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.
We thank C. Klämbt, T. Lecuit, B. McCartney, D. Montell, T. Schu¨pbach, E. Wieschaus and J. Zallen for generously providing reagents, and F. Robin and E. Munro for assistance with near-total internal reflection fluorescence microscopy. We are also grateful to E. Ferguson and members of the Horne-Badovinac lab for insightful comments on the manuscript and to N. Ellis for illustrations. This work was supported by NIH T32 GM007183 to M.C., a NSF Graduate Research Fellowship to G.R.R-SJ., an ACS Postdoctoral Fellowship (PF-12-135-01-CSM) and an award from the AHA to L.L., an NSERC Alexander Graham Bell Canada Graduate Scholarship to M.J.F., a Canadian Institutes of Health Research operating grant (MOP-272122) to G.T., a Burroughs Wellcome Career Award, American Asthma Early Excellence Award and funding from the Packard Foundation to M.L.G., and grants from the Edward Mallinckrodt, Jr Foundation and NIH (R01 GM094276) to S.H.-B.
Membrane protrusions extend from the leading edge of each follicle cell.Membranes of a stage seven egg chamber expressing Nrg and Indy GFP protein traps. follicle cellswere imaged at the basal surface. Laser-scanning confocal images. 25.5 mins/ 30 sec interval. 190x play back rate. Scale bar,10 μm.
Abi depletion in all follicle cells blocks rotation. Nrg and Indy GFP protein traps mark follicle cell membranes in a stage eight egg chamber. Control egg chambers rotate normally (left) while rotation is blocked when Abi RNAi is expressed in all follicle cells (right). Laser-scanning confocal images. 17.5 mins/ 30 sec interval. 210x play back rate. Scale bar,10 μm.
A small clone expressing Abi RNAi is carried along within the migrating epithelium. The follicle cell membranes of a stage seven egg chamber are marked with the Nrg GFP protein trap. Follicle cells expressing Abi RNAi are marked with RFP. The clone is carried along by wild-type neighbors. Laser-scanning confocal images. 45 mins/1 min interval. 450x play back rate. Scale bar, 10μm
Egg chambers rotate prior to stage five. Stage one, three, and four egg chambers expressing a nuclear marker, His2Av RFP, rotate. A row of cells in each egg chamber is indicated by colored dots through time. Spinning disk confocal images. 180 mins/ 1 min interval. 600x play back rate. Scale bar,10 μm.
fat2 mutant egg chambers do not rotate at early stages. A fat2n103-2 ovariole containing stage one, two, three and four egg chambers that do not rotate during a twenty minute time lapse. FM4-64 dye was used to label cell membranes. Spinning disk confocal images. 20 mins/ 1 min interval. 600x play back rate. Scale bar,10 μm
Global actin bundle alignment of a stage four egg chamber increases as follicle cells migrate. TIRF microscopy of an egg chamber expressing the actin binding domain of Utrophin fused to GFP to visualize F-actin live. Basal actin bundles are initially not well aligned. As the follicle cells migrate, the global alignment of the actin bundles fluctuates. At the end of the time series, the basal actin bundles are globally aligned. 60 mins/ 1 min interval. 515x play back rate. Scale bar,10 μm.
Follicle cell migration is blocked by the Arp2/3 inhibitor. Membranes of a stage seven egg chamber expressing the Indy GFP protein trap. The inhibitor (CK-666, right) or control molecule (CK-689, left) was added at the 20-minute time point (blank frame). The control egg chamber continues to rotate while the inhibitor-treated egg chamber stops rotating within an hour. Spinning disk confocal images. 80 mins/ 1 min interval. 370x play back rate. Scale bar,15 μm.
Acutely blocking follicle cell migration in a stage seven egg chamber with the Arp2/3 inhibitor does not alter global actin bundle alignment. An Arp2/3 inhibitor-treated egg chamber expressing the actin binding domain of Utrophin fused to GFP. The egg chamber is not rotating but the basal actin bundles maintain their global alignment. Images were acquired on a laser-scanning confocal. 21 mins/ 30 sec interval. 250x play back rate. Scale bar,10 μm.