1. Developmental Biology and Stem Cells
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Jak-Stat pathway induces Drosophila follicle elongation by a gradient of apical contractility

  1. Hervé Alégot
  2. Pierre Pouchin
  3. Olivier Bardot
  4. Vincent Mirouse Is a corresponding author
  1. Université Clermont Auvergne - CNRS UMR 6293- INSERM U1103, France
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Cite as: eLife 2018;7:e32943 doi: 10.7554/eLife.32943

Abstract

Tissue elongation and its control by spatiotemporal signals is a major developmental question. Currently, it is thought that Drosophila ovarian follicular epithelium elongation requires the planar polarization of the basal domain cytoskeleton and of the extra-cellular matrix, associated with a dynamic process of rotation around the anteroposterior axis. Here we show, by careful kinetic analysis of fat2 mutants, that neither basal planar polarization nor rotation is required during a first phase of follicle elongation. Conversely, a JAK-STAT signaling gradient from each follicle pole orients early elongation. JAK-STAT controls apical pulsatile contractions, and Myosin II activity inhibition affects both pulses and early elongation. Early elongation is associated with apical constriction at the poles and with oriented cell rearrangements, but without any visible planar cell polarization of the apical domain. Thus, a morphogen gradient can trigger tissue elongation through a control of cell pulsing and without a planar cell polarity requirement.

https://doi.org/10.7554/eLife.32943.001

Introduction

Tissue elongation is an essential morphogenetic process that occurs during the development of almost any organ. Therefore, uncovering the underlying molecular, cellular and tissue mechanisms is an important challenge. Schematically, tissue elongation relies on at least three determinants. First, the elongation axis must be defined by a directional cue that usually leads to the planar cell polarization (pcp) of the elongating tissue. Second, a force producing machinery must drive the elongation, and this force can be generated intrinsically by the cells within the elongating tissue and/or extrinsically by the surrounding tissues. Finally, such force induces tissue elongation via different cellular behaviors, such as cell intercalation, cell shape modification, cell migration or oriented cell division. This is exemplified by germband extension in Drosophila embryos where Toll receptors induce Myosin II planar polarization, which drives cell rearrangements (Bertet et al., 2004; Irvine and Wieschaus, 1994; Blankenship et al., 2006; Paré et al., 2014).

In recent years, Drosophila egg chamber development has emerged as a powerful model to study tissue elongation (Bilder and Haigo, 2012; Cetera and Horne-Badovinac, 2015). Each egg chamber (or follicle) consists of a germline cyst that includes the oocyte, surrounded by the follicular epithelium (FE), a monolayer of somatic cells. The FE apical domain faces the germ cells, while the basal domain is in contact with the basement membrane. Initially, a follicle is a small sphere that progressively elongates along the anterior-posterior (AP) axis, which becomes 2.5 times longer than the mediolateral axis (aspect ratio [AR] = 2.5), prefiguring the shape of the fly embryo.

All the available data indicate that follicle elongation relies on the FE. Specifically, along the FE basal domain, F-actin filaments and microtubules become oriented perpendicularly to the follicle AP axis (Gutzeit, 1990; Viktorinová and Dahmann, 2013). The cytoskeleton planar polarization depends on the atypical cadherin Fat2, which acts via an unknown mechanism (Viktorinová et al., 2009; Viktorinová and Dahmann, 2013; Chen et al., 2016). Fat2 is also required for a dynamic process of collective cell migration of all the follicle cells around the AP axis until stage 8 of follicle development. This rotation reinforces F-actin planar polarization and triggers the polarized deposition of extracellular matrix (ECM) fibrils perpendicular to the AP axis (Haigo and Bilder, 2011; Lerner et al., 2013; Viktorinová and Dahmann, 2013; Cetera et al., 2014; Isabella and Horne-Badovinac, 2016; Aurich and Dahmann, 2016). These fibrils have been proposed to act as a molecular corset, mechanically constraining follicle growth along the AP axis during follicle development (Haigo and Bilder, 2011). In addition, Fat2 is required for the establishment of a gradient of basement membrane (BM) stiffness at both poles at stage 7–8 (Crest et al., 2017). This gradient also depends on the morphogen-like activity of the JAK-STAT pathway, and softer BM near the poles would allow anisotropic tissue expansion along the A-P axis (Crest et al., 2017). After the end of follicle rotation, F-actin remains polarized in the AP plane during stages 9–11 and follicular cells (FCs) undergo oriented basal oscillations that are generated by the contractile activity of stress fibers attached to the basement membrane ECM via integrins (Bateman et al., 2001; Delon and Brown, 2009; He et al., 2010).

Nonetheless, in agreement with recently published observations, we noticed that a first phase of follicle elongation does not require fat2 and the planar polarization of the basal domain (Aurich and Dahmann, 2016). We therefore focused on this phase, addressing main three questions which are: how the follicle elongation axis is defined, what the molecular motor triggering elongation in a specific axis is, and how FCs behave during this phase.

Results

Polar cells define the axis of early elongation

We analyzed the follicle elongation kinetics in fat258D mutants, which block rotation and show a strong round-egg phenotype. Follicle elongation is normal in fat2 mutants during the first stages (3–7) with an AR of 1.6 (Figure 1a–d). Thus, at least two mechanistically distinct elongation phases control follicle elongation, a first phase (stages 3–7), which is independent of fat2, rotation and ECM basal polarization, and a later phase (stages 8–14), which requires fat2. This observation is consistent with the absence of an elongation defect of clonal loss-of-function of vkg before stages 7–8 (Bilder and Haigo, 2012).

Figure 1 with 1 supplement see all
Polar cells determine the axis of early elongation.

(a) WT ovariole illustrating follicle elongation during the early stages of oogenesis (stages 2–6). (b) Optical cross-section of a stage 7 WT follicle stained with FasIII, a polar cell marker (white) and F-actin (red). (c) Stage seven fat2 mutant follicle stained with FasIII (white) and DE-Cad (red). (d) Elongation kinetics of WT and fat2 mutant follicles (n > 6 for each point). (e) Z-projection of a Pak mutant ovariole. Round follicles have only one cluster of polar cells (stage 5 and 8 follicles) or two non-diametrically opposed clusters (stage 3 follicle). (f) Removing a copy of Pvr restores early elongation and polar cell position in Pak mutants. (g) Elongation coefficient of Pak6/Pak11, Df(Pvr)/+ follicles, affecting (n = 23) (1) or not (n = 13) (2) polar cell positioning. (h,i) View of a mys mutant clone (GFP-negative) in a mosaic follicle showing (h) normal polar cell positioning and no elongation defect and (i) abnormal polar cell positioning and an early elongation defect. (j) Elongation coefficient of follicles containing mutant clones for mys affecting (n = 34) (3) polar cell positioning or not (n = 31) (4). Full details of the genotypes and sample sizes are given in the supplementary files. (p *<0.05, **<0.01, ***<0.001.) Scale bars are 10 μm throughout.

https://doi.org/10.7554/eLife.32943.002

To try to identify the mechanism that regulates the early phase of follicle elongation, we first analyzed trans-heterozygous Pak mutant follicles, which never elongate (Conder et al., 2007) (Figure 1e). The Pak gene encodes a Pak family serine/threonine kinase that localizes at the FE basal domain. Pak mutants also show many other abnormalities, such as the presence of more than one germline cyst and abnormal interfollicular filaments ([Vlachos et al., 2015] and not shown). Interfollicular cells derive from prepolar cells that also give rise to the polar cells, which prompted us to analyze the distribution of the latter using the specific marker FasIII (Bastock and St Johnston, 2008; Horne-Badovinac and Bilder, 2005). Polar cells are pairs of cells that differentiate very early and are initially required for germline cyst encapsulation (Grammont and Irvine, 2001). They also have a role as an organizing center for the differentiation of FC sub-populations during mid-oogenesis (Xi et al., 2003). In WT follicles, polar cells are localized at the follicle AP axis extremities (Figure 1b). Conversely, in Pak mutants, we observed a single polar cell cluster or two clusters close to each other (Figure 1e). This suggests that Pak is required for polar cell positioning, although a role in the specification or survival of these cells cannot be excluded, which in turn could play a role in defining the elongation axis. Some dominant suppressors of the Pak elongation defect have been identified, including PDGF- and VEGF-receptor related (Pvr), although the reason for this suppression is unknown (Vlachos and Harden, 2011). By using flies that are heterozygous for a Pvr allele and mutant for Pak, we observed that the normal positioning of polar cells is frequently but not always restored (Figure 1f and Figure 1—figure supplement 1c). We quantitatively compared the elongation of those two situations, normal or abnormal polar cells, by plotting the long axis as a function of the short axis for previtellogenic stages (before stage 8) and determined the corresponding regression line (Figure 1—figure supplement 1d). We defined an elongation coefficient that corresponds to the slope of this line and for which a value of 1 means no elongation. This method allows us to quantify elongation independently of any bias that could be introduced by stage determination approximation due to aberrant follicle shape or differentiation. Moreover, focusing on previtellogenic stages allows the exclusion of genotypes that affect only the late elongation phase. It is exemplified by a fat2 mutant that does not induce significant defects if we include only stage 3–7 follicles (previtellogenic), but does show a difference if we include stage 8 follicles (Figure 1—figure supplement 1a,b). The statistical comparison of the elongation coefficients clearly shows that restoring polar cell position by removing one copy of Pvr in Pak mutants strongly rescues follicle elongation (Figure 1g and Figure 1—figure supplement 1c,d).

Although not been fully demonstrated in this context, Pak often works as part of the integrin signaling network, and mosaic follicles containing FC clones that are mutant for myospheroid (mys), which encodes the main fly β-integrin subunit, also show a round follicle phenotype at early stages (Haigo and Bilder, 2011). We noticed that in some follicles containing mys mutant clones, polar cells are mispositioned, a defect generally observed when at least one polar cell is mutant. As in Pak mutants, the two polar cell clusters are not diametrically opposed (Figure 1—figure supplement 1e), or a single cluster is observed (Figure 1i, Video 1). Importantly, the polar cell positioning defect is associated with the round follicle phenotype (Figure 1j, and Figure 1—figure supplement 1f). Conversely, in mosaic follicles in which polar cell positioning was not affected, the round egg phenotype is never observed at early stages, even with large mutant clones (Figure 1h and Figure 1—figure supplement 1f, Video 2). In agreement, the elongation coefficient of mosaic follicles that have normal polar cell positioning is much higher than that for those with abnormal polar cells (Figure 1j). Thus, together, these results indicate that pak and mys mutants are not required for the early phase of elongation once polar cells are well-placed and thus affect this phase indirectly. The results also strongly suggest that polar cells are required to define the follicle elongation axis.

Video 1
Full z-stack of a follicle with a mys mutant clone that affects polar cells.
https://doi.org/10.7554/eLife.32943.004
Video 2
Full z-stack of a follicle with a mys mutant clone that does not affect polar cells.
https://doi.org/10.7554/eLife.32943.005

A JAK-STAT gradient from the poles is the cue for early elongation

Once the follicle is formed, polar cells are important for the differentiation of the surrounding FCs. From stage 9 of oogenesis, FCs change their morphology upon activation by Unpaired (Upd), a ligand for the JAK-STAT pathway, which is exclusively produced by polar cells throughout oogenesis (Silver and Montell, 2001; Xi et al., 2003; McGregor et al., 2002). To identify the FCs in which the JAK-STAT pathway is active, we used a reporter construct in which GFP transgene expression is controlled by STAT binding repeat elements in the promoter (Bach et al., 2007). During the early stages of oogenesis, the pathway is active in all the main body FCs (Figure 2b). Moreover, we observed differences in GFP expression level (and thus STAT activity) between the poles and the mediolateral region, starting at about stage 3, concomitantly with the beginning of elongation (Figure 2b,h). At later stages (5–7), these expression differences lead to the formation of a gradient of STAT activity, as indicated by strong GFP expression at each pole and very weak or no signal in the large mediolateral part of each follicle (Figure 2b,h, and Figure 2—figure supplement 1a). Thus, the spatiotemporal pattern of JAK-STAT activation is consistent with a potential role of this pathway in follicle elongation.

Figure 2 with 1 supplement see all
Upd is a polarizing cue for early elongation.

(a) Optical cross-section of a WT stage 7 follicle stained with FasIII (polar cell marker, white) and F-actin (red). (b) Expression of the 10xStatGFP reporter showing the progressive formation of a STAT gradient at each pole. Early elongation is affected by (c) knocking down upd in polar cells or (d) the expression of Stat92E in the anterior and posterior follicular cells. Early elongation is also affected by (e) clonal ectopic expression of upd (GFP-positive cells) and by (f) expression of a Hop gain of function mutant in all follicular cells. (g) Quantification of the elongation coefficient in WT and the different JAK-STAT loss- and gain-of-function genotypes — corresponding to Upd:GAL4 and Upd-RNAi, Fru:GAL4 and STAT92E-RNAi, and Tj:GAL4 and UAS:Hoptum — during early and intermediate stages of elongation (D, Driver; U, UAS line). (For each point n > 30; p *<0.05, **<0.01, ***<0.001.) (h) Quantification of the Stat activity gradient at stages 3, 5 and 7 using the 10XSTATGFP reporter. A gradient is already visible at stage 3 and becomes more visible until stage 7. Scale bars are 10 μm throughout. Relative intensity = intensity at a given position/mean intensity of measured signal. Full details of the genotypes and sample sizes are given in the supplementary files.

https://doi.org/10.7554/eLife.32943.006

The key role of JAK-STAT signaling during follicle formation precluded the analysis of elongation defects in large null mutant clones (McGregor et al., 2002). Therefore, we knocked-down by RNAi the ligand upd and the most downstream element of the cascade, the transcription factor Stat92E, both efficiently decreasing the activity of the pathway in the follicular epithelium (Figure 2a,c,d,g and Figure 2—figure supplement 1). Upd knockdown was performed using either upd:GAL4 that is specifically expressed in the polar cells (Khammari et al., 2011) or tj:GAL4 that is expressed in all FCs, and then analyzed only in follicles that contained one germline cyst and correctly placed polar cells. During the early stages, with both drivers, such follicles are significantly rounder than control follicles (Figure 2a,c,g and Figure 2—figure supplement 1e). This indicates a role for JAK-STAT pathway in early elongation and confirms the causal link between polar cells and early elongation. Moreover, knockdown of Stat92E using a driver that is specifically expressed at the poles (Fru:GAL4) also affects early elongation (Figure 2d,g and Figure 2—figure supplement 1e), suggesting a transcriptional control of elongation by JAK-STAT (Borensztejn et al., 2013). These results are the first examples of loss of function with an effect only on early elongation and independent of polar cells position, and indicate that both Upd secreted by the polar cells and JAK-STAT activation in FCs arerequired for follicle elongation. Moreover, clonal ectopic upd overexpression completely blocks follicle elongation, without affecting polar cell positioning (Figure 2e), demonstrating that Upd is not only a prerequisite for the elongation but the signal that defines its axis (n = 20). Similarly, general expression of HopTum, a gain-of-function mutation of fly JAK, that disrupts the pattern of JAK-SAT activation also affects follicle elongation (Figure 2f,g and Figure 2—figure supplement 1e). Thus, spatial control of JAK-STAT pathway activation is required for follicle elongation. Altogether, these results show that Upd secretion by polar cells and the subsequent gradient of JAK-STAT activation act as developmental cues that define the follicle elongation axis during the early stages of oogenesis.

MyosinII activity drives apical pulses and early elongation

Once the signal for elongation had been identified, we aimed to determine the molecular motor driving this elongation, which in many morphogenetic contexts is MyosinII (MyoII)(Heisenberg and Bellaïche, 2013; Lecuit et al., 2011). The knockdown in all FCs of spaghetti squash (sqh), the MyoII regulatory subunit, leads to a significant decrease in the elongation coefficient and in follicle AR from stage 4 (Figure 3a–b and Figure 3—figure supplement 1b,c), indicating that MyoII is the motor of early elongation. We have shown that the rotation and the planar polarization of the basal actomyosin is not involved in early elongation. Moreover, at these stages, MyoII is strongly enriched at the apical cortex, suggesting that its main activity is on this domain of the FCs (Figure 3—figure supplement 1a and Figure 5c) (Wang and Riechmann, 2007). We therefore looked at MyoII on living follicles, focusing on the apical side, and found that it is highly dynamic (Video 3). In Drosophila, transitory medio-apical recruitment of actomyosin usually drives apical pulses (Martin et al., 2009; Martin and Goldstein, 2014). Accordingly, using a GFP trap line for Bazooka (Baz-GFP), which concentrates at the zonula adherens and marks the periphery of the apical domain, we observed that the transient accumulation of MyoII is associated with a contraction of this domain, which is followed by a relaxation when the MyoII signal decreases (Figure 3c–e, Video 4). Although we did not find a clear period because cells can pause for a variable time between two contractions, the approximate duration of a pulse was about three minutes. Cross-correlation analysis on many cells from several follicles (n = 86) confirms the association between MyoII and pulses, and reveals that Sqh accumulation slightly precedes the reduction of the apical surface, arguing that it is the motor responsible for these contractions (Figure 3f). Inhibiting the activity of Rho kinase (rok), the main regulator of MyoII, using Y-27632, reduces follicle cells' surface variation by ~30% (Figure 3g). Thus, MyoII drives apical pulsing during early stages. Consequently, we asked whether and how apical pulses could induce elongation. From stage 9, basal pulses, which are important for the second phase of elongation, have been shown to be anisotropic (He et al., 2010). However, quantification of axis length variations showed that the apical pulses were isotropic, both in the mediolateral and polar regions (Figure 3h). Tissue elongation is often associated with tissue planar cell polarization, we therefore investigated whether Myosin II and Baz showed exclusive cortical planar polarization, as demonstrated for instance during germband extension (Bertet et al., 2004; Zallen and Wieschaus, 2004). Consistent with the isotropic nature of the pulses, we failed to detect any oriented enrichment of these proteins, indicating the absence of noticeable apical planar cell polarization of the motor that generates early elongation (Figure 3i,j). Altogether, these data indicate that MyoII induces apical pulses and early elongation. Nonetheless, neither the isotropic nature of the pulses nor MyoII localization explains how the pulses could induce elongation.

Figure 3 with 1 supplement see all
Myosin II is required for early elongation and apical pulses.

(a) WT and (a′) sqh knockdown stage 7 follicles stained for F-actin (red) and FasIII (white). (b) Elongation coefficient of WT or sqh knockdown follicles during the early elongation phase (D, Driver; U, UAS line) (n > 30). (c) Fluorescence video-microscopy images of a stage 4 WT follicle that expresses BAZ-GFP and Sqh-mCherry. (d) Higher magnification of the area highlighted in (c) showing a pulsing cell. (e) Quantification of the cell apical surface (green) in the cell shown in (d) and of Sqh signal intensity in the apical area (red) over time. (f) Cross-correlation analysis over time of apical surface and Sqh apical signal intensity based on 86 cells from six follicles at stages 3–4. (g) Incubation with the Rok inhibitor Y-27632 strongly reduces pulse activity in stages 3 to 5 WT follicles. Each dot corresponds to one follicle, and at least 10 cells per follicle were analyzed. Red bars represent mean and ± SD (n ≥ 7 follicles). (h) Quantification of the length variation of the follicle cell AP and mediolateral axes during pulses indicates that pulses are isotropic (n > 13 follicles, at least 10 cells per follicle were analyzed). (i) Quantification of the relative BAZ-GFP and Sqh-mCherry signal intensity in cell bounds in function of their angle relative to the AP axis (n = 41 follicles). Relative intensity is given over the mean bond intensity. (j) Fixed stage 7 WT follicle that expresses GFP-Baz and Sqh-mCherry. Scale bars are 10 μm throughout. (p ***<0.001.) Full details of the genotypes and sample sizes are given in the supplementary files.

https://doi.org/10.7554/eLife.32943.008
Video 3
Stage 3 follicle expressing Sqh-GFP.

The pool of apical myosinII is very dynamic.

https://doi.org/10.7554/eLife.32943.010
Video 4
Zoom in on a cell of a stage 3 follicle expressing Baz-GFP and Sqh-mCherry.

Apical MyosinII enrichment occurs at the same time as the apical cell domain contracts.

https://doi.org/10.7554/eLife.32943.011

JAK-STAT induces a gradient of apical pulses

Our previous results suggest that pulses do not provide an explanation for elongation at a local cellular scale, and we therefore analyzed their spatiotemporal distribution at the tissue scale to determine whether they present a specific tissue pattern. On the basis of the JAK-STAT activity gradient, we hypothesized that cells in the mediolateral part of the follicles should progressively change their behavior during follicle growth. We therefore monitored the mediolateral region of stage 3 and 7 follicles. At stage 3, cells undergo contractions and relaxations asynchronously (Figure 4a, Video 5). At stage 7, cells were much less active (Figure 4c, Video 6). This difference was confirmed by monitoring the variation of the relative apical surface of individual cells (Figure 4e) or a whole population (Figure 4f) (40% of mean variation at stage 3 and only about 15% in the equatorial part at stage 7). Quantification of the average variation of the apical cell surface in a series of follicles indicates that the pulsing amplitude gradually decreases in the mediolateral region from stage 3 to stage 8 (Figure 4—figure supplement 1a). This correlation between JAK-STAT activity and pulsing activity in the mediolateral region prompted us to develop a method to visualize the poles of living follicles, which has never been done before (see Materials and methods). We managed to image the poles of stage 3–4 and stage 7–8 follicles, and in both cases the pulse activity is high (Figure 4b,d–f, Videos 7 and 8). Finally, the analysis of slightly tilted stage 7–8 follicles clearly revealed a gradient of pulse intensity emanating from the pole (Figure 4g and Figure 4—figure supplement 1b). Thus, the pulse intensity distribution is similar in space and time to the JAK-STAT activity gradient. Moreover, the cell pulse amplitude is significantly reduced in the mediolateral region of stage 3–4 follicles and near the poles of stage 7–8 upd RNAi follicles (Figure 4h,i, Videos 9 and 10), indicating that JAK-STAT regulates FC apical pulsatory activity. Finally, we found that clonal ectopic activation of JAK is sufficient to increase pulse intensity in the mediolateral region of stage 7–8 follicles when compared to similar control clones (Figure 4j, Videos 11 and 12). Together, these results show that the JAK-STAT pathway has an instructive role in controlling the intensity of FC apical pulses, leading to a specific spatiotemporal pattern breaking follicle symmetry in each hemisphere.

Figure 4 with 1 supplement see all
JAK-STAT induces a double gradient of pulses.

(ad) Images from movies of the mediolateral region of (a) stage 3 and (c) stage 7 BAZ-GFP expressing follicles, or of the area near the polar cells (red arrowheads) of (b) stage 3 and (d) stage 7 follicles. Scale bars: 10 μm. (e) Surface variation of individual cells (examples shown in [a–d]) as a function of time (ML, mediolateral). The surface of each cell is divided by its average surface over time. (f) Mean percentage of apical surface variation depending on stage and position (n ≥ 9 follicles). (g) Color-coding of the pulse intensity of a representative stage 7 follicle (tilted view from the pole, see schematic image in insert) reveals an intensity gradient from the polar cells (in green) to the mediolateral region. (h–j) Mean percentage of apical surface variation in the mediolateral region of (h) stage 3–5 follicles and (j) stage 7 to 8 follicles, and (i) at the pole of stage 7–8 follicles for the indicated genotypes. (h, i) n ≥ 9, (j) n ≥ 5. (p **<0.01, ***<0.001, red bars represent mean and ± SD). Full details of the genotypes and sample sizes are given in the supplementary files.

https://doi.org/10.7554/eLife.32943.012
Video 5
Stage 3 follicle expressing Baz-GFP.

Cells in the mediolateral part undergo apical pulsations.

https://doi.org/10.7554/eLife.32943.014
Video 6
Stage 7 follicle expressing Baz-GFP.

The apical surface variation is strongly reduced on the mediolateral part compared with stage 3 follicles.

https://doi.org/10.7554/eLife.32943.015
Video 7
Stage 7 follicle expressing Baz-GFP observed from the pole.

Polar cells are indicated on the corresponding Figure 5d (red arrowheads). The pulse intensity remains high in these cells compared with Video 4. The rotation is visible and occurs around the polar cells.

https://doi.org/10.7554/eLife.32943.016
Video 8
Stage 3 follicle expressing Baz-GFP observed from the pole.

Polar cells are indicated on the corresponding Figure 5b (red arrowhead).

https://doi.org/10.7554/eLife.32943.017
Video 9
Stage 3 upd knockdown follicle expressing Baz-GFP.

The intensity of the pulse is reduced compared with a WT stage 3 follicle (Video 3).

https://doi.org/10.7554/eLife.32943.018
Video 10
Stage 7 upd knockdown follicle expressing Baz-GFP and observed from the pole.

The intensity of the pulse is reduced compared with a WT stage 7 follicle (Video 6).

https://doi.org/10.7554/eLife.32943.019
Video 11
Stage 7 follicle expressing ectopically Baz-mCherry.

The intensity of the pulse is low.

https://doi.org/10.7554/eLife.32943.020
Video 12
Stage 7 follicle expressing ectopically Baz-mCherry and Hoptum.

Activation of the JAK-STAT pathway is sufficient to increase the pulsing.

https://doi.org/10.7554/eLife.32943.021

Myosin II is required at the poles but is not controlled by JAK-STAT

Since the JAK-STAT pathway and MyoII are both important for apical pulses, we studied their functional relationship. The apical level of the Myosin II active form, visualized by its phosphorylation, is significantly reduced by 18% in STAT92E null mutant clones on young follicles when compared to WT surrounding cells (n = 17 clones, p<0,001), which may suggest that MyoII activity is regulated by JAK-STAT signaling (Figure 5a). However, clonal gain of function of JAK in the region where the JAK-STAT pathway is normally inactive (mediolateral at stage 7–8) does not increase the apical phosphorylation level of MyoII (Figure 5b). Moreover, analysis of the global pattern of apical MyoII phosphorylation does not reveal any gradient between the poles and the mediolateral regions (Figure 5c,d). Altogether,these data indicate that MyoII activation by phosphorylation is independent of JAK-STAT signaling and that JAK-STAT regulates pulses by another means, which might be required for efficient apical recruitment of MyoII. Thus, although JAK-STAT and Myosin II are both required for early elongation, they control pulses in parallel.

Myosin II is not controlled by JAK-STAT but is required at the poles.

(a) Apical level of phosphorylated Sqh (pSqh, white and [a′]) is reduced in a mutant Stat92E clone (RFP-negative) in a stage 3 follicle (z-projection of the superior half of the follicle). (b) Clonal overexpression of Hoptum (green cells) on a stage 7 follicle is not sufficient to increase the expression of apical pSqh (white and [b′]) z-projection of the superior half of the follicle). (c) pSqh staining in the middle plane of a wildtype stage 7 follicle. (d) Quantification of the intensity of apical pSqh along the AP axis of stage 6–7 follicles. n = 5 follicles. Baseline value = mean apical pSqh per follicle. (e) Illustration of extrapolated Aspect Ratio (eAR) calculation based on width measure of a pole at 25% of AP axis length. (f) Quantification of the extrapolated aspect ratio (eAR) of stage 4–7 WT follicles or follicles with a sqhAX3 or Rok2 clone covering the anterior pole (n ≥ 10). In WT follicles, the anterior is significantly more curved than the posterior, whereas the tendency is opposite with sqh and Rok clones. (p ***<0.001.) (g,h,I,j) representative images of (g) WT, (h) mediolateral sqhAX3 clone, (i) anterior sqhAX3 clone and (j) anterior Rok2 clone with the corresponding eARs. Full details of the genotypes and sample sizes are given in the supplementary files.

https://doi.org/10.7554/eLife.32943.022

If the gradient of apical pulses induces early elongation and explains MyoII involvement in this process, then MyoII function should be required at the poles. We generated mutant clones for a null allele of sqh to analyze where MyoII is required for elongation. As previously shown (Wang and Riechmann, 2007), such clones reach a limited size, probably explaining why it is rare to obtain a clone that covers poles, especially after stage 5. We focused on clones covering the anterior pole. To quantify the effect of mutant clones on semi-follicles, we measured extrapolated Aspect Ratio (eAR) of each semi-follicle, which means, the ratio of the corresponding full ellipse (see Materials and methods and Figure 5e). For a WT follicle, the anterior eAR is equal or superior to the posterior eAR, as the anterior pole is normally more pointed than the posterior (Figure 5g). Analysis of the eAR of the poles containing such mutant clones indicates that Myosin II loss of function specifically affects the elongation of this pole, compared to the opposite WT posterior poles (n = 10) (Figure 5f,i). Moreover, we never observed clones in the mediolateral regions inducing elongation defects (n = 35) (Figure 5h). Finally, we also performed similar experiment with Rok null mutant clones. Such clones have a weaker effect on cell morphology (Figure 5j and Wang and Riechmann, 2007), but still affect elongation when situated at the pole (Figure 5f,j). Thus, MyoII and Rok are required specifically at the poles to induce early elongation. These results strongly argue that the gradient of apical isotropic FC pulses is the force-generating mechanism that drives early elongation.

Early elongation is associated with cell constriction and cell intercalation

Independently of the upstream events, we asked which cellular behavior was associated with early elongation. The simplest possibility would be that cells are stretched along the AP axis. However, cells are actually slightly elongated perpendicularly to the axis of elongation and this morphology did not change significantly over time, indicating that this parameter does not contribute to follicle elongation during early stages (Figure 6—figure supplement 1a,b). Tissue elongation can be also associated with oriented cell divisions. A movie of mitosis in the FE showed that this orientation is really variable through the different steps of mitosis (Figure 6—figure supplement 1c). We therefore quantified the orientation of cytokinesis figures, which did not highlight any bias towards the AP axis (Figure 6—figure supplement 1d). Finally, we asked whether early elongation could be associated with cell intercalation. Analysis of fluorescence video-microscopy images gave inconclusive results because such events are probably rare and slow, and because follicle rotation precludes their reproducible observation (Video 13). We therefore used an indirect method. As follicle cells from stage 6 onwards stop dividing and their number remains constant, we counted the number of cells in the longest line of the AP axis (i.e., the follicle plane that includes the polar cells). This number significantly increases between stage 6 and 8, showing that cells intercalate in this line (Figure 6a–d). This number is also correlated with the follicle AR (Figure 6e), indicating that follicle early elongation is associated with cell intercalation along the AP axis. Cell intercalation can be powered at a cellular scale by the polarized enrichment of Myosin II in the cells that rearrange their junctions (Bertet et al., 2004). However, we have already shown that MyoII does not show such a pattern in FCs (Figure 3i,j). Alternatively, intercalation can be promoted at a tissue scale. For instance, apical cell constriction in the wing hinge induces cell intercalations in the pupal wing (Aigouy et al., 2010). We observed that the cell apical surface is lower at the poles than in more equatorial cells, and that this difference increases during the early elongation phase (Figure 6f,g,h). Such a difference could be explained by cell shape changes or by a differential cell growth. Cell height is significantly larger at the poles, indicating that the changes in apical surface are linked to cell morphology, as previously shown during mesoderm invagination for instance (Figure 6i) (He et al., 2014). However, cells at the poles have a lower volume than those in the mediolateral region at stage 7 (Figure 6—figure supplement 1e). This difference of volume is nonetheless proportionally weaker than the change in apical surface, suggesting the cell shape changes induce the reduction of volume rather than the opposite. Thus, early elongation is associated with a moderate cell constriction in the polar regions. sqh mutant FCs are stretched by the tension coming from germline growth, a defect opposite to cell constriction (Figure 5g,h) (Wang and Riechmann, 2007). Interestingly, FCs that are mutant for Stat92E are also flattened, with a larger surface and a lower height, compared to WT surrounding cells (Figure 6j–m). Moreover, the apical cell surface at the poles of stages 7–8 is increased by the loss of function of Upd (Figure 6h). Hence, these results link JAK-STAT and the morphology of the follicle cells in a coherent manner with an involvement of apical pulses for the cell constriction observed at the poles.

Figure 6 with 1 supplement see all
Localized apical cell constriction and oriented cell intercalation occur during early elongation.

(a–c) AR and number of cells along the polar cell plane in representative (a) stage 6, (b) stage 7 and (c) stage 8 follicles stained for DE-Cad. (d–e) Number of follicular cells in the plane of polar cells based on DE-Cad staining of stage 6–8 follicles depending on (d) the stage and (e) the aspect ratio of the follicle. (f) Heat map of the cell apical surface of a representative stage 7 follicle imaged as on Figure 4g. Arrowhead shows polar cells. (g) Quantification of the relative apical cell surface (smallest cell = 1) as a function of the distance from polar cells (n = 10 stage 7 follicles, 1487 cells). (h) Apical cell surface and (i) cell height depending on stage, position and genotype (ML, mediolateral). (j) Representative top view and (l) section view of Stat92E397 mutant clones at stage 3. Mutant cells have (k) a larger apical surface and (m) a lower cell height than do wildtype cells. Each dot corresponds to the mean value obtained for a clone. (n) Schematic figure showing the progressive restriction of JAK-STAT signaling (green) and of cell constriction to the follicle poles. p *<0.05, **<0.01, ***<0.001. Full details of the genotypes and sample sizes are given in the supplementary files.

https://doi.org/10.7554/eLife.32943.023
Video 13
Movie representing a stage 7 DE-Cad-GFP follicle imaged over two hours.

One cell is tracked (red line). No intercalation occurs during this period.

https://doi.org/10.7554/eLife.32943.025

Altogether these results indicate that two cell behaviors occur during the early phase of elongation: oriented cell intercalation towards the A-P axis and apical cell constriction at the poles.

Discussion

The first main conclusion of this work is that follicle elongation can be subdivided into at least two main temporal and mechanistic phases: an early one (stages 3–6) that is independent of Fat2, rotation, and ECM and F-actin basal polarization, and a second one (stages 7–14) that requires Fat2. This is reminiscent of germband extension where different elongation mechanisms have been described (Lye et al., 2015; Collinet et al., 2015; Rauzi et al., 2010; Blankenship et al., 2006; Sun et al., 2017). In the case of the follicle, it is still not clear how overlapping and interconnected these different mechanisms are.

Fat2 has no role in early elongation. Nevertheless, Fat2 is required as early as the germarium for the correct planar polarization of the microtubule cytoskeleton and for follicle rotation, which takes place during the early elongation phase (Viktorinová and Dahmann, 2013; Chen et al., 2016). The rotation reinforces the basal pcp of the F-actin during stages 4–6, and thus probably participates in the late phase in this way (Cetera et al., 2014; Aurich and Dahmann, 2016). Rotation is also necessary for the deposition of ECM fibrils, although their specific role in elongation has not been clearly elucidated yet. Another mechanism that participates in elongation is the ECM stiffness gradient (Crest et al., 2017). However, its contribution begins only at stage 7–8. This is in agreement with the fact that the ECM stiffness gradient depends on Fat2 and that vkg (ColIV) loss-of-function follicles elongate correctly up to stage 8, showing that the ECM is required only in the second elongation phase (Crest et al., 2017; Haigo and Bilder, 2011). Thus, the setting up of the elements required for this second elongation phase fully overlaps with the first elongation phase, but these two phases are so far unrelated at the mechanistic level. Notably, the early elongation phase requires elements of the apical side of follicle cells, whereas the second phase involves the basal side. Mirroring our observations, a recent report nicely shows that the fly germband extension, which was thought to depend exclusively on the apical domain of the cells, also involves their basal domain (Sun et al., 2017). As both Fat2 and the gradient of BM stiffness are involved in the elongation at stage 8 and as apical pulses are still observed at this stage, it appears that the apical and basal domain contributions may slightly overlap. Moreover, both the gradients of apical pulses and of BM stiffness are under the control of JAK-STAT, indicating that this pathway has a pleiotropic effect on follicle elongation.

We have also shown that integrin and Pak contribute to early elongation in an indirect manner through their impact on the positioning, the differentiation or the survival of the polar cells. In this respect, Pak and mys mutants belong to a new phenotypic class that could also comprise the Laminin β1 subunit (LanB1) and the receptor-like tyrosine phosphatase Lar (Díaz de la Loza et al., 2017; Frydman and Spradling, 2001). We do not yet know how the A-P position of those cells is established and maintained. Interestingly, Pak mutants also have an altered germarium structure leading to abnormal follicle budding, suggesting that polar cell mispositioning might be linked to this primary defect (Vlachos et al., 2015). However, it is worth noticing that Pak mutant follicles do not elongate at all, whereas they still have a cluster of polar cells. Thus, Pak might also be required for early elongation in a more direct manner than polar cell positioning, downstream of or in parallel to the JAK-STAT pathway, but independently of basal planar polarization.

We found that polar cells define the elongation axis of each follicle during early elongation by secreting the Upd morphogen and by forming a gradient from each pole, which in turn induces apical pulses. The isotropic nature of these pulses does not provide an evident link with tissue elongation, unlike the oriented basal pulses going on in later stages (He et al., 2010). Moreover, the absence of planar polarization of MyoII in apical regions, which is the driving force of early elongation, and the non-requirement for ‘basal pcp’ strongly argue against a control of this elongation phase by a planar cell polarity working at a local scale. Instead, several strong arguments propose that early elongation relies on pulses working at a tissue scale (Figure 6n). First, the pulses are distributed in a gradient from the poles, suggesting that this distribution can orient the elongation in each hemisphere. Also, our data indicate that JAK-STAT does not directly regulate MyoII activity, and, thus, that they probably work in parallel to control pulses. The convergence of requirement for JAK-STAT and myosin II activities for both pulses and early elongation argues for a causal link between these two processes. To date, JAK-STAT has no other known morphogenetic function before stage 8. Similarly, the only other known function of MyoII is linked to the rotation, which is not involved in early elongation, and MyoII is very concentrated at the apical cortex, emphasizing the role of this domain. Moreover, though present all around the follicle, MyoII is required for early elongation at the poles. Thus, the apical localization and the spatiotemporal requirement of MyoII are coherent with the action of apical pulses as the driving force for early elongation.

JAK-STAT has already been implicated in the elongation of different tissues in flies and in vertebrates. For instance, Upd works as the elongation cue for the hindgut during fly embryogenesis, a process also associated with cell intercalation, although the underlying mechanism is unknown (Johansen et al., 2003). Maybe more significantly, JAK-STAT is involved in the extension-convergence mechanism during zebrafish gastrulation (Yamashita et al., 2002). Moreover, JAK-STAT also participates in other morphogenetic events, such as tissue folding in the fly gut and wing disc (Wells et al., 2013). All these roles are potentially linked to a control of apical cell pulses. As our results indicate that this control is not through MyoII activation, identifying the transcriptional targets of STAT that explain its impact on apical actomyosin will be relevant for many developmental contexts.

How the apical pulses precisely drive early elongation remains a question that will require further investigations. Nonetheless, we determined that early elongation is associated with apical cell constriction close to the poles and oriented cell intercalations. Cell constriction is probably a direct consequence of apical pulses, as has been shown in many other contexts, because both myosin II and JAK-STAT loss of function affect pulse and induce an increase of the apical surface (Wang and Riechmann, 2007; Martin and Goldstein, 2014). Thus, as during tissue invagination, cell constriction may accentuate the curvature at the poles and thus promote elongation. Intercalation can be induced at a tissue scale by long-range anisotropic tensions in the tissue, as exemplified by the development of pupal wings or mammalian limb bud ectoderm (Aigouy et al., 2010; Lau et al., 2015). In the wing, elongation is due to contraction of the hinge, which corresponds to an apical constriction of the cells. Here, the apical pulses could act in a similar way via the constriction, acting as a pulling force at each pole. Thus, intercalations may correspond to a passive response, bringing plasticity to the tissue and hence stabilizing its elongation. Although the respective contribution of these two cell behaviors - apical constriction at the poles and cell intercalation along the AP axis – and their potential links remain to be determined, together they probably recapitulate at the cellular scale the elongation observed at the tissue scale. Importantly, such a mechanism does not require any planar cell polarization, in agreement with our observations. A gradient of randomly oriented cell migration contributes to vertebrate AP axis elongation and is, to our knowledge, the only other example of a tissue elongation mechanism instructed by a signaling cue and independent of pcp (Bénazéraf et al., 2010), in contrast to the many examples where pcp controls cell-movements that induce axis elongation in vertebrates. Our work proposes an alternative mechanism to explain how a morphogen gradient can induce elongation solely through transcription activation, and without any requirement for a polarization of receiving cells. This simple mechanism may apply to other tissues and other morphogens.

Materials and methods

Genetics

All the fly stocks with their origin and reference are described in Supplementary file 1. The detailed genotypes, temperature and heat-shock conditions are given in Supplementary file 2.

Immunostaining and imaging

Dissection and immunostaining were performed as described previously (Vachias et al., 2014) with the following exceptions: ovaries were dissected in Supplemented Schneider, each of the ovarioles was separated before fixation to obtain undistorted follicles. Primary antibodies were against pMyoII (1/100, Cell Signaling #3675), DE-Cad (1/100, DHSB #DCAD2), Dlg (1/200 DHSB #4F3), and FasIII (1/200, DHSB #7G10). Images were taken using a Leica SP5 or SP8 confocal microscope. Stage determination was performed using unambiguous reference criteria, which are independent of follicle shape (Spradling, 1993).

For live imaging, ovaries were dissected as described previously (Prasad et al., 2007) with the following exceptions: each ovariole was separated on a microscope slide in a drop of medium and transferred into a micro-well (Ibidi BioValey) with a final insulin concentration of 20 µg/ml. Samples were cultured for less than 2 hr before imaging with a Leica SP8 confocal using a resonant scanner. Follicles were incubated with Y-27632 (Sigma) (diluted in PBS to 250 µM) for 10–30 min before image acquisition. To image the poles, glass beads were added into the well to form a monolayer (Sigma-Aldrich, G4649 for stage 6–8 or G1145 for earlier stages). Ovarioles were added on top of the beads and follicles falling vertically between the beads were imaged.

Cell pulse analysis was performed using the Imaris software and a MATLAB homemade script to segment and measure the cell surface on maximum intensity projections of 40 stacks taken every 15 s. The intensity of one cell pulsation corresponds to: (maximum surface of the cell – min surface)/(mean surface). The isotropy of one cell pulse is measured by dividing the AP and ML bounding box (best fit rectangle) axis length at cell maximal area by the AP and MP bounding box axis length, respectively, at the cell’s minimal area. For each follicle, at least 10 cells were analyzed. For visualization (images presented in Figure 4a,c,d and the attached movies), the original files were deconvolved, but all the analyses were carried out using the raw files.

The Fiji software was used to measure the length of the long and short axis of each follicle on the transmitted light channel, and then to determine the aspect ratio in WT and mutant follicles. Cells in the longest line of the AP axis were counted manually using Fiji on the DNA and DE-Cadherin channels. Bazooka-GFP and MyosinII-mCherry enrichment were analyzed using the Packing Analyser software (Aigouy et al., 2010). Cells were semi-automatically segmented on the basis of the Baz-GFP channel that was used as common pattern to calculate the intensity of each bond for both channels.

Fiji was used to measure the intensity of the pSqh signal and the 10XStatGFP signal. A 15-pixel wide line was drawn using the freehand tool, either within the cells (10X StatGFP) or at the apical level of the cells (pSqh), from the anterior to the posterior of cross-sectional images of follicles.

The extrapolated aspect ratio (eAR) was estimated for each pole by measuring the width of the follicle at 25% of its total length: for any given ellipse, this value corresponds to 3/2 times its total width. Therefore, this measure allows the extrapolation of a width and an aspect ratio for each pole. Follicles with gaps in the epithelium were excluded on the basis of Dlg staining.

To measure cell elongation, images of DE-Cadherin-GFP-expressing follicles were semi-automatically segmented using the Packing Analyser software, and for each follicle, the elongation tensor was calculated. The elongation tensor was defined by the mean elongation of all the segmented cells (elongation magnitude) and the mean orientation.

The rose diagrams were generated with Packing Analyser; each bin represents a 10° range and the bin size is proportional to the number of acquired data. Cell volume was obtained by the multiplication of the mean surface and the mean height of the cells.

Figures were assembled using ScientiFig (Aigouy and Mirouse, 2013).

Statistical analysis

For all experiments, sample size is indicated in the figure legends or in Supplementary file 3. No statistical method was used to predetermine sample size. Results were obtained from at least two independent experiments, and for each experiment, multiple females were dissected. No randomization or blinding was performed. For each experimental condition,variance was low. Matlab software has been used to perform analysis of covariance to determine the elongation coefficient, and multiple pairwise comparison tests were run to determine the p-value between different conditions (aoctool and multicompare, Statistic and Machine Learning Toolbox). The normality of the samples was calculated using a D'Agostino and Pearson normality test. The unpaired t-test was used to compare samples that had a normal distribution. The unpaired Mann-Whitney test was used to compare samples that were not normally distributed. For comparison of eAR of anterior and posterior poles, a two-way ANOVA test with repeated measures was conducted on both poles and for two genotypes. The post-hoc analysis (two pair-wise Bonferroni tests) was performed. When shown, error bars represent SD. For all figures, p *<0.01, **<0.005, ***<0.001.

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Decision letter

  1. Elisabeth Knust
    Reviewing Editor; Max Planck Institute of Cell Biology and Genetics, Germany

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Jak-Stat pathway induces Drosophila follicle elongation by a gradient of apical contractility" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Both reviewers agreed that there is a lot of new, important information contained within the manuscript, particularly describing the new process of elongation at early stages of oogenesis, but that there is definitely more work to do to make the data presented more solid. We would welcome a substantially revised paper in the future, but the authors should address all of the reviewers' concerns before beginning a new submission.

Additionally:

In the Abstract the authors write that elongation occurs "without planar cell polarity requirement". However, they showed lack of planar polarization just for MyoII and Baz (subsection “MyosinII activity drives apical pulses and early elongation”).

Subsection “Polar cells define the axis of early elongation”, second paragraph: In Pak mutants, the authors often observe a single polar cell cluster (it should be Figure 1E, not 1D) and conclude, that Pak is required for polar cell "positioning". If it is just involved in positioning, I would expect two follicle cell clusters in each case. For me it looks that Pak is required either for polar cell specification or for polar cell survival.

I also had some difficulties to find the link between the JAK-STAT activity gradient and the morphogenetic behavior (intercalation, stretching) described in the second part.

Reviewer #1:

The manuscript by Alegot et al. focuses the elongation of the Drosophila follicle. Previous work established that this morphogenesis depends on a whole-tissue rotation; however, this paper reports that the follicle completes the earliest phase of elongation when rotation is blocked. The authors use two conditions in which the follicle remains spherical (loss of Pak and Mys) to show that the early elongation defect correlates with defects in the positioning of the polar cells. They then perform several experiments that suggest that an Upd signal from the polar cells is required for early elongation. Finally, they present data and propose a model suggesting that a gradient of myosin-based apical contraction emanating from the poles causes a convergence and extension type process in the epithelium.

The ovarian follicle has emerged as a powerful system to identify and study novel mechanisms contributing to tissue elongation. As such, the introduction of a new mechanism that feeds into this process represents an important contribution to this field. Moreover, if the follicle cells really are intercalating with one another in a directional manner in the absence of a planar polarized cue, this observation is likely to be of broad interest to the morphogenesis community. As it stands, however, I have concerns about some of the experiments and their interpretations.

Statistical analyses need to be reported for the graph in Figure 1D. This will likely require increasing the n for many of the fat2 measurements as some of them are currently very low (including one stage that is zero).

Figures 1G and J report aspect ratios using a range of stages (4-8). The range is problematic. If one group has more stage 8 follicles and the other has more stage 4 follicles, it is possible to obtain a false positive result. These types of comparisons can only be made when all of the follicles are at a single stage.

Figure 1K purports to show that the polar cells are mis-positioned because they are not adjacent to the stalk. However, the stalk moves away from the polar cells during mid-oogenesis, a phenomenon that can be seen in the oldest follicle in Figure 1A. The oocyte appears to be mis-localized to the anterior in this follicle, but I am unconvinced that the polar cells are mis-positioned based on the criteria given. Also, the authors state that this follicle has a single small polar cell cluster that contained both WT and eya mutant cells. Are the authors saying that a clone of eya cells in one part of the epithelium eliminates the normal polar cells on the other side? I understand why the authors wanted to do the eya experiment, but given the confusing results, it might be better to remove these data from the paper.

The paper makes extensive use of RNAi transgenes without any controls or references showing the specificity and/or effectiveness of these reagents. At the very least, the authors should confirm that the jak/stat pathway RNAis in Figure 2 reduce the expression of the 10xStatGFP reporter, and the extent to which their sqh RNAi reduces myosin levels via pSqh staining.

The authors mention a Gal4 driver that only drives expression at the follicle poles. In the text it is called Ft-Gal4, whereas in the figures it is called Fru-Gal4. Which notation is correct? Also the authors should either cite a reference showing that this driver is exclusive to the terminal domains or show it themselves with a UAS-GFP reporter.

The authors claim that Figure 2F shows a specific effect of a hop RNAi clone on the posterior half of the follicle. While the anterior is less round than the posterior by eAR analysis, it is far from normal, as the anterior typically has a sharp point at this stage, as shown in Figure 2A. The authors should tone down their claims here.

In Figure 2G, the authors show that over-expressing Upd in the center of the epithelium disrupts follicle elongation. If over-expressing Upd using the Upd-Gal4 driver is sufficient to hyper-elongate the follicle, this result would provide even stronger support for the authors' model. This is an easy experiment that should be attempted.

In Figure 5G it looks as if there are gaps in the epithelium in the sqh clone, a phenotype that was previously documented by Wang and Riechmann (2007). If this is true, it makes it very difficult to interpret the results of this experiment. The authors should confirm that the epithelium is fully intact for all follicles assayed.

Reviewer #2:

In this study the authors analyze the change in shape (elongation) that egg chambers experienced between the early stages (3-7). They also describe changes at the cell and tissue level that happen in that period, and try to understand the causal link between these cellular and tissue changes with the early elongation. It is an original and quite comprehensive study of morphological and molecular changes from stages 3-8, but there are some problems in the study and interpretation that make it difficult for me to believe that the causal link is actually established.

1) Most of the conclusions about the effects on elongation are based on the fact that stage 5-7 mutant egg chambers show a rounder shape than stage 5-7 wildtype egg chambers. Because of this, it is crucial that stage 5-7 are properly identified, especially in mutants, as a mis-identification of an early egg chamber (e.g., stage 4) for an older one (stage 6-7) would have a huge impact in the interpretation of the phenotypes. How are the stages defined? This point is important not only for the mutant egg chambers, but also for wildtype ones. How are the authors staging so precisely stage 3, stage 4, stage 5, and so on? It is not an easy task, as addressed in this paper Sci Rep. 2016 Jan 6;6:18850. doi: 10.1038/srep18850. Automatic stage identification of Drosophila egg chamber based on DAPI images. Jia D et al.)

In several experiments, a mutant egg chamber is defined to be one precise stage, but I doubt that these stagings could have been achieved morphologically, as the mutant egg chambers would also present defects in cell numbers, cell shape, cell fate, egg chamber shape, etc..– that would strongly affect the staging. The authors need to clarify how the staging was done, and also need to characterize stages by molecular markers. I would suggest starting with markers of terminal fate, as well as PH3 stainings, as this would show the mitotic state of cells (follicle cells exit mitosis at stage 6); Staufen, which starts being localized to the posterior at stage 7-8, and maybe for other ideas see the paper I referred to above.

For example: Figure 2C – this egg chamber is to me obviously younger that the control in A), as it can be seen by the shape of the oocyte anterior membrane (a V shape in C), while a straighter membrane in A)), but they are both defined as stage 7.

2) There is a clear correlation between polar cells (PCs) positioning and egg chamber early elongation, and this is an original thought, but I do not think the causal relationship has been proven, and I understand this to be a difficult task. Two of the best experiments for the possible causal link are in Figure 1K and Figure 2F, and for this reason, it should be included a comprehensive description and quantification of the results: What percentage of clones show this phenotype and how many have been analyzed (this is specially missing in Figure 2F)?

3) Regarding the myo2 function in elongation: The authors do a nice job in characterizing actomyo behavior in the apical membrane, and relating this to apical surface changes. I especially liked the original approach to filming the poles. However, the link with elongation is again a hard one to establish: since reducing myo2 activity results in such a huge effect on cell numbers, it is very difficult to conclude that it is the lack of myo2 what is responsible for the defects in elongation. I think the authors need to manipulate the myo2 pathway by other means that might have less of an impact in cell number. For example, manipulating the activity of Rho, Rock, myo2 phosphatases, Myo2LC, etc., I would also like to suggest that the activities of these components of the pathway are both reduced (ag., mutants, dominant negatives), as well as increased (over-expression, dominant active forms, etc.), when possible.

4) Regarding Jak/stat: since the interpretation is that the jak/stat gradient impacts on myo2 and then on elongation, and since the gradient is present in both anterior and posterior poles, the same findings described for posterior pole should be analyzed in anterior poles. It would be ideal to get an idea of the volume and apical surface changes in the anterior pole, as well as on the myo2 behavior there. Also, when studying the effect of the Jak/Stat pathway on the stage 3-7 cellular changes, they need to include analysis not only at stage 7, but stage 3-7. In fact, the described defects at stage 7 do not show much of an effect, and further analysis is required. For example, reducing upd in polar cells or inactivating jak/stat in terminal follicle cells at stage 7 should eliminate the high surface variation in both poles and stage 7 poles should then be similar to stage 7ML. The authors need to check this.

Also, does the jak/stat pathway impact on the gradient of actomyosin contraction at the poles? If possible this needs to be checked, as the surface variation gradient, which may be affected by jak/stat gradient, is not necessarily a complete reflection of actomyo contraction.

Furthermore, no experiment shows that the production of upd by the PCs is required, as there is no experiment showing than when this secretion is affected, elongation is aberrant. The authors need to eliminate, or at least reduce, upd in the PCs to answer this question. In the TjG4, updRNAi experiment, upd is reduced in all follicle cells, but I am not sure it is reduced on PCs, could this be described, please? And if the PCs are the source of upd, why is the reduction of upd in all other follicle cells giving a phenotype? And related to this, why would overexpression of upd in the follicle cells that are not the source, result in defects in elongation?

https://doi.org/10.7554/eLife.32943.033

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

[…] In the Abstract the authors write that elongation occurs "without planar cell polarity requirement". However, they showed lack of planar polarization just for MyoII and Baz (subsection “MyosinII activity drives apical pulses and early elongation”).

There are two ideas associated with this sentence. First, the elongation that we observed is independent of Fat2, which controls the only pcp pathway known the be active in this tissue. Second, apical MyoII is not planar polarized thought it is the motor of elongation. We prefer to maintain this sentence as it is in the Abstract because of the lack of space but we tried to give better explanations in the Discussion: “Moreover, the absence of planar polarization of MyoII in apical, the driving force of early elongation, and the nonrequirement for “basal pcp” strongly argues against a control of this elongation phase via a planar a cell polarity working at a local scale.”

Subsection “Polar cells define the axis of early elongation”, second paragraph: In Pak mutants, the authors often observe a single polar cell cluster (it should be Figure 1E, not 1D) and conclude, that Pak is required for polar cell "positioning". If it is just involved in positioning, I would expect two follicle cell clusters in each case. For me it looks that Pak is required either for polar cell specification or for polar cell survival.

We agree with this comment. We initially wrote that it “suggests that Pak is required for polar cell positioning” because our observation of this phenotype suggests that there are initially two clusters per follicle but that the persistence of stalk cells along follicles (and not only between) that contact the clusters induces somehow their convergence and fusion. However, it is indeed overstated at the level of description that we provide and we therefore modified the sentence accordingly.

I also had some difficulties to find the link between the JAK-STAT activity gradient and the morphogenetic behavior (intercalation, stretching) described in the second part.

JAK-STAT induces a gradient of pulsing from the poles. These pulses induce an apical constriction at the poles, as shown by the reverse phenotype of STAT mutant (cell stretching) and in agreement with what happens in many tissues where pulses induce constriction. Then, the full understanding of the link between JAK-STAT and pulses will require to identify the transcriptional targets of STAT in this tissue and analyse their function. We will definitively try this in the future.

The link between JAK-STAT and intercalations is indeed more difficult to establish. We mentioned these intercalations because they probably explain why the cells are not stretched in the AP axis whereas the follicle elongates. However, there is no indication that they are instrumental for elongation, and they might rather reflect a passive response of the tissue to relax the tensions due to the elongation. Thus, we assume that demonstrating a link between JAK-STAT and intercalation is not essential for our work at this stage.

Reviewer #1:

[…] Statistical analyses need to be reported for the graph in Figure 1D. This will likely require increasing the n for many of the fat2 measurements as some of them are currently very low (including one stage that is zero).

We have increased our sample size and performed a proper statistical analysis. The stage with 0 follicle in fat2 mutant was due to the fact that the WT stages 10A and 10B were plotted separately. We apologise for this mistake.

Figures 1G and J report aspect ratios using a range of stages (4-8). The range is problematic. If one group has more stage 8 follicles and the other has more stage 4 follicles, it is possible to obtain a false positive result. These types of comparisons can only be made when all of the follicles are at a single stage.

This representation was initially chosen because, in both cases, one of the two categories were very rarely observed and it was therefore not possible to provide statistics for each stage. Nonetheless, we agree with this formal criticism, though we had it in mind and paid attention that it actually did not apply to our samples.

We therefore significantly increased our sample size. However, it is still impossible to plot these follicles according to their stages and performed a statistical test for each stage and each category. Rather, we plotted the long axis as a function of the short axis, a method that offers the advantage of no bias due to stage determination (see response to point #1 reviewer #2), and statistically compared the slope of the regression lines, which we defined as “elongation coefficient” (Figure 1 and Figure 1—figure supplement 1). This approach confirms our previous conclusions.

Figure 1K purports to show that the polar cells are mis-positioned because they are not adjacent to the stalk. However, the stalk moves away from the polar cells during mid-oogenesis, a phenomenon that can be seen in the oldest follicle in Figure 1A. The oocyte appears to be mis-localized to the anterior in this follicle, but I am unconvinced that the polar cells are mis-positioned based on the criteria given. Also, the authors state that this follicle has a single small polar cell cluster that contained both WT and eya mutant cells. Are the authors saying that a clone of eya cells in one part of the epithelium eliminates the normal polar cells on the other side? I understand why the authors wanted to do the eya experiment, but given the confusing results, it might be better to remove these data from the paper.

We agree that our data on eya were much more complicated than expected, based on published description of the phenotype. We therefore also agree to remove these data. Also, we added the effect on elongation of Upd:gal4, Upd:RNAi, which, has a significant effect on early elongation (Figure 2). Upd:gal4 being only expressed in polar cells (Khammari et al., 2011), this result causally links polar cells and early elongation.

The paper makes extensive use of RNAi transgenes without any controls or references showing the specificity and/or effectiveness of these reagents. At the very least, the authors should confirm that the jak/stat pathway RNAis in Figure 2 reduce the expression of the 10xStatGFP reporter, and the extent to which their sqh RNAi reduces myosin levels via pSqh staining.

We performed these controls with STAT-GFP for STAT92E and Upd RNAi and observed a strong diminution of the signal (Figure 2—figure supplement 1). Surprisingly, we did not observed an effect of the RNAi against Hop on STAT-GFP. We checked the identity of this line by sequencing and it was ok. It is therefore unclear why this line induced the same kind of elongation and pulsing defects than STAT or Upd RNAi but has no effect on STAT-GFP. We therefore removed all the data obtained with Hop RNAi and replaced all with experiments with STAT92E and/or Upd RNAi, with the exception of the previous Figure 2F. Importantly it did not change our conclusions. We also looked at pSqh in sqh RNAi and it is strongly reduced (Figure 3—figure supplement 1).

The authors mention a Gal4 driver that only drives expression at the follicle poles. In the text it is called Ft-Gal4, whereas in the figures it is called Fru-Gal4. Which notation is correct? Also the authors should either cite a reference showing that this driver is exclusive to the terminal domains or show it themselves with a UAS-GFP reporter.

Fru is the proper abbreviation, sorry for the mistake. Fru:Gal4 expression profile is described in Borensztejn et al., 2013, and we now mentioned it in the text.

The authors claim that Figure 2F shows a specific effect of a hop RNAi clone on the posterior half of the follicle. While the anterior is less round than the posterior by eAR analysis, it is far from normal, as the anterior typically has a sharp point at this stage, as shown in Figure 2A. The authors should tone down their claims here.

This figure corresponded to a flip-out experiment, in which the Hop knocked-down cells are marked by the GFP and are therefore at the anterior, and not the posterior of the follicle. However, this experiment, obtained with Hop RNAi, has been removed. We tried to reproduce it with stat RNAi but we did not manage to find proper conditions.

Nonetheless, it is worth noticing that we have other results indicating that JAK-STAT acts in cell autonomous manner at each pole. STAT has a cell-autonomous effect on apical surface, cell height and phospho-MyoII recruitment (Figure 5 and 6) and MyoII is specifically required at each pole for elongation (Figure 5).

In Figure 2G, the authors show that over-expressing Upd in the center of the epithelium disrupts follicle elongation. If over-expressing Upd using the Upd-Gal4 driver is sufficient to hyper-elongate the follicle, this result would provide even stronger support for the authors' model. This is an easy experiment that should be attempted.

We tried to overexpress Upd with Upd Gal4 but did not observed any effect on elongation. Of notice, we placed STAT-GFP reporter in such flies and did not observed any change in JAK-STAT activation gradient. Thus, this result suggests a mechanism buffering Upd production by polar cells, which might be interesting to look at in the future but is beyond the scope of this article on tissue elongation.

In Figure 5G it looks as if there are gaps in the epithelium in the sqh clone, a phenotype that was previously documented by Wang and Riechmann (2007). If this is true, it makes it very difficult to interpret the results of this experiment. The authors should confirm that the epithelium is fully intact for all follicles assayed.

We agree that gaps in the epithelium could be an issue. It was actually why, for this experiment, we stained for Dlg and not just F-actin. It allowed us to get a better view of the small lateral domains of the cells, and follicles with gaps were excluded from the analysis. This point is now specified in the methods.

Of notice, we now provide data obtained with rok mutant clones, in which the cells are less flatten and gaps are not observed, with the same effect on elongation than with sqh clones.

Reviewer #2:

In this study the authors analyze the change in shape (elongation) that egg chambers experienced between the early stages (3-7). They also describe changes at the cell and tissue level that happen in that period, and try to understand the causal link between these cellular and tissue changes with the early elongation. It is an original and quite comprehensive study of morphological and molecular changes from stages 3-8, but there are some problems in the study and interpretation that make it difficult for me to believe that the causal link is actually established.

1) Most of the conclusions about the effects on elongation are based on the fact that stage 5-7 mutant egg chambers show a rounder shape than stage 5-7 wildtype egg chambers. Because of this, it is crucial that stage 5-7 are properly identified, especially in mutants, as a mis-identification of an early egg chamber (e.g., stage 4) for an older one (stage 6-7) would have a huge impact in the interpretation of the phenotypes. How are the stages defined? This point is important not only for the mutant egg chambers, but also for wildtype ones. How are the authors staging so precisely stage 3, stage 4, stage 5, and so on? It is not an easy task, as addressed in this paper Sci Rep. 2016 Jan 6;6:18850. doi: 10.1038/srep18850. Automatic stage identification of Drosophila egg chamber based on DAPI images. Jia D et al.)

We have indeed seen this very interesting method and we will try to use it in the future. The first figure of this article actually nicely sums up what was already described in Spradling, 1993 (based on King 1970) in which stage definitions is only based on white light (allowing seeing the presence of vitellus or not) and DAPI staining. We used the same criteria as indicated in the Materials and methods.

In several experiments, a mutant egg chamber is defined to be one precise stage, but I doubt that these stagings could have been achieved morphologically, as the mutant egg chambers would also present defects in cell numbers, cell shape, cell fate, egg chamber shape, etc. – that would strongly affect the staging.

As mentioned previously, stage definition was assessed by criteria that are independent of cell number, cell shape or egg chamber shape.

The authors need to clarify how the staging was done, and also need to characterize stages by molecular markers. I would suggest starting with markers of terminal fate, as well as PH3 stainings, as this would show the mitotic state of cells (follicle cells exit mitosis at stage 6); Staufen, which starts being localized to the posterior at stage 7-8, and maybe for other ideas see the paper I referred to above.

DAPI staining is sufficient to detect the arrest of cell division. Moreover, it would be impossible to define a combinatory of markers that allows the unambiguous determination of all the stages. Finally, as mentioned before, the original definition of the stages has been done without any markers excepted DAPI.

However, we understand your concern and we therefore conducted two simple tests:

1) We compared all the mutant genotypes at a stage n to stage n-1 wild-type follicles. Doing so, there is still a significant difference for most of tested genotypes, including actors of the JAK-STAT pathway and MyosinII. In other words, even though we had systematically underestimated the stage of mutant follicles and not the wild-type ones, which I am sure it is not the case, the main conclusions would not be affected.

2) More significantly, we plot the long axis as a function of the short axis for previtellogenic stages and make the regression line as shown on Figure 1—figure supplement 1. We determined the slope of the lines which corresponds to what we defined as an “elongation coefficient”. Importantly, this method is independent of stage determination. We validated this method using fat2 mutant (Figure 1—figure supplement 1A, B). Statistical comparison of the elongation coefficients confirmed our previous results (Figure 1, 2, 3).

For example: Figure 2C – this egg chamber is to me obviously younger that the control in A), as it can be seen by the shape of the oocyte anterior membrane (a V shape in C), while a straighter membrane in A)), but they are both defined as stage 7.

We would have the same visual impression based on V shape versus straight membrane. However, both can be observed at stage 7 (i.e. WT controls on Figure 1 and 2), even on the same follicle at 2µm of differences in the z axis as depicted on pictures of a same follicle (Author response image 1).

2) There is a clear correlation between polar cells (PCs) positioning and egg chamber early elongation, and this is an original thought, but I do not think the causal relationship has been proven, and I understand this to be a difficult task. Two of the best experiments for the possible causal link are in Figure 1K and Figure 2F, and for this reason, it should be included a comprehensive description and quantification of the results: What percentage of clones show this phenotype and how many have been analyzed (this is specially missing in Figure 2F)?

As proposed by reviewer #1, to avoid any confusion due to these results, eya data were removed. Figure 2F was performed with Hop RNAi and was also therefore removed. We tried to reproduce such phenotype with STAT RNAi but for some reasons probably linked to the role of JAK-STAT during follicle budding, we could not defined proper conditions. However, thanks to a later suggestion, we now provided data that clearly link polar cells and elongation. Upd is only expressed in polar cells and Upd:Gal4 reproduces this pattern (Khammari et al., 2011). Knocking-down Upd with this driver affects early elongation and thus polar cells are required for early elongation.

3) Regarding the myo2 function in elongation: The authors do a nice job in characterizing actomyo behavior in the apical membrane, and relating this to apical surface changes. I especially liked the original approach to filming the poles. However, the link with elongation is again a hard one to establish: since reducing myo2 activity results in such a huge effect on cell numbers, it is very difficult to conclude that it is the lack of myo2 what is responsible for the defects in elongation. I think the authors need to manipulate the myo2 pathway by other means that might have less of an impact in cell number. For example, manipulating the activity of Rho, Rock, myo2 phosphatases, Myo2LC, etc., I would also like to suggest that the activities of these components of the pathway are both reduced (ag., mutants, dominant negatives), as well as increased (over-expression, dominant active forms, etc.), when possible.

The question of the primary effect of MyoII on elongation is indeed really important. First of all, one could emphasize that we already affected MyoII by two different means:

- A drastic one with a null mutant, but in clones, which allows us to show a specific role of MyoII at the poles (Figure 5), which is coherent with the proposed role of the pulses in these regions.

- A more subtitle one, with a RNAi in the whole epithelium, which only moderately affects cell shape but still shows an elongation defect (Figure 4).

Nonetheless, as suggested, we also looked at the involvement of the Rho kinase using null mutant clones. These mutant clones recapitulate the effect on elongation observed with sqh, though the flattening of the mutant cells was weaker (Figure 5E, I). Thus, this result confirms both the involvement of the Rho pathway and the primary effect of this pathway on early elongation.

The MyoII gain of function was a very interesting suggestion. We tried by overexpression of the phosphomimetic form SqhEE (and SqhDD) in all the follicle cells, with tj:gal4, or at the poles, with Fru:gal4. In both cases we observed no effect on tissue elongation. These results are actually consistent with the fact the Sqh phosphorylation is homogenous along AP axis whereas pulses are not. Thus, the spatiotemporal control of the pulses by JAKSTAT is not at the level of Myosin II activation and it is therefore logical that Sqh phosphorylation is necessary but not sufficient to modulate tissue elongation. Alternatively, the absence of effect could be due to the fact that these mutant forms of Sqh are not active as proper phosphorylated protein as it has been very recently shown (Vasquez et al., eLife, 2016). Thus, such experiments appear inconclusive and we did not add them in the new version of the article, but it could be done upon request.

An alternative approach for a gain of function of MyoII would have been to use a mutant for the myosin phosphatase. However, it has been recently shown that such mutant follicle cells prematurely undergo basal polarized oscillations (Valencia-Expósito, Nat Com, 2016), which normally start at stage 9 and are likely the driving force for the second elongation phase (He et al., Nat Cell Biol 2010). Consistently, we observed that this mutation leads to a dramatic increase of pSqh staining at the basal domain but not at the apical one during early stages (see the two examples in Author response image 2). The effect on elongation of such mutant might be therefore confusing and was not analysed.

4) Regarding Jak/stat: since the interpretation is that the jak/stat gradient impacts on myo2 and then on elongation, and since the gradient is present in both anterior and posterior poles, the same findings described for posterior pole should be analyzed in anterior poles. It would be ideal to get an idea of the volume and apical surface changes in the anterior pole, as well as on the myo2 behavior there.

We agree that it would be ideal but it turn out that the trick that we used to film the poles cannot be applied to the anterior pole for technical reasons. Each ovariole is initially in a muscle sheath that has to be removed for imaging. To do so, we pull on the anterior tip of an ovariole whereas we maintain the rest of the ovary. Either it leads to the complete removal of the ovariole from the muscle sheath, or it leads to a break of an interfollicular filament and we release only part of an ovariole, with the posterior pole of the last follicle that can be easily observed when it is placed between beads. However, such approach does not allow to obtain “free” anterior poles. The solution would be a mechanical dissection between follicles but it could be then almost impossible to confidently assess whether the poles would be damaged or not.

Therefore, the only new data that we can provide about the anterior pole is the height of the cells. It is also increased, compared to younger stages and to lateral cells, as observed at the posterior (Figure 6I). Since an increase in cell height is classically associated with cell constriction and since both JAK-STAT and MyoII affect cell height in the follicular cells, we assume that similar events are taking place at each poles.

Also, when studying the effect of the Jak/Stat pathway on the stage 3-7 cellular changes, they need to include analysis not only at stage 7, but stage 3-7. In fact, the described defects at stage 7 do not show much of an effect, and further analysis is required. For example, reducing upd in polar cells or inactivating jak/stat in terminal follicle cells at stage 7 should eliminate the high surface variation in both poles and stage 7 poles should then be similar to stage 7ML. The authors need to check this.

Also, does the jak/stat pathway impact on the gradient of actomyosin contraction at the poles? If possible this needs to be checked, as the surface variation gradient, which may be affected by jak/stat gradient, is not necessarily a complete reflection of actomyo contraction.

We agree that it is an important point. We now provide data showing that Upd RNAi:

- reduces pulse intensity at the poles at stage 7-8 (Figure 4I)

- increases cell surface at the poles at stage 7-8 (Figure 6H)

Furthermore, no experiment shows that the production of upd by the PCs is required, as there is no experiment showing than when this secretion is affected, elongation is aberrant. The authors need to eliminate, or at least reduce, upd in the PCs to answer this question. In the TjG4, updRNAi experiment, upd is reduced in all follicle cells, but I am not sure it is reduced on PCs, could this be described, please? And if the PCs are the source of upd, why is the reduction of upd in all other follicle cells giving a phenotype? And related to this, why would overexpression of upd in the follicle cells that are not the source, result in defects in elongation?

As mentioned before, Upd, and Upd:Gal4 are expressed only in the polar cells whereas Tj:gal4 is expressed in all FCs including PCs. We now provided experiments with Upd:gal4 and we obtained similar phenotype.From our point of view, once we have shown that Upd is required for early elongation, the fact that its ectopic expression blocks elongation is actually the best argument that it needs to be expressed in a specific pattern and thus delivering a spatial cue for elongation.

https://doi.org/10.7554/eLife.32943.034

Article and author information

Author details

  1. Hervé Alégot

    GReD Laboratory, Université Clermont Auvergne - CNRS UMR 6293- INSERM U1103, Clermont-Ferrand, France
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    Competing interests
    No competing interests declared
  2. Pierre Pouchin

    GReD Laboratory, Université Clermont Auvergne - CNRS UMR 6293- INSERM U1103, Clermont-Ferrand, France
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon 0000-0003-3858-3152
  3. Olivier Bardot

    GReD Laboratory, Université Clermont Auvergne - CNRS UMR 6293- INSERM U1103, Clermont-Ferrand, France
    Contribution
    Resources, Data curation, Formal analysis, Validation, Visualization, Methodology
    Competing interests
    No competing interests declared
  4. Vincent Mirouse

    GReD Laboratory, Université Clermont Auvergne - CNRS UMR 6293- INSERM U1103, Clermont-Ferrand, France
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    vincent.mirouse@u-clermont1.fr
    Competing interests
    No competing interests declared
    ORCID icon 0000-0001-5823-342X

Funding

Fondation ARC pour la Recherche sur le Cancer (ATIP-Avenir)

  • Vincent Mirouse

Institut National de la Santé et de la Recherche Médicale (ATIP-Avenir)

  • Vincent Mirouse

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank R Basto, M Crozatier, C Dahmann, M Grammont, D Harrison, A-M Pret and E Wieschaus for fly stocks or reagents. This work was funded by the ATIP-Avenir program, Association pour la Recherche contre le Cancer (ARC) and the Auvergne Region. We also thank the confocal imaging facility of Clermont-Ferrand (ICCF) and team members for comments on the manuscript.

Reviewing Editor

  1. Elisabeth Knust, Reviewing Editor, Max Planck Institute of Cell Biology and Genetics, Germany

Publication history

  1. Received: November 9, 2017
  2. Accepted: January 19, 2018
  3. Version of Record published: February 8, 2018 (version 1)

Copyright

© 2018, Alégot et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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