Morphogenetic forces planar polarize LGN/Pins in the embryonic head during Drosophila gastrulation

  1. Jaclyn Camuglia
  2. Soline Chanet
  3. Adam C Martin

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Abstract

Spindle orientation is often achieved by a complex of Partner of Inscuteable (Pins)/LGN, Mushroom Body Defect (Mud)/Nuclear Mitotic Apparatus (NuMa), Gαi, and Dynein, which interacts with astral microtubules to rotate the spindle. Cortical Pins/LGN recruitment serves as a critical step in this process. Here, we identify Pins-mediated planar cell polarized divisions in several of the mitotic domains of the early Drosophila embryo. We found that neither planar cell polarity pathways nor planar polarized myosin localization determined division orientation; instead, our findings strongly suggest that Pins planar polarity and force generated from mesoderm invagination are important. Disrupting Pins polarity via overexpression of a myristoylated version of Pins caused randomized division angles. We found that disrupting forces through chemical inhibitors, depletion of an adherens junction protein, or blocking mesoderm invagination disrupted Pins planar polarity and spindle orientation. Furthermore, directional ablations that separated mesoderm from mitotic domains disrupted spindle orientation, suggesting that forces transmitted from mesoderm to mitotic domains can polarize Pins and orient division during gastrulation. To our knowledge, this is the first in vivo example where mechanical force has been shown to polarize Pins to mediate division orientation.

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  1. Version published to 10.7554/elife.78779 on eLife
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    Reply to the reviewers

    __We thank the reviewers for carefully reading our manuscript. We found their comments to be incredibly thoughtful and constructive and greatly appreciate their feedback. We are confident that addressing the reviewers’ concerns has strengthened our manuscript. __

    Reviewer #1 (Evidence, reproducibility and clarity (Required)): Camuglia, Chanet and Martin investigate the mechanisms that control cell division orientation in vivo, using the mitotic domains (MDs) in the head of the Drosophila embryo as their main model system. They find that cells in the head mitotic domains rotate and align their spindles within 30 degress of the anterior-posterior axis of the embryo. The Pins protein, implicated in spindle orientation in other systems, is planar polarized in mitotic cells. Pins polarization precedes spindle rotation and is correlated with the division angle (but cell shape is not, violating Hertwig's rule). Overexpression of myristoylated Pins results in uniform Pins distribution on the membrane and affects spindle orientation. alpha-catenin RNAi (but not canoe RNAi) disrupts Pins polarity and spindle orientation in MDs 1, 3 and 5. Low dose CytoD injections (which should disrupt force transmission) also result in defective Pins polarity and spindle orientations. Finally, mechanical isolation by laser ablation also disrupts spindle orienttion. The authors find that preventing mesoderm invagination by snail dsRNA disrupts Pins polarity and spindle orientation in the head. MAJOR 1. Is there a certain chirality in the rotation of the spindles? From Movie 1, it seems like in MDs 1 and 3 at least, a majority of spindles on the right side of the embryo rotate clockwise, while spindles on the left side rotate counter-clockwise? Is that so, and in that case, are there geometric/molecular considerations that could explain that chirality?

    We thank the reviewer for pointing this out. They are correct in that there is a tilt to the spindle orientation relative to the AP axis. To illustrate this tilt, we performed our spindle analysis separately on the right and left sides of MD1 and found that spindles on the left side align with an average division angle of about 30from the AP axis whereas spindles on the right side align with an average division angle of -30 __from the AP axis. To determine whether spindles on either side rotated with a certain chirality, we found there was no preference in rotating clockwise or counterclockwise on the left and right sides (on the left side of MD1 53% of measured spindles rotated counterclockwise and 47% rotated clockwise, on the right side 46% rotated counterclockwise and 54% clockwise). We have added this data as Fig. 1I-J and discussed in the Results lines 134-145. __

    1. The authors are experts in mesoderm invagination, and understandably concentrate on the role that forces from that process may have in the orientation of head MD divisions. However, the cephalic furrow forms much closer to the head MDs, and in an orientation that might also explain the alignment of spindles in the head. Is cephalic furrow formation important for Pins polarity and spindle orientation in the head MDs?

    __This was certainly a possibility, but our experimental results strongly argues that mesoderm invagination is most relevant. __

    __1) Perturbing the ventral furrow (e.g. by Snail depletion) does not block the cephalic furrow (Vincent et al., 1997; Leptin and Grunewald, 1990), but does block mesoderm invagination. Snail depletion strikingly disrupted spindle orientation and Pins localization, which suggests mesoderm is most important. __

    __2) In addition, depletion of -catenin blocks ventral furrow invagination but not cephalic furrow formation. We see a disruption in spindle orientation and Pins localization in -catenin RNAi, which suggests cephalic furrow itself cannot orient spindles. __

    __3) Furthermore, light sheet imaging of the Drosophila embryo has shown that the head region of the embryo undergoes tissue movement in the direction of the cell division and that this is associated with mesoderm invagination (Streichan et al., 2018; Stern et al., 2022). __

    __See movies here: https://www.youtube.com/watch?v=kC11Upr30JY __

    __To further test the importance of mesoderm invagination, we will perform additional ablation experiments trying to disrupt forces transmitted to the mitotic domains from distinct directions. Once we get this experimental result we will include language in the Discussion that will summarize the experimental results and the weight of the evidence for the roles of either ventral or cephalic furrow. __

    1. Does expression of myristoylated Pins affect mesoderm invagination (or cephalic furrow formation)? From Table S1 it seems that a maternal Gal4 driver was used to express myristoylated Pins, which could affect other tissues in the embryo. So it is in principle possible that effects of myristoylated Pins on mesoderm internalization/cephalic furrow formation could affect cell division orientation much like sna loss of function does, but in a mechanism that does not depend on Pins polarity. There is definitely an effect on mesoderm invagination in alpha-catenin RNAi (but not in canoe RNAi) embryos, so I wonder if the effect could be consistently through defects in mesoderm invagination (or cephalic furrow formation), and Pins polarity is really dispensable for spindle orientation. Are there head-specific Gal4 drivers that could be used to drive myristoylated Pins exclusively in the head?

    __We apologize that we did not clarify this in the text. Maternal overexpression of myr-Pins does not obviously disrupt mesoderm internalization/cephalic furrow formation. But, we do see that targeted disruption of mesoderm internalization via a Snail depletion affects the orientation of division. Note that our paper demonstrates the effect of force transmission on Pins polarity and division orientation, which is new and the main conclusion. The role of these divisions in morphogenesis is more complicated and is beyond the scope of this study. __

    __In response to this comment we: 1) added language in the Results that states that gastrulation proceeds in myr-Pins expressing embryos (lines 206-208), 2) Added to the Discussion of the role of these oriented divisions to morphogenesis (lines 443-449), and 3) will add a figure showing ventral furrow and cephalic furrow formation in embryos ectopically expressing the myr-Pins. __

    1. Related to the previous point, does mechanical isolation by laser ablation (Figure 6I-N) affect Pins polarity? This experiment could alleviate some of my concerns above, as it certainly does not (should not?) disrupt neither mesoderm invagination nor cephalic furrow formation.

    __We agree that it would be useful to look at Pins polarity in laser ablated embryos. Currently, we have been unable to analyze Pins polarity after laser ablation, because the ablation to fully isolate the mitotic domain has bleached our Pins::GFP signal. Also, we have shown that Pins polarity is disrupted by 1) alpha-catenin-RNAi, 2) low dose CytoD injection, and 3) Snail depletion, all of which are expected to disrupt force generation and transmission through tissues. __

    __In response to the reviewer comment, we will determine if Pins::GFP can be analyzed in less aggressive (directional) laser ablations. Again, remember that myr-Pins does not affect mesoderm internalization and that Snail depletion affects Pins polarity. __

    MINOR 1. Figure S5: I am a bit confused about the role of Toll 2, 6, 8 in orienting spindle orientation. In Figure S5D it seems that dsRNA treatment against these genes does not disrupt spindle orientation, but Figure S5F shows quite a significant (p=0.0057) effect in triple mutants. The authors favor the idea that Toll receptors do not affect spindle orientation, but the difference with the mutant should be addressed. Furthermore, what happens in MDs 3, 5 and 14 (if the germband extension defect does not affect those divisions)? Is there a difference between dsRNA and triple mutant embryos in these other MDs?

    __We think this is a great point. We stated in the text that TLRs are not solely responsible (line 247) for spindle orientation as they do not recapitulate the random pattern of division seen in the myr-Pins expression condition. We acknowledge the differences between the dsRNA injection and TLR triple mutant in the manuscript (lines 242-247), but our data show a greater importance for the role of force transmission. We favor the idea that other mechanisms contribute to spindle orientation because of the small effect of mutating all three Tolls and the dramatic effects of depleting AJs, inhibiting actin (with CytoD), laser ablation, and blocking mesoderm invagination. The planned laser ablation experiments (described above) will also contribute to addressing this point. __

    1. No statistical analysis is provided for any of the differences in polarity between Pins and Gap43, and this should be done to demonstrate the significance of the polarization of Pins. Also, particularly for MD14, they should compare anterior vs. posterior polarity, as based on the images in Figure 2H it is not clear that there is a difference between the anterior and posterior side of cells.

    __We thank the reviewer for this point. We have added the statistical comparison. __

    1. Figure 2A-D: the authors propose that Pins localizes preferentially to the posterior end of cells (instead of both anterior and posterior ends) in MDs 1, 3 and 14 (and anterior in MD 5). How is the asymmetry in the distribution of Pins along the AP axis accomplished, and is there any significance to it? This should be discussed in a bit more detail (currently no potential mechanisms provided in the discussion, just an acknowledgment of the question).

    __We agree the localization of Pins to the posterior end of cells in MDs 1, 3, and 14 and anterior end in MD 5 is of great interest. The details and further mechanism of this preferential localization are beyond the scope of this paper, but we have added an acknowledgment of the question and discuss possible models that could explain the result (lines 458-460). __TYPOS 1. Line 49: "one daughter cells" should be "one daughter cell". 2. Line 193: "rotation. (Figure 3E-F)." should be "rotation (Figure 3E-F)." 3. Lines 232-237: please review. 4. Line 238: "epithelia cells" should be "epithelial cells".

    __We thank the reviewers for carefully reading our manuscript. We have fixed the typos mentioned. __

    Reviewer #1 (Significance (Required)): This is the first study to my knowledge that demonstrates the role of mechanical forces in polarizing Pins, and provides a nice model to further investigate how mechanical forces generated in one tissue may affect cell division orientation in distant ones. The paper is clear, well written, and quantitative analysis is present for most results. I have some issues with the statistics (or lack thereof) for a couple of results, and potential alternative interpretations for some experiments that in my opinion should be addressed prior to publication. Specifically, it is not clear to me if Pins polarity is at all necessary for spindle orientation in any of the examined MDs.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): Overview: In this manuscript, Camuglia et al. show Pins/LGN, which is understood to drive spindle orientation, can localize asymmetrically (with respect to the tissue plane) in the Drosophila embryo. Experimental work (including drug treatments, laser ablation, and knockdowns) lead the authors to propose that this asymmetry is driven by tissue-level tension. The findings are quite interesting and the manuscript is well-written overall. Major Comments: • The authors propose that localization is driven by tissue-level tension, but the direction of the tension isn't clear from the experimental work. For example, the laser ablation experiments cut around the entire perimeter of the mitotic domain, rather than along just one tension axis. Similarly, the finding that disruption of the ventral furrow (by Snail RNAi) interferes with spindle orientation in the head is very puzzling; the furrow is A) outside the embryonic head and B) runs in the parallel direction to the divisions considered. The authors need to address the directionality of tension experimentally.

    __We thank the reviewer for this comment and agree that better defining the direction of tension would strengthen our manuscript. We showed that blocking mesoderm invagination with Snail depletion disrupts spindle orientation, despite Snail not being required for cephalic furrow formation (refs). Recent light sheet data has shown that mesoderm invagination is associated with global movements throughout the embryo. Furthermore, the ventral furrow extends into the head region just past the anterior of MD5. To address the reviewer’s comments, we plan to: 1) Perform directional laser ablations to determine the directionality of the tension that orients the spindle, 2) Analyze strain rates in the mitotic domains prior to and during division, and 3) Add to our Discussion more about what is said in the literature about the movements that occur in the head during mesoderm invagination. __

    • As acknowledged in the text, the asymmetric enrichment of Pins in MD14 is fairly weak. Since the cells being examined here border a divot in the tissue, and might therefore be curving relative to the focal plane, it would be good to rule out the possibility that some of the asymmetry in Pins intensity is just a consequence of cell/tissue geometry. One way this could be achieved is by showing multiple focal planes.

    __Good point. We do not think that the asymmetric Pins enrichment in MD14 is due to tissue geometry or junction tilt. 1) MD14 divides ~10-15 minutes after mesoderm invagination is completed, so the cells do not border a divot (as seen with Gap43::mCh, Fig. 2I). The cells do round up, which can be seen as gaps between cells (Fig. 3E). 2) We compare Pins to GapCh and only see an enrichment with Pins (Fig. 2H-K). If the enrichment was due to tissue curvature or junction orientation relative to imaging axis, we would see the same enrichment in GapCh. 3) Expression of myr-Pins randomizes spindle orientation in MD14 (Fig. 3M, N). __

    • In Figure 3I (and 3M?), it appears that there are fewer cell divisions in the presence of myr-Pins. Is this the case? Since cell shapes change during division, and cell shapes influence tissue tension, an increase in cell divisions could lead to a change in tissue tension. This would be important to address, since tissue tension plays an important role in the proposed model.

    __These images are not taken at the same point of MD1 division ‘wave’, there are the same number of divisions in each condition. These mitotic domains exhibit a ‘wave’ of cell division (Di Talia and Wieschaus, 2012), and so the number of divisions in each image reflect the timing at which we captured the image. Quantifications involved divisions throughout this wave, but we have chosen images for figures which are most representative of what we see. We will add this to the text in the final version of the manuscript. __

    • The alpha-catenin and Canoe results are a bit confusing: - The rose plot in Figure 4D doesn't show a random distribution of spindle angles, but rather a modest change; most spindles still orient in the normal range. The p value in the figure legend (0.0012) is very different from the one in the figure (5.8284e-04). - Alpha-catenin is the strongest way to disrupt AJs, but A) the epithelium appears to be intact in the knockdown condition and B) spindle orientation is impacted but not randomized. Does this mean that the knockdown is incomplete? Or is Cadherin-mediated adhesion (in which alpha-catenin participates) only partially responsible for force transduction?

    We acknowledge that perturbation using ____alpha-__cat RNAi does not recapitulate the complete disruption of division orientation seen in embryos expressing myr-Pins. This is likely due to the variability in the strength of RNAi knockdown, which is observed for most RNAi lines that we use. To address the reviewer’s comment, we have added rose plots for individual embryos showing extremes in the severity of division orientation disruption (Fig. 4E and F). For the main plot (Fig. 4D), we have included all the data that we took because we obviously did not want to pick and choose which embryos were used for analysis. So Fig. 4D includes all the variability. __

    • Given that previous studies implicate Canoe in Pins localization, it seems important to lock down the question of whether Canoe is participating in the mechanism described in this paper. How do the authors know the extent of Canoe knockdown? As suggested by the alpha-catenin results (described above), is it possible that Canoe knockdown is simply not strong enough to impact spindle orientation? Aren't there genetic nulls available? __We thank the reviewer for bringing these points to our attention. There are certainly genetic nulls available (Sawyer et al., 2009), but the experiment suggested by the reviewer would not establish the necessity of Canoe in mitotic domain cells. This is because Canoe nulls severely disrupt mesoderm invagination (Sawyer et al., 2009; Jodoin et al., 2015), as well as affecting junctions in the ectoderm during germband extension (Sawyer et al., 2011). Therefore, we would not be able to distinguish what effect of Canoe would be responsible for the spindle orientation using a null mutation. We did better experiments, we used 1) a mutant which specifically compromised mesoderm invagination (*snail), *2) laser isolation to show the importance of external force transmission in orienting mitotic domain divisions, and 3) RNAi to deplete Canoe so that mesoderm invagination initiates and pulls on the ectoderm, but where there is clearly compromised Canoe function. This treatment did not cause any effect on spindle orientation arguing against a role of Canoe in this case. In response to the reviewers comment, we added language to the Results to indicate that it is possible that the Canoe knockdown is not strong enough and our rationale for why we did not perform the experiment in a Canoe null (lines 279-282). __

    Minor Comments:

    • It can be difficult to interpret some of the spindle orientation data since the AP axis is vertical in the diagrams but horizontal in the rose plots. Can one of these be flipped so they go together?

    __We thank the reviewer for this suggestion and have flipped the rose plots so they match the images. Note that because of the large size of the figures, we have had to consistently orient anterior towards the top, which we establish at the beginning of the Results. __

    • Figure S3 is important information for the reader and should be ideally moved into the main paper. - Protein localizations referred to in text should be annotated on images, as they can be hard to see.

    __We disagree that S3 should be included in the main paper. The myr-Pins reagent has been used previously so the information in S3 is not new (Chanet et al., 2017). __

    • There are some discrepancies between figures, legends and text. - p-values differ between figures, legends, and/or text. - Fluorescent markers are labelled differently in figures and legend (CLIP170 in Figure 1) - Graphs appear to show that MD3 polarizes on posterior side, but figure legend says anterior in Figure S1. Vice versa for MD5.

    __We thank the reviewer for catching these typos. We have fixed these issues. __

    • Ideally, multichannel image overlays should be shown along with individual channels (b/w). However, it is appreciated that the fluorescent signals are exceptionally weak in this study, presenting a challenge to presentation and to quantification.

    __We agree the overlays would be nice. However, the Pins::GFP signal is weak compared to the tubulin and Gap43 signals, the merge does not provide more clarity, and the figures are already quite large. Therefore, we have only included the separated the images. __

    • Graph axes depicting spindle orientation would be more clear if shown in degrees, instead of normalized or in radians.

    __We thank the reviewer for this suggestion. We have changed the graph axes to be in degrees. __

    Reviewer #2 (Significance (Required)): Several recent studies have demonstrated that division orientation (in the tissue plane) is governed by tissue level tension. Remarkably, it appears that diverse mechanisms link tension with spindle orientation. Here the authors provide the first in vivo evidence connecting tension to the asymmetric localization of Pins, an important and evolutionarily conserved spindle orientation factor.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)): This beautiful manuscript uncovers a role for planar polarized PINS/LGN in orienting the mitotic spindle in Drosophila epithelia. In response to morphogenetic forces acting on adherens junctions, PINS/LGN localises to junctions in a planar polarized fashion to orient the spindle, and de-polarization of PINS/LGN prevents planar spindle orientation. The experiments are very well performed and the findings are robust. The conclusions are well supported by the data. Reviewer #3 (Significance (Required)): These important findings mirror previous work in human cell culture, but crucially reveal that the same phenomenon occurs in vivo in the Drosophila embryo. Thus, the findings underscore the highly conserved nature and in vivo relevance of this phenomenon.

    __We thank this reviewer for reading the manuscript and their encouraging words. __

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    Referee #3

    Evidence, reproducibility and clarity

    This beautiful manuscript uncovers a role for planar polarized PINS/LGN in orienting the mitotic spindle in Drosophila epithelia. In response to morphogenetic forces acting on adherens junctions, PINS/LGN localises to junctions in a planar polarized fashion to orient the spindle, and de-polarization of PINS/LGN prevents planar spindle orientation. The experiments are very well performed and the findings are robust. The conclusions are well supported by the data.

    Significance

    These important findings mirror previous work in human cell culture, but crucially reveal that the same phenomenon occurs in vivo in the Drosophila embryo. Thus, the findings underscore the highly conserved nature and in vivo relevance of this phenomenon.

    Referees cross-commenting

    this session contains comments of all reviewers

    Reviewer 2

    My biggest concern was that the direction of tension isn't obvious. I was particularly puzzled over the ventral furrow experiments, since I'm not clear on how that manipulation impacts the head. I agree with Reviewer #1 that it makes more sense to disrupt the cephalic furrow, but I'm not sure how to do that.

    Reviewer 1

    Agreed. I guess the question is whether there are cephalic furrow mutants in which mesoderm invagination is not affected. If so, those would be ideal.

    Reviewer 3

    Hi both. I understand your comments, but I felt that the direction of tension was apparent from the spindle orientation and the cell division axis itself. So, I wasn't concerned about using the snail mutant to prevent gastrulation and thus abolish forces generally.

    Reviewer 2

    I see. Well I certainly suspect that you and the authors are correct - and I'm enthusiastic about that! - but I'm concerned that using the direction of division to define the direction of tension is getting a little bit circular with the argument. I noticed that their ablation experiments aren't directional; instead they isolate the entire MD. Reviewer 1, as an expert in ablations, do you think it would make sense to make cuts that are only AP or DV?

    Reviewer 1

    I agree with Reviewer 2 about the circularity of the argument. I was going to propose AP vs DV cuts in sna mutants,with the idea that the wound healing response to those would pull in specific directions. My concern is that It won't be an effect of the same magnitude as the entire mesodermal placode going in, but maybe worth trying?

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    Referee #2

    Evidence, reproducibility and clarity

    Overview:

    In this manuscript, Camuglia et al. show Pins/LGN, which is understood to drive spindle orientation, can localize asymmetrically (with respect to the tissue plane) in the Drosophila embryo. Experimental work (including drug treatments, laser ablation, and knockdowns) lead the authors to propose that this asymmetry is driven by tissue-level tension. The findings are quite interesting and the manuscript is well-written overall.

    Major Comments:

    • The authors propose that localization is driven by tissue-level tension, but the direction of the tension isn't clear from the experimental work. For example, the laser ablation experiments cut around the entire perimeter of the mitotic domain, rather than along just one tension axis. Similarly, the finding that disruption of the ventral furrow (by Snail RNAi) interferes with spindle orientation in the head is very puzzling; the furrow is A) outside the embryonic head and B) runs in the parallel direction to the divisions considered. The authors need to address the directionality of tension experimentally.
    • As acknowledged in the text, the asymmetric enrichment of Pins in MD14 is fairly weak. Since the cells being examined here border a divot in the tissue, and might therefore be curving relative to the focal plane, it would be good to rule out the possibility that some of the asymmetry in Pins intensity is just a consequence of cell/tissue geometry. One way this could be achieved is by showing multiple focal planes.
    • In Figure 3I (and 3M?), it appears that there are fewer cell divisions in the presence of myr-Pins. Is this the case? Since cell shapes change during division, and cell shapes influence tissue tension, an increase in cell divisions could lead to a change in tissue tension. This would be important to address, since tissue tension plays an important role in the proposed model.
    • The alpha-catenin and Canoe results are a bit confusing:
      • The rose plot in Figure 4D doesn't show a random distribution of spindle angles, but rather a modest change; most spindles still orient in the normal range. The p value in the figure legend (0.0012) is very different from the one in the figure (5.8284e-04).
      • Alpha-catenin is the strongest way to disrupt AJs, but A) the epithelium appears to be intact in the knockdown condition and B) spindle orientation is impacted but not randomized. Does this mean that the knockdown is incomplete? Or is Cadherin-mediated adhesion (in which alpha-catenin participates) only partially responsible for force transduction?
      • Given that previous studies implicate Canoe in Pins localization, it seems important to lock down the question of whether Canoe is participating in the mechanism described in this paper. How do the authors know the extent of Canoe knockdown? As suggested by the alpha-catenin results (described above), is it possible that Canoe knockdown is simply not strong enough to impact spindle orientation? Aren't there genetic nulls available?

    Minor Comments:

    • It can be difficult to interpret some of the spindle orientation data since the AP axis is vertical in the diagrams but horizontal in the rose plots. Can one of these be flipped so they go together?
    • Figure S3 is important information for the reader and should be ideally moved into the main paper.
      • Protein localizations referred to in text should be annotated on images, as they can be hard to see.
    • There are some discrepancies between figures, legends and text.
      • p-values differ between figures, legends, and/or text.
      • Fluorescent markers are labelled differently in figures and legend (CLIP170 in Figure 1)
      • Graphs appear to show that MD3 polarizes on posterior side, but figure legend says anterior in Figure S1. Vice versa for MD5.
    • Ideally, multichannel image overlays should be shown along with individual channels (b/w). However, it is appreciated that the fluorescent signals are exceptionally weak in this study, presenting a challenge to presentation and to quantification.
    • Graph axes depicting spindle orientation would be more clear if shown in degrees, instead of normalized or in radians.

    Significance

    Several recent studies have demonstrated that division orientation (in the tissue plane) is governed by tissue level tension. Remarkably, it appears that diverse mechanisms link tension with spindle orientation. Here the authors provide the first in vivo evidence connecting tension to the asymmetric localization of Pins, an important and evolutionarily conserved spindle orientation factor.

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    Referee #1

    Evidence, reproducibility and clarity

    Camuglia, Chanet and Martin investigate the mechanisms that control cell division orientation in vivo, using the mitotic domains (MDs) in the head of the Drosophila embryo as their main model system. They find that cells in the head mitotic domains rotate and align their spindles within 30 degress of the anterior-posterior axis of the embryo. The Pins protein, implicated in spindle orientation in other systems, is planar polarized in mitotic cells. Pins polarization precedes spindle rotation and is correlated with the division angle (but cell shape is not, violating Hertwig's rule). Overexpression of myristoylated Pins results in uniform Pins distribution on the membrane and affects spindle orientation. alpha-catenin RNAi (but not canoe RNAi) disrupts Pins polarity and spindle orientation in MDs 1, 3 and 5. Low dose CytoD injections (which should disrupt force transmission) also result in defective Pins polarity and spindle orientations. Finally, mechanical isolation by laser ablation also disrupts spindle orienttion. The authors find that preventing mesoderm invagination by snail dsRNA disrupts Pins polarity and spindle orientation in the head.

    Major

    1. Is there a certain chirality in the rotation of the spindles? From Movie 1, it seems like in MDs 1 and 3 at least, a majority of spindles on the right side of the embryo rotate clockwise, while spindles on the left side rotate counter-clockwise? Is that so, and in that case, are there geometric/molecular considerations that could explain that chirality?
    2. The authors are experts in mesoderm invagination, and understandably concentrate on the role that forces from that process may have in the orientation of head MD divisions. However, the cephalic furrow forms much closer to the head MDs, and in an orientation that might also explain the alignment of spindles in the head. Is cephalic furrow formation important for Pins polarity and spindle orientation in the head MDs?
    3. Does expression of myristoylated Pins afect mesoderm invagination (or cephalic furrow formation)? From Table S1 it seems that a maternal Gal4 driver was used to express myristoylated Pins, which could affect other tissues in the embryo. So it is in principle possible that effects of myristoylated Pins on mesoderm internalization/cephalic furrow formation could affect cell division orientation much like sna loss of function does, but in a mechanism that does not depend on Pins polarity. There is definitely an effect on mesoderm invagination in alpha-catenin RNAi (but not in canoe RNAi) embryos, so I wonder if the effect could be consistently through defects in mesoderm invagination (or cephalic furrow formation), and Pins polarity is really dispensable for spindle orientation. Are there head-specific Gal4 drivers that could be used to drive myristoylated Pins exclusively in the head?
    4. Related to the previous point, does mechanical isolation by laser ablation (Figure 6I-N) affect Pins polarity? This experiment could alleviate some of my concerns above, as it certainly does not (should not?) disrupt neither mesoderm invagination nor cephalic furrow formation.

    Minor

    1. Figure S5: I am a bit confused about the role of Toll 2, 6, 8 in orienting spindle orientation. In Figure S5D it seems that dsRNA treatment against these genes does not disrupt spindle orientation, but Figure S5F shows quite a significant (p=0.0057) effect in triple mutants. The authors favor the idea that Toll receptors do not affect spindle orientation, but the difference with the mutant should be addressed. Furthermore, what happens in MDs 3, 5 and 14 (if the germband extension defect does not affect those divisions)? Is there a difference between dsRNA and triple mutant embryos in these other MDs?
    2. No statistical analysis is provided for any of the differences in polarity between Pins and Gap43, and this should be done to demonstrate the significance of the polarization of Pins. Also, particularly for MD14, they should compare anterior vs. posterior polarity, as based on the images in Figure 2H it is not clear that there is a difference between the anterior and posterior side of cells.
    3. Figure 2A-D: the authors propose that Pins localizes preferentially to the posterior end of cells (instead of both anterior and posterior ends) in MDs 1, 3 and 14 (and anterior in MD 5). How is the asymmetry in the distribution of Pins along the AP axis accomplished, and is there any significance to it? This should be discussed in a bit more detail (currently no potential mechanisms provided in the discussion, just an acknowledgment of the question).

    Typos

    1. Line 49: "one daughter cells" should be "one daughter cell".
    2. Line 193: "rotation. (Figure 3E-F)." should be "rotation (Figure 3E-F)."
    3. Lines 232-237: please review.
    4. Line 238: "epithelia cells" should be "epithelial cells".

    Significance

    This is the first study to my knowledge that demonstrates the role of mechanical forces in polarizing Pins, and provides a nice model to further investigate how mechanical forces generated in one tissue may affect cell division orientation in distant ones. The paper is clear, well written, and quantitative analysis is present for most results. I have some issues with the statistics (or lack thereof) for a couple of results, and potential alternative interpretations for some experiments that in my opinion should be addressed prior to publication. Specifically, it is not clear to me if Pins polarity is at all necessary for spindle orientation in any of the examined MDs.

  6. Version published to 10.1101/2022.01.07.475359 on bioRxiv