Abstract
Left and right symmetry breaking is involved in many developmental processes that are important to form bodies and organs. One of them is the epithelial rotation of developing organs. However, how epithelial cells move, how they break symmetry to define common direction of their collective movement and what function rotational epithelial motions have in morphogenesis remain elusive. Here, we identified a dynamic actomyosin network with preferred retrograde contractility at the basal side of the rotating follicle epithelium in Drosophila oogenesis. We provide evidence that unidirectional epithelial rotation is a result of actomyosin asymmetry cue transmission onto a tissue plane synchronized by the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis. We found that Fat2 directs actomyosin contractility to move the epithelial tissue in order to provide directed elongation of follicle cells. In contrast, loss of Fat2 results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and deform cell shape, tissue and Drosophila eggs. Our data indicate that directed elongation of follicle cells is critical for proper Drosophila egg morphogenesis. Together, we demonstrate the importance of atypical cadherins in the control of cell mechanics, left/right symmetry breaking and its propagation onto the tissue scale to facilitate proper organ morphogenesis. This process may be evolutionarily conserved in rotating animal organs.
Functional organ morphogenesis1-3 has been linked to left and right (LR) turns and rotations of epithelial sheets4,5,6,7,8,9,10 relative to the organ or body anterior-posterior (AP) axis. A primary determinant of this LR chirality has been associated with the cytoskeleton in different species11,12,13,14,15. In rotating Drosophila organs such as hindgut10 and male genitalia6, the consistent handedness of epithelial rotation depends on myosinID (myoID) and utilizes asymmetric cellular intercalations. Completely different rotational movement was recently identified in the Drosophila ovary8, where organ-like structures called egg chambers display rotation of an edgeless, monolayered follicle epithelium together with underlying germline cells (called nurse cells and the oocyte), which rotate through the surrounding rigid extracellular matrix (ECM)8 (Figure 1a). In contrast to the Drosophila hindgut and male genitalia where cell membranes adopt a specific form of asymmetry called planar cell chirality (PCC), the follicle epithelium displays no membrane PCC (Extended Data Fig. 1a, Online Methods) and different egg chamber units in one animal can rotate clockwise or anti-clockwise performing more than three full rotations around their AP axis during early and mid oogenesis8,16. This suggests that an alternative, possibly myoID-independent, mechanism drives this collective cell behaviour. Interestingly, the basal side of each follicle cell displays clear local chirality of actin-rich protrusions and chiral localization of several planar cell polarity (PCP) molecules genetically implicated in egg chamber rotation17-20,9,21,22,23. Epithelial rotation is initially slow during early oogenesis (stages 1-5: average speed ~ 0.2 μm/min)19 accelerates in mid oogenesis (stages 6-8: average speed ~ 0.5-0.6 μm/min)8,9,19,24 and stops at stage9, 8. It has been shown that microtubules (MTs) predict the direction of epithelial rotation in early and mid-oogenesis and their global alignment is regulated by the atypical cadherin Fat225,24. Fat2 is a key PCP regulator of the actin cytoskeleton25, basement membrane components26,20 and its function is required for epithelial rotation and elongation of Drosophila egg chambers25,9. The Fat2 asymmetric planar polarized pattern on the basal lagging-membrane side of a follicle cell depends on MTs during fast epithelial rotation9. There is no evidence that MTs represent the active force-generating mechanism that drives epithelial rotation, which has been recently shown to involve the actin-rich protrusions19,23. However, non-muscle myosin II (Myo-II) that generally provides contractility and force generation to actin cytoskeleton is missing on actin-rich protrusions19. Therefore, motivated by the observation that pharmacological depletion of non-muscle myosin II (Myo-II) leads to no epithelial rotation9, we hypothesized that the basal actin filaments that contain Myo-II are better candidates to fulfill the force generating function. To test this hypothesis, we investigated the function of Myo-II, its connection to the PCP pathway in Drosophila epithelial rotation and the role of their interplay in egg chamber morphogenesis.
RESULTS
Highly dynamic Myo-II behaviour at the basal side of the follicle epithelium
In order to understand Myo-II function in epithelial rotation, we first investigated the behaviour of the Myo-II regulatory light chain (MRLC, called Spaghetti Squash, sqh in Drosophila) fused to GFP (MRLC::GFP) in a null sqhAX3 mutant27 at the basal cortex of the follicle epithelium using ex vivo live imaging. We analyzed Myo-II behaviour in three different situations: slow (stage 4), fast (stage 7) and no epithelial rotation (stage 7 of fat258D/103C mutants in a null sqhAX3 background since fat2 mutant egg chambers display no epithelial rotation9,19) (Extended Data Fig. 1b). High-speed confocal live imaging of MRLC::GFP uncovered a very dynamic pattern of Myo-II in a thin layer (≤1000 nm) at the basal cortex of the follicle epithelium during slow, fast and no rotation (Figure 1b and Movies 1, 2, 3). We distinguished individual MRLC::GFP dot-like signals with an average size of 363 nm ± 0.05 nm (n = 136) and an average speed of 2.12 μm/min ± 0.8 (n = 101) for slow, 2.44 μm/min ± 0.96 (n = 105) for fast and 1.99 μm/min ± 0.62 (n = 100) for no epithelial rotation. This speed was consistent with the speed of anterograde flow of actomyosin during zebrafish gastrulation28. We also observed large intense MLRC::GFP dots (1.01um ± 0.14 um, n= 50, Figure 1b and Movie 2) close to the lagging end of migrating follicle cells, which were lost in the fat2 mutant follicle epithelium (Figure 1b and Movie 3), suggesting an unknown function in epithelial rotation. Taken together, we discovered highly dynamic behaviour of Myo-II at the basal cortex of the Drosophila follicle epithelium.
Global actomyosin retrograde movement is regulated by atypical cadherin Fat2 in the follicle epithelium
To find out whether the small (~360nm) MRLC::GFP dots moved in a specific direction with respect to the egg chamber axis, we quantified directions of MRLC::GFP movement expressed as angles ranging from 0° to 360° where 0° represented the anterior and 180° the posterior of egg chambers (Extended Data Fig. 1c and Online Methods). In contrast to the situation with no epithelial rotation, which showed no clear preference in direction of MRLC::GFP movement (Figure 1b), we observed that during slow rotation, MRLC::GFP showed weak preferred movement perpendicularly to the AP axis of egg chambers (Figure 1b) that was strongly reinforced during fast rotation (Figure 1b and Extended Data Fig. 1d). Similarly, labeling actin filaments with a LifeAct29 molecule fused to GFP (LifeAct::GFP) showed strong preference in LifeAct::GFP movement perpendicularly to the AP axis of egg chamber during fast rotation (Extended Data Fig. 1d). This preference of small MRLC::GFP dots to move perpendicularly to the AP axis of egg chambers as well as the loss of their preferred direction in fat2 mutant egg chambers observed in live imaging was corroborated by the analysis of MRLC::GFP signal in fixed wild type and fat2 mutant egg chambers during early and mid-oogenesis (Extended Data Fig. 2).
Having established the global trend of Myo-II movement, we next asked whether individual MRLC::GFP dots moved randomly along their preferred direction (perpendicular to the AP axis of egg chambers) during slow and fast epithelial rotation. To this end, we calculated how frequently MRLC::GFP dots moved within defined angle range of four (90 degrees) quadrants: Anterior (315°≤45°), Up (45°≤135°), Posterior (135°≤225°) and Down (225°≤315°). The data revealed preferred movement within Up and Down quadrants and an asymmetry in movement of MRLC::GFP dots within these quadrants in individual egg chambers (Figure 1b). This asymmetry was initially small during slow rotation and became prominent during fast rotation. In contrast, rather weak to no asymmetry has been detected in fat2 mutant egg chambers (Figure 1b).
Next we asked in what way this MRLC::GFP movement asymmetry relates to the direction of epithelial rotation. In order to define the average percentage of MRLC::GFP dots moving with or against epithelial rotation, we unified the direction of epithelial rotations in the direction Up for all analyzed egg chambers and detected that on average 59% and 77% MRLC::GFP dots moved against epithelial rotation during slow and fast rotation, respectively (Figure 1c and Extended Data Fig. 3a). This was not true for the fat2 mutant egg chambers (Figure 1c and Extended Data Fig. 3a), where no preferred global direction was identified in individual egg chambers. We also observed that actin molecules preferably moved (78%) against fast epithelial rotation, based on LifeAct-GFP, and this movement was comparable to the MRLC::GFP movement during fast rotation (Extended Data Fig. 3b and Figure 1c). Thus, we discovered that MRLC::GFP dots preferred to move against epithelial rotation during slow and fast rotation.
Contractile actomyosin rings, Myo-II pulses and Myo-II asymmetric localization on the cell membrane represent features that have been previously linked to tissue movements during epithelial morphogenesis28,30,31,6. However, we observed neither of these, indicating that known mechanisms of actomyosin mediated collective epithelial movement do not play a role in this system.
Taken together, our results revealed novel temporally regulated and spatially coordinated Fat2-depenent actomyosin movement at the basal side of the follicle epithelium in the direction opposite to epithelial rotation (retrograde movement).
Fat2 regulates actomyosin retrograde movement in individual follicle cells
Next, we sought to uncover how Fat2 coordinates retrograde Myo-II movement to break the Myo-II symmetry in the follicle epithelium in order to define the direction of epithelial rotation around the AP axis of egg chambers and how it increases Myo-II retrograde movement during the onset of fast epithelial rotation (Figure 1c). We have shown here that Fat2 regulates global alignment of Myo-II perpendicularly to the AP axis of egg chambers based on live imaging and fixed tissue (Figure 1 and Extended Data Fig. 2). We additionally observed that Fat2 is not required for local (intracellular) alignment of MRLC::GFP in individual follicle cells of fixed egg chambers during slow and fast epithelial rotation (Extended Data Fig. 2). Based on this, we hypothesized that artificial global alignment of individual fat2 mutant follicle cells perpendicularly to the AP axis of egg chambers should be in principle sufficient to break the symmetry in the follicle epithelium. To simulate such situation, we developed a computational angular correction approach (Figure 2a and Online Methods), in which we assumed that such epithelial remodeling (either by dissolving/re-establishment of adherens junctions or cell intrinsic reorientation of actomyosin cytoskeleton) happens in the wild-type situation with minimal movement of the respective components (i.e. cells or cytoskeleton would rotate 60 rather than 120 degrees to align). To mimic this, individual fat2 mutant follicle cells were angularly corrected for their main MRLC::GFP direction within the smallest possible angle (i.e. ≤ 90°) to reach perpendicular alignment to the AP axis of the individual egg chambers (Figure 2a and Online Methods).
When angular correction was applied to our data obtained from live imaging, MRLC::GFP dots tended to move perpendicularly to the AP axis in the fat2 mutant follicle epithelia of stage 7 (Figure 2a), however, only to the extent observed in the angularly corrected control stage 4 (slow epithelial rotation) and far less when compared to the original control stage 7 (fast epithelial rotation, no angular correction in Figure 1b). This indicated an additional Fat2-dependent mechanism that is required to increase number of MRLC::GFP dots moving perpendicularly to the AP axis of egg chambers. In addition, as fixed follicle cells at control stage 4 did not display proper perpendicular alignment of the MRLC::GFP pattern to the AP axis of egg chambers (Extended Data Fig. 2), we expected an increase in the movement of MRLC::GFP dots perpendicularly to the AP axis of living egg chambers after the angular correction, however, instead we observed that angular corrected control stage 4 did not change (Figure 2a) compared to the original situation (Figure 1b), indicating that the movement of MRLC::GFP dots needed to be additionally refined with the onset of fast epithelial rotation.
Taken together, this analysis revealed that in addition to the requirement of Fat2 to globally align Myo-II of follicle cells perpendicularly to the AP axis of egg chambers, Fat2 is also required to refine Myo-II alignment and reinforce this directed movement of Myo-II in the follicle epithelium to reach fast epithelial rotation (compare to Figure 1b).
To find out whether the global artificial alignment of fat2 mutant follicle cells is sufficient to break the Myo-II symmetry in the follicle epithelium, we further calculated frequency of MRLC::GFP dots moving in the preferred directions (quadrants Up and Down) in individual egg chambers (Figure 2b and Online Methods). We found that five out of ten egg chambers would clearly break Myo-II symmetry similarly to the control stage 4, three displayed only slight asymmetry and two remained symmetrical (Figure 2a, b), suggesting that global alignment of Myo-II alone could in theory lead to the Myo-II symmetry breaking in the follicle epithelium, however, to reach the Myo-II asymmetry comparable to the control stage 4, an additional Fat2-dependent mechanism was required in ~50% of the egg chambers.
To further understand why only 50% of egg chamber clearly broke Myo-II symmetry after applying the angular correction (Figure 2b), we analyzed behaviour of MRLC::GFP dots in individual follicle cells of control and fat2 mutant egg chambers. To this end, we plotted the direction of MRLC::GFP dots as the weighted ratio of those moving against epithelial rotation (retrograde MRLC::GFP dots) versus those moving with it (anterograde MRLC::GFP dots) (Figure 2c). The analysis revealed that individual follicle cells displayed different ratios of retrograde/anterograde MRLC::GFP dots, but on average during slow rotation with the magnitude around 1-2 while during fast rotation with the significantly increased magnitude of 3 or more (Figure 2d and Extended Data Fig. 4a,b). Notably, we identified one or more follicle cells of individual egg chambers with symmetric MRLC::GFP movement or with anterograde direction during slow rotation. This was never the case during fast rotation when all egg chambers displayed only follicle cells with retrograde MRLC::GFP movement (Figure 2c). Moreover, we observed the same, exclusively retrograde movement with LifeAct::GFP in individual follicle cells during fast rotation (Extended Data Fig. 4c,d), indicating that MRLC::GFP movement reliably reflects LifeAct::GFP behaviour. Coming back to our question why only 50% of egg chambers clearly broke the Myo-II symmetry, we found that in the angularly corrected fat2 mutant follicle cells the magnitude of the retrograde/anterograde ratio resembled those found during slow rotation. Importantly, the fat2 mutant egg chambers with no clear Myo-II asymmetry (Figure 2b) contained one or more follicle cells with strong preferred anterograde MRLC::GFP movement (Figure 2c) and that occurrence was on average more frequent in fat2 mutant egg chambers than in the control ones during slow rotation (Figure 2d).
Thus, this data uncovered that next to global Fat2 function to align actomyosin cytoskeleton perpendicularly to the AP axis of egg chambers and to define preferred direction of actomyosin contractility by breaking the Myo-II symmetry, Fat2 functions also within individual follicle cells to reorient the anterograde Myo-II movement and to refine and reinforce retrograde Myo-II in individual follicle cells to promote fast epithelial rotation (Figure 2e). In addition, we point out that the weak Myo-II asymmetries detected in the fat2 mutant follicle cells (Figure 2c) indicate existence of an alternate, initially Fat2-independent mechanism of the Myo-II symmetry breaking in the follicle cells.
Altogether, our data provide evidence that Fat2 propagates the initial, intracellular actomyosin-based asymmetry onto the tissue level to facilitate epithelial rotation of Drosophila egg chambers.
Fat2-dependent epithelial rotation directs elongation of follicle cells
Because loss of Fat2 leads to static and round egg chambers9, we next wished to understand what role Fat2-dependent epithelial rotation plays in egg chamber morphogenesis. Epithelial rotation has been clearly linked to proper PCP of basement membrane and actin filaments, which is required for egg chamber elongation along the AP axis18,19. In addition, integrin-based adhesions to ECM can modulate speed of epithelial rotation and impact shape/stretching of follicle cells18. Thus, we wondered whether epithelial rotation could define the shape of follicle cells and so measured their roundness (Online Methods). Indeed, we found that follicle cells are on average significantly more elongated during fast rotation than follicle cells in static fat2 mutant egg chambers and less elongated during slow rotation (Figure 3a). We also pharmacologically perturbed epithelial rotation by using actin-depleting drug (Latrunculin A) and adhesion-depleting drug (CK-666, depleting Arp2/3 complex) that have been shown to stop epithelial rotation9,19. Both experiments resulted in static follicle cells and similar round shape of fat2 mutant follicle cells (Figure 3a and Extended Data Fig. 5a), indicating that epithelial rotation is required for elongation of follicle cells.
Next, we wished to know whether epithelial rotation could define the direction of elongation of follicle cells. We therefore quantified the alignment of cell elongation expressed as angular direction in 20 degree bins through a range of 0°-180° during slow, fast and no epithelial rotation. We found that follicle cells elongated mainly perpendicularly to the AP axis of egg chambers during fast epithelial rotation, which was weaker during slow rotation (Figure 3b). To understand how the elongation of follicle cells relates to the global alignment of Myo-II (the predicted active force generator together with actin in this tissue) in individual cells, we calculated the relative angle between the global alignment of follicle cell elongation and the MRLC::GFP pattern (Online Methods). We revealed that MRLC::GFP followed follicle cell elongation in 65% during slow epithelial rotation and increased to 100% during fast epithelial rotation (Figure 3b). Interestingly, the global alignment of MRLC::GFP pattern preceded the one of follicle cell elongation during slow epithelial rotation, indicating that epithelial movement prefigures elongation of follicle cells. In contrast, fat2 mutant follicle cells rather displayed random global alignment of their elongation, the MRLC::GFP pattern and their relative angle.
This data show that Fat2-dependent epithelial rotation is required for proper elongation of follicle cells and the global alignment of their elongation in the direction of epithelial rotation (henceforth called directed elongation). This explains why it does not effectively matter whether egg chambers rotate clockwise or anti-clockwise as long as the epithelial rotation is around the AP axis of egg chambers. In addition, supported by the recent findings that shape of follicle cells can determine cellular alignment of MTs in Drosophila oogenesis32, we further speculate that such directed elongation leads to local and global alignment of MTs at the basal side of follicle cells. Not surprisingly, static egg chambers do not align MTs9,26.
Fat2 suppresses anisotropic and premature Myo-II pulses
We next asked what role directed elongation plays in egg chamber morphogenesis. Interestingly, when we analyzed individual fat2 mutant follicle cells, besides weak Myo-II asymmetries and rounder cell phenotype, we also observed spatially unequal (anisotropic) MRLC::GFP pulses (Figure 4a, b, c and Extended Data Fig. 5b) with constant remodeling and deformation of cellular membranes, impacting area size (Figure 4d and Extended Data 5b). This pulsatile behaviour was missing in the corresponding control (stage 7) during fast epithelial rotation (Figure 4a). This membrane deformation resulted in significant basal area contractions in fat2 mutant follicle cells compared to the corresponding control (Figure 4d). However, average area stayed unchanged, indicating that these area contractions were asynchronous among neighboring fat2 mutant follicle cells (Figure 4d). We observed that the reduction in basal area followed ~6s after the increase of MRLC::GFP (Figure 4e) based on a cross-correlation coefficient ((Figure 4f and Online Methods). Thus, our data provide evidence that Fat2 suppresses anisotropic Myo-II pulses and cellular membrane contractions/relaxations to prevent cell and tissue deformations (Figure 4g). This quantification of live imaging data also extends our previous observation of basally deformed follicle cells in the fixed follicle epithelium of fat2 mutant egg chambers25. Notably, similar physiological Myo-II pulses have been observed during mid-late Drosophila oogenesis (starting stage 9 with the peak at stage 10), which have been shown to properly elongate follicle cells and whole egg chambers along their AP axis33.
In summary, we propose that Fat2 synchronizes the initial default LR Myo-II asymmetry in one direction in the plane (the global Fat2 function), reorients and reinforces actin cytoskeleton within follicle cells (the local Fat2 function), resulting in transmission of actomyosin-based LR asymmetry onto tissue scale. This facilitates epithelial rotation, directed elongation of follicle cells and cell-shape-dependent local and global alignment of MTs. As MTs have been reported to be relevant for turn-over of the Fat2 (zig-zag) asymmetric pattern during fast epithelial rotation9, we further hypothesize that Fat2 uses epithelial rotation to align MTs for establishment of its planar polarized asymmetric pattern in a positive-feedback amplification mechanism9. This mechanism thus provides a robust Fat2-MTs-dependent platform that guarantees stable global and local alignment of actomyosin and MTs cytoskeleton during fast epithelial rotation (stage 6-8), which upon its establishment and maintenance9 likely becomes independent of epithelial rotation as observed in19. We envision that this is further critically required for globally aligned, physiological Myo-II pulses at the basal side of follicle cells in order to properly elongate follicle cells and egg chambers along their AP axis in later Drosophila oogenesis (Figure 5).
Discussion
One of the best understood processes that involves actomyosin flow in LR symmetry breaking is single and four cell C. elegans embryo15, where active torque generates force that leads to LR symmetry breaking and embryo asymmetry. Another example, where actomyosin anterograde and retrograde flow are involved, is the yolk cell during spreading of the enveloping cell layer over it by a process that involves unidirectional movement of strong actomyosin contractile ring in zebrafish embryo28. Besides invertebrate and vertebrate embryos2, single migrating cells also need to break symmetry to polarize in order to define direction of their motion. For example, actomyosin retrograde flow has been found in non-adhesive cells, allowing pushing of the cell mass forward34. In contrast, classical Myosin II-driven actin retrograde flow on the leading edge is used in_single pulling (ECM-adhesive) cells as originally found in neuronal growth cones35, however, little is known about dynamics of central stress fibers that retract the majority of the cell mass in direction of cell migration. Here, we identified preferred retrograde Myo-II movement on the basal actin stress-like fibers in ECM-adhesive follicle cells of the rotating Drosophila follicle epithelium. We therefore propose directed basal actomyosin contractility a candidate force-generating mechanism that actively moves the cell mass of follicle cells in collective manner and drives epithelial rotation of Drosophila egg chambers.
Little is known about the mechanistic control of LR symmetry breaking in tissue morphogenesis. It has been only recently discovered that atypical cadherins can act via force generating molecules such as unconventional myosins Dachs and MyoID36,37. Our data show that another atypical cadherin Fat2 can control LR symmetry breaking by regulation of cell mechanics via conventional non-muscle Myo-II. It appears that the atypical cadherin subfamily likely developed a prominent function to shape tissues in two ways dependent on tissue character: i) in migratory tissue via retrograde Myo-II movement and ECM-dependent mechanism (Fat2-Myo-II in Drosophila egg chambers in this work) and ii) in moving but non-ECM-migratory tissue via intercalations (Dachsous-MyoID in the Drosophila hindgut37, Fat-Dachsous-Dachs in the Drosophila wing38). As Fat2 close homologs, namely Fat1-3, exist in vertebrates39, it is likely that similar conserved mechanisms are used to move and sculpt tissues and organs in vertebrates.
AUTHOR CONTRIBUTIONS
I.V. made the observation of retrograde movement and pulsed contractility, planned the project and performed all experiments. I.V. analyzed all data. I.V. and I.H. analyzed the pulse contractility data together and I. H. helped with the R scripts for data analysis. P.T. generously supported this work. I.V. wrote the manuscript and created figures with help from P.T. and I.H. All authors commented on the manuscript.
AUTHOR INFORMATION
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to viktorin{at}mpi.cbg.de and tomancak{at}mpi-cbg.de.
MOVIES
Movie 1
Time-lapse movie of MRLC::GFP (green) signals moving at the basal cortex of young slowly rotating egg chamber (control stage 4). Membrane marker stains cell outlines (red). Frame interval = 6s. Scale bar = 5 μm. Anterior is on the left.
Movie 2
Time-lapse movie of MRLC::GFP (green) signals moving at the basal cortex of mid-oogenesis fast rotating egg chamber (control stage 7). Membrane marker stains cell outlines (red). Note the MRLC::GFP directed movement that is perpendicularly to the AP axis of the egg chamber. Preferred direction against the epithelial rotation was revealed only upon an angular quantification (see Online Methods and Figure 1 and Figure 2). Large MRLC::GFP dots always position towards the lagging side of follicle cells. Frame interval = 6s. Scale bar = 5 μm. Anterior is on the left.
Movie 3
Time-lapse movie of MRLC::GFP (green) signals moving at the basal cortex of the static fat258D/103C mutant mid-oogenesis egg chamber (stage 7). Membrane marker stains cell outlines (red). Note that MRLC::GFP is locally (within a cell) polarized (directed subcellular movement shown in Figure 2) but its global polarity is lost (Figure 1). Large MRLC::GFP dots were also lost. Cell outlines display deformations linked to MRLC::GFP increased intensity (Figure 4). Frame interval = 6s. Scale bar = 5 μm. Anterior is on the left.
Movie 4
Time-lapse movie of MRLC::GFP (green) signals at the basal cortex of static, actin-depleted (Latrunculin A) mid-oogenesis egg chamber (control stage 7). Membrane marker stains cell outlines (red). Note that MRLC::GFP movement is ceased and MRLC::GFP large dots affected in their position. Frame interval = 6s. Scale bar = 5 μm. Anterior is on the left.
Movie 5
Time-lapse movie of MRLC::GFP (green) signals at the basal cortex of CK666 (ARrp2/3 inhibitor) treated mid-oogenesis egg chamber of stage 7, which staled the epithelial rotation. Membrane marker stains cell outlines (red). Note that MRLC::GFP signal movement centers as well as MRLC::GFP large dots cross-link each other and position towards the center of follicle cells. Frame interval = 6s. Scale bar = 5 μm. Anterior is on the left.
Movie 6
Time-lapse movie of Act5Gal4>UAS-LifeAct::GFP signals at the basal cortex of control stage 7-8. Direction of the epithelial rotation is downwards. Note that the driver used allowed patched expression of LifeAct::GFP to identify front (leading edge) actin protrusions of follicle cells. Frame interval = 6s. Scale bar = 5 μm. Anterior is on the left.
ONLINE METHODS
Fly stocks and genetics
Drosophila MRLC (myosin regulatory light chain of the non-muscle conventional Myosin II), encoded by spaghetti-squash (sqh), was visualized by MRLC fused with eGFP27 under the sqh promoter in a null sqhAX3 or sqhAX3/sqhAX3;;fat258D/fat2103C mutant background to avoid competition with the endogenous protein. The following stocks: sqhAX3/ sqhAX3; sqh-MRLC::GFP/ sqh-MRLC::GFP (on the II. chromosome) and newly established sqhAX3/sqhAX3;sqh-MRLC::GFP/ sqh-MRLC::GFP; fat258D/fat2103C, were used in all figures except of Extended Data Figure 2, where sqh-MRLC::GFP/ sqh-MRLC::GFP; fat258D/fat2103C line was used for fat2 mutant egg chambers. To visualize actin filaments by time-lapse life imaging, we used Act5C-Gal4 (BL4414, on II. chromosome)>UAS-LifeAct::GFP (on II. chromosome) shown in Movie 6. To label cell membranes we used CellMask™ Deep Red (Invitrogen).
Time lapse imaging
Egg chambers were cultured and life imaging performed as described9. An inverted LSM 700 Zeiss confocal microscope was used with 63x/1.45 water immersion lens. Time-lapse movies were taken with an interval of 6s for 300s-600s.
Fixation and immunohistochemistry
Adult fly ovaries were dissected in 1×PBS and fixed with 4% p-formaldehyde for 20minutes. Immunostaining followed standard protocols. We used polyclonal GFP tag antibody conjugated with Alexa Fluor 488 (Molecular Probes) in dilution of 1:100 and rhodamine-phalloidin in dilution of 1:200. Images were acquired on inverted LSM700 Zeiss confocal microscope with 63x/1.45 oil immersion lens.
Drug treatments
To inhibit polymerization of actin filaments, Latrunculin A (10 μΜ in 1 % DMSO, Enzo Life Sciences) was used for ca. 10mins before direct imaging. To deplete actin protrusions and follicle cell adhesion to the ECM, we used Arp2/3 inhibitor (CK-666, 250 μΜ, Sigma) for ca. 1h as described19.
Image processing, data analysis and statistics
Measurement of direction of MRLC::GFP (small dot-like signals) movement
To measure the direction of MRLC::GFP movement in individual follicle cells and globally in the epithelial tissue, we measured angle relatively to the AP axis of egg chambers (Extended Data Figure 1c) with ‘Angle’ tool in Fiji. Before angle measurement, time-lapse movies were corrected for bleaching and cell membranes were registered for their movement to make them static. Angles were then measured on time (60s) projections of MRLC::GFP signals. Altogether, we measured movement of MRLC::GFP relatively to the cell membrane.
To unify fat2 mutant egg chambers with no epithelial rotation in order to compare with the weak and fast epithelial rotation data, the higher value of MRLC::GFP movement identified for Up (45°<135°) and Down (225°<315°) quadrants was artificially assigned to the Down quadrant to mimic as if all egg chambers rotated upwards, i.e. the preferred MRLC::GFP movement was against (retrograde) epithelial rotation (used in Figure 1c and Figure 2b,c,d).
Measurement of Myo-II size and velocity
The size of small and big MRLC::GFP signal was measured as a diameter over 5 and 10 independent egg chambers, respectively. Myo-II velocity was measured on a displacement of Myo-II signals in 30s-60s in original time-lapse movies over 10 independent egg chambers.
Measurement of velocity of epithelial rotation
The velocity was defined as an average velocity over 3 independent measurement of cell membrane movement in the most central part of the confocal plane.
Angular correction
To define a direction of the MRLC::GFP movement within follicle cells in fat2 mutant egg chambers, time projected MRLC::GFP pattern served as a definition of the main MRLC::GFP arrays that were reoriented within the existing smallest angle (≤90°) to achieve the perpendicular orientation to the AP axis (used in Figure 2). We hypothesized that Fat2 reorients Myo-II of follicle cells either by spatial regulation of the intracellular actomyosin dynamics or via remodeling of adherens junctions. Thus, in both cases, we concluded that it will be an energy-demanding process and angularly corrected follicle cell perpendicularly to the AP axis of egg chambers (based on the time projected Myo-II pattern) in the smallest possible angle (e.g. as shown in Figure 2a, 45° clockwise and not 135° anti-clockwise).
Quantification of global Myo-II and actin filament alignment
To measure global alignment of MRLC::GFP and actin filaments in fixed tissues, we used Fiji software ‘Directionality’ http://imagej.net/Directionality.
Measurement of follicle cell shape and elongation direction
Outlines of follicle cells were used to measure Roundness parameter (Shape descriptors>Roundness: 4×[Area]/π×[Major axis]2, Uses the heading Round) in Fiji. Angles for definition of direction of elongation of follicle cells (longest axis) were measured with ‘Angle’ tool in Fiji. Angle for definition of the Myo-II direction was defined on time projected MRLC::GFP pattern.
Measurement of Myo-II intensity
MRLC::GFP intensity was measured as a mean intensity in the defined cell outline of follicle cells. We developed following pipeline: To measure MRLC::GFP intensity and area of follicle cells, cell segmentation was performed using a custom macro from IRB (Barcelona, Spain) http://adm.irbbarcelona.org/image-j-fiji. Afterwards, cells between subsequent time frames were associated with each other if they overlapped by more than 60 % according to the Jaccard index metric. Determination of mean intensity, cell area and cell roundness was done using an in-house developed plugin utilizing the Analyze Particles Plugin http://imagej.net/ParticleAnalysis in Fiji41. Analyses were done on time-lapse movies (6s frame interval), for each frame one plane in z-axis was imaged. Bleach correction was applied in Fiji.
Amplitude of MRLC::GFP intensity change and cell area change was measured in the way that a mean of MRLC::GFP intensity and cell area was plotted over time (360s) of individual cells of a type (control vs. fat2 mutant), smooth curves were fitted, detrended and a value (based on detrended max.-min. values) calculated for each individual cell of a type and averaged over all cells of the same type.
The time series were smoothened with a Gaussian filter with window of 10 data points (i.e. 1min.) in R Studio http://www.rstudio.com. Rate of MRLC::GFP change was calculated as a first derivative of measured intensity. Rate of basal area reduction was the inverse value of the first derivative of basal area.
Cross-correlation efficiency was calculated with time shift from −6 to +6 min in R Studio. Rate of MRLC::GFP and area was used to calculate the cross-correlation coefficient. The average correlation coefficient was result of averaging smooth correlation data for individual cells.
MRLC::GFP intensity was measured in the most central follicle cells of focal plane of time-lapse movies.
Measurement of planar cell chirality (PCC)
One frame of time-lapse movies was selected and the AP axis of egg chambers was turned 90° clockwise or anti-clockwise in order to unify direction of epithelial rotation to the right. Oblique angle (θ) was measured for all cell membranes between 0°-90° (right) and −90°-0° (left) range, right and left frequencies were summarized to define to what side (left or right) cell membranes prefer to tilt as described 6.
Data plotting, statistics and cartoons
Rose diagrams, quadrant plots, histograms, bar and box plots were assembled in R studio http://www.rstudio.com. using various packages 42,43,44,45. Error bars represent standard error of the mean (s. e. m.). An unpaired two-sided t-test was used. Cartoons were made in Adobe Illustrator CS 5.1.
ACKNOWLEDGEMENTS
We thank to Thomas Lecuit for AX3; Sqh::GFP fly stock, to Daiki Umetsu for LifeAct::GFP fly stock and to Bloomington Stock Center for the line BL4414. We also thank the Scientific Computing Facility at MPI-CBG (Robert Haase) for developing an in-house Fiji Plugin. We are grateful to Stephan Grill for comments on the manuscript.