter-mCherry-marked microtubules. Similar vesicles start to be visible in the neighbouring follicle cells at the same time.

    Minor comments

    1. In Figure 4A-C, it is not clear what area has been ablated
    2. The authors should provide a simple 1-6 numbering for Video files

    Significance

    The observation that the PFC are required to maintain oocyte polarity at stage 9 is significant, but not very surprising, given the recent observation by Doerflinger et al that the posterior localisation of Par-1 requires continuous myosin activation, demonstrating that the antagonism between anterior and posterior Par proteins is not sufficient to maintain polarity once established. The authors must improve the quality of the live imaging to support this conclusion.

    The conclusion that phosphorylation of Bazooka by Par-1 is not sufficient to exclude Bazooka from the posterior cortex is not novel (see Doerflinger et al 2010, 2022).

  5. Version published to 10.1101/2022.03.03.482911v2 on bioRxiv
  6. Version published to 10.1101/2022.03.03.482911v1 on bioRxiv