Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber elongation

Abstract

Tissues use numerous mechanisms to change shape during development. The Drosophila egg chamber is an organ-like structure that elongates to form an elliptical egg. During elongation the follicular epithelial cells undergo a collective migration that causes the egg chamber to rotate within its surrounding basement membrane. Rotation coincides with the formation of a 'molecular corset', in which actin bundles in the epithelium and fibrils in the basement membrane are all aligned perpendicular to the elongation axis. Here we show that rotation plays a critical role in building the actin-based component of the corset. Rotation begins shortly after egg chamber formation and requires lamellipodial protrusions at each follicle cell's leading edge. During early stages, rotation is necessary for tissue-level actin bundle alignment, but it becomes dispensable after the basement membrane is polarized. This work highlights how collective cell migration can be used to build a polarized tissue organization for organ morphogenesis.

Figure 1

Overview of key concepts in egg chamber elongation. (a) Illustration of an ovariole, a developmental array of egg chambers. Egg chambers are spherical when they bud from the germarium and then lengthen along their anterior-posterior axes as they develop. (b) Blowup of the boxed region in (a) highlighting the apical-basal axis of the follicle cell epithelium. (c) The “molecular corset” consists of parallel arrays of actin bundles at the basal epithelial surface (stage nine) and fibril-like structures in the adjacent basement membrane (stage seven). Laser-scanning confocal images. Scale bar, 10 μm. (d) Transverse section through a stage seven egg chamber, as shown by the dashed line in (a). The egg chamber rotates within the surrounding basement membrane (illustration adapted from Rørth, 2012).

Figure 2

Filopodia and lamellipodia form the leading edge of each migrating follicle cell. Laser-scanning confocal images were taken at the basal surface of stage seven and eight egg chambers. Scale bars, 10 μm. (a) The actin-binding domain of Moesin (MoeABD mCherry) was expressed in a clone of follicle cells (red) and the total F-actin population was labeled with phalloidin (green). Protrusions extend from only one side of each cell (yellow arrow) and not the other (green arrow). (b) Neuroglian (Nrg) and I’m not dead yet (Indy) GFP protein traps were used to visualize follicle cell membranes. Live imaging shows that membrane protrusions extend at the leading edge of the follicle cells. A single leading edge is indicated by the yellow arrow at 0 and 10 minutes. The arrows show that the protrusive activity and migration both occur in a downward direction. (c) Endogenous Ena localizes to the tips of filopodia. (d) Endogenous SCAR localizes more broadly within the leading edge protrusions, revealing the lamellipodia. (e) In a SCAR^Δ37 mosaic egg chamber, the leading actin networks underlying the protrusions are absent in follicle cells lacking SCAR (white arrows). Control cells (blue) maintain SCAR localization with the leading edge protrusions (yellow arrow). (f) Clonal expression of AbiRNAi (blue) also eliminates SCAR enrichment and protrusions at the leading edge.

Figure 3

SCAR-dependent lamellipodia promote egg chamber rotation. (a) Live imaging was used to determine the contributions of lamellipodia (Abi) and filopodia (Ena) to follicle cell (FC) migration. Expressing AbiRNAi in all follicle cells throughout oogenesis with the traffic jam-Gal4 (tj-Gal4) driver blocks the rotation of stage seven-eight egg chambers, whereas enaRNAi has no effect. Individual data points, mean ± SEM, t-test: P*** = 1.7×10⁻⁷. (b) AbiRNAi egg chambers fail to elongate properly beginning at stage six, whereas enaRNAi does not disrupt elongation. The aspect ratio is the length of the egg chamber divided by its width. For stages 4–10 n ≥ 6, stages 11–13 n ≥ 3, stage 14 n ≥ 25 (exact n values are in Supplementary Table 2). Data represent mean ± SD, t-test: **P < 0.003, ***P < 0.0008. (c) The basement membrane fails to become polarized when rotation is blocked by Abi depletion. Basement membrane structure is visualized using a GFP protein trap in the Collagen IV (Col IV) α2 chain, Viking (Vkg GFP). (d) Expression of AbiRNAi with tj-Gal4 eliminates leading edge structures and disrupts the tissue-level alignment of the basal actin bundles. (c, d) Laser-scanning confocal images of stage seven-eight egg chambers. Scale bars, 10 μm.

Figure 4

Egg chamber rotation begins at stage one. (a) SCAR is enriched at one side of each follicle cell during early rotation stages. Scanning confocal image of the basal epithelial surface of a stage two egg chamber. (b) Stills from a time series of His2Av RFP-expressing egg chambers that show rotation occurring at stages one (0.10 μm/min), three (0.34 μm/min) and four (0.32 μm/min). The colored dots mark the same set of nuclei through each 30-minute interval. Spinning disk confocal images. (c) Quantification of follicle cell (FC) migration rate. Each data point represents and individual egg chamber with mean ± SD. Scale bars, 10 μm.

Figure 5

Method for determining the global alignment of the basal bundles in a given epithelium. (a) Representative image of a stage five egg chamber where the actin is labeled with phalloidin (left panel). The pattern obtained by segmenting the epithelium into individual cells (center panel). To analyze actin bundle alignment, individual cells are broken into subcellular windows of 6.74 μm² (right panel, windows are not shown to scale). (b) Representative image of the basal actin bundles in a subcellular window as defined in (a). Scale bar, 2 μm. The 2D Fast Fourier Transform (FFT) of the actin image where the red line indicates the principal direction of the FFT, orthogonal to the orientation of actin bundles (center panel, green line). Director indicating the local alignment of the actin image (left panel). (c) Magnification of the epithelial region within the green rectangle in (a). Directors (green and magenta lines) indicate the local alignment of actin bundles within each cell. Directors deviating from the mean by more than a standard deviation (magenta lines) were discarded (See Methods). (d) Director field showing the mean orientation (yellow lines) of the green directors within each cell shown in (c). (e) The order parameter (S) is calculated by comparing the relative angle between all directors in an epithelium.

Figure 6

Egg chamber rotation maintains the tissue-level alignment of the basal actin bundles. (a) Representative images of basal actin bundle alignment in control vs. AbiRNAi epithelia at different stages with the corresponding order parameter value (S). Yellow lines represent the mean orientation of the basal actin bundles in each cell. Spinning disk confocal images. (b) Graph showing the average order parameters for control (blue line) vs. AbiRNAi (red line) epithelia at stages one through eight. For stages 1–6 n ≥ 8, stage 7 n ≥ 5, and stage 8 n ≥ 4 (exact n values are in Supplementary Table 2). Data points represent mean ± SEM, t-test: **P < 0.003, ***P < 1.95×10⁻⁷. A t-test compares the difference between control and AbiRNAi epithelia at each stage. (c) Graph showing the average order parameters for wild-type (blue) vs. fat2^n103-2 (red) epithelia at stages one through eight. For stage 1 n ≥ 4, 2–5 n ≥ 7, 6 n ≥ 5, 7 n ≥ 8, 8 n ≥ 4 (exact n values are in Supplementary Table 2). Data points represent mean ± SEM, t-test: *P = 0.012, ***P < 5.96×10⁻⁷. A t-test compares the difference between wild-type and fat2^n103-2 epithelia at each stage. (d) Basal actin bundles within the germarium are globally aligned in control, AbiRNAi, wild-type and fat2 conditions. Spinning disk confocal images. (e) Stills from a sixty-minute near TIRF time series showing the basal actin bundles in a stage four egg chamber. The actin binding domain of Utrophin fused to GFP marks F-actin. The order parameter fluctuates, but there is a trend toward global actin bundle alignment. The colored lines show the mean actin bundle orientation for each cell. Rows of cells sharing the same color can be followed through time to visualize the downward migration of the epithelium. Scale bars, 10 μm.

Figure 7

Rotation is specifically required to maintain tissue-level actin alignment during early stages. (a) Late expression of AbiRNAi using mirror-Gal4 (mirr-Gal4) does not affect the basal actin pattern at stage six but by stage eight, the leading edge actin network is eliminated without altering basal actin bundle alignment. One copy of SCAR was also removed in this genetic background. Laser-scanning confocal images. (b) Kymographs of rotating egg chambers where the follicle cell membranes are marked with Indy GFP. The slopes of the individual lines indicate the rate at which the cell membranes are moving over time. After 20 minutes, the samples were treated with either with the Arp2/3 inhibitor CK-666 or the control molecule CK-689 (asterisk). The inhibitor stops follicle cell migration within one hour. n = 5 for each condition. Spinning disk confocal images. (c) Representative images of the basal actin bundle alignment after one hour of drug treatment during each of the given stages with the corresponding order parameter value (S). Yellow lines represent the mean orientation of the actin bundles in each cell. Spinning disk confocal images. (d) Graph showing the average order parameter for the basal actin bundles for stages one through eight after one hour of drug treatment. The value is reduced in inhibitor-treated samples compared to controls only during early stages. During stages six to eight, the two values are indistinguishable. For stage 1 n ≥ 2, stages 2–3 n ≥ 6, stages 4–7 n ≥ 10, stage 8 n ≥ 4 (exact n values are in Supplementary Table 2). Data points represent mean ± SEM. The t-test compares the difference between the basal actin bundle order parameter in control and inhibitor-treated samples at each stage (**P = 0.009, ***P = 7.90×10⁻⁵). When stars are not shown, there was no statistically significant difference observed. (e) Representative images of basement membrane polarization (Col IV, Vkg GFP) with the corresponding order parameter value. Spinning disk confocal images. (f) Overlay of graphs from Fig. 7d and Supplementary Fig. S5e showing that the period when rotation becomes dispensable for the tissue-level actin alignment coincides with the period of highest basement membrane polarization. Scale bars, 10 μm.

Figure 8

Model for the formation of the actin-based component of the molecular corset for egg chamber elongation. Tissue-level alignment of the basal actin bundles is established in the germarium. Slow rotation occurs between stages one and five. This phase of rotation is required to maintain tissue-level actin bundle alignment. Fast rotation occurs between stages six and eight but is dispensable for the maintenance of the global actin pattern. This phase correlates with the polarization of the basement membrane, which may function to stabilize the actin pattern. Tissue-level polarization of the actin bundles and the basement membrane is maintained after migration stops at stage nine and the oscillating actin bundle contractions begin.