Fly stocks and genetics
The following stocks containing fluorescent fusion proteins were used: Spider-GFP
(95–1) and Resille-GFP
(117–2) (Morin et al., 2001
), membrane-mCherry (this paper), myosin-GFP (sqh-GFP
; Royou et al., 2002
), and myosin-mCherry (sqh
; Martin et al., 2009
). To examine cell shape in embryos devoid of a-p polarity, we used the stock w;
Resille-GFP; bicoidE1 nanosL7 torso-like146
/TM3; Sb. We analyzed embryos from mothers that were homozygous for bicoidE1 nanosL7 torso-like146.
To generate armM/Z
mutants, we created arm043A01
germ-line clones using the FLP-DFS system (Chou and Perrimon, 1992
). We visualized myosin II in armM/Z
mutants by generating a stock that was arm043A01 FRT101/FM7; sqh-GFP
. We crossed females of this genotype to w ovoD FRT101
males to obtain arm043A01 FRT101/w ovoD FRT101
/+ females. These females were heat shocked as larvae for 2 h at 37°C each day to induce mitotic recombination in the germ line. We imaged embryos from the following cross: arm043A01 FRT101/w ovoD FRT101
/+ females x FM7/+; flp-138
/+ males. Half of these embryos showed loss of cell–cell adhesion, which is consistent with half being rescued zygotically.
Construction of membrane-mCherry
A membrane-mCherry marker was created by fusing the N-terminal 20 amino acids of the rat Gap43 gene, which contains a myristoylation sequence, to mCherry. This membrane-mCherry fusion was cloned into a pBluescript vector containing the sqh
promoter and 3′ untranslated region (Martin et al., 2009
). The 3-kilobase KpnI/XbaI sqh5′
fragment was then inserted into a transformation vector containing the attB site (pTiger, courtesy of S. Ferguson, State University of New York at Fredonia, Fredonia, NY). The resulting construct was sent to Genetic Services, Inc., for integration into the attP2 site using the phiC31 integrase system (Groth et al., 2004
Live cell imaging
To prepare embryos for live imaging, embryos were dechorionated with 50% bleach, washed with water, and mounted ventral side up on a slide covered with embryo glue (Scotch tape resuspended in heptane) between two No. 1.5 coverslip spacers. A coverslip was placed over the embryo and Halocarbon 27 oil was added to the resulting chamber. The spacers prevented the overlying coverslip from contacting the ventral surface of the embryo such that the embryo was not compressed. Embryos imaged under these conditions developed normally and subsequently hatched, demonstrating that our imaging conditions had minimal impact on development. All imaging was performed in Halocarbon 27 oil at room temperature (~23°C).
Spider-GFP, Resille-GFP, sqh-mCherry;Spider-GFP, and sqh-GFP; membrane-mCherry videos were obtained with a confocal microscope (SP5; Leica), a 63×/1.3 NA glycerine-immersion objective (Leica), an argon ion laser, and a 561-nm diode laser. Images were acquired using a pinhole setting from 1–2 airy units. For multichannel imaging, we set the excitation bandpass to 495–550 nm to detect GFP and 578–650 nm to detect mCherry. sqh-GFP single-channel videos were obtained using the aforementioned imaging system or with a spinning disk confocal microscope (Ultraview; PerkinElmer) controlled with Volocity Acquisition software (Improvision), a 60×/1.4 NA oil-immersion objective (Nikon), an argon/krypton laser, and an electron-multiplying charge-coupled device camera (C9100-13; Hamamatsu).
Embryos were prepared for live imaging and were imaged using an Ultraview spinning disk confocal microscope equipped with a 63×/1.4 NA oil-immersion lens controlled with MetaMorph software (Universal Imaging). An N2 Micropoint laser (Photonics Instruments) tuned to 365 nm was focused on sqh-GFP structures on the ventral surface of the embryo to ablate actomyosin structures. Point ablations were performed by ablating isotropic sqh-GFP spots with a point ~1 µm in diameter, which took ~670 ms. Line ablations were performed by making nine sequential point ablations to make a 20-µm incision, which took ~6.4 s. Z stacks were acquired immediately before and after ablation in order to measure displacement of myosin II structures upon release of tension.
Image processing and analysis
Images presented were processed using ImageJ (http://rsb.info.nih.gov/ij/
) and Photoshop CS (Adobe Systems, Inc.). A Gaussian smoothing filter with a radius of one pixel was used to reduce noise in published images. Myosin II images presented in all figures are maximum intensity z projections of the apical ~5 µm of cells in the middle of the image. Because myosin II is almost entirely present on the apical surface of cells, these images represent a surface projection of the embryo. Images of cell outlines are z slices ~2 µm below the apical surface.
Image segmentation was performed using custom MATLAB (MathWorks) software. Raw images were bandpass filtered with effective cutoff wavelengths of ~1.4 µm (low pass) and ~16 µm (high pass). Images were then thresholded and skeletonized to reduce the width of the membranes to one pixel. Cells were then indexed and tracked based on the distance between cell centroids at subsequent time points. We manually removed cells with errors in the segmentation to ensure that all cells in the dataset were correctly identified. Aspect ratio and anisotropy were calculated by using the “regionprops” function in MATLAB to measure major axis length, minor axis length, and orientation for individual cells. We measured the intensity of cortical myosin II in individual cells using three-dimensional time-lapse videos that were ~5 µm in depth. To separate cortical myosin II structures from the diffuse cytoplasmic staining, we smoothed sqh-GFP images using a Gaussian smoothing filter with a three-pixel kernel size, s = 0.5 pixels, and clipped intensity values three standard deviations above the mean. We then made maximum-intensity z projections of myosin II (averaging the two highest-intensity values) and integrated the intensity of all the pixels in a given cell. Data for apical area, myosin intensity, and anisotropy were smoothed using a Gaussian smoothing filter (σ = 18–24 s, three time points) to remove noise.
Recoil of sqh-GFP
structures after laser ablation was quantified using custom software in which myosin spots were hand selected and tracked from the pre-ablation frame to the post-ablation frame (Fernandez-Gonzalez et al., 2009
To measure the continuity of the supracellular meshwork in control-injected and twiRNAi embryos, we thresholded maximum-intensity projections of sqh-GFP images using the mean pixel intensity as a cutoff. We then used the “bwmorph” function in MATLAB to identify connected objects and identified the largest object at each time point in a time-lapse video.
Embryo fixation and staining
To visualize cytoskeletal structures, embryos were fixed with 8% paraformaldehyde/heptane for 30 min, manually devitellinized, stained, and mounted in AquaPolymount (Polysciences, Inc.). Endogenous sqh-GFP fluorescence was used to visualize myosin II, and Alexa Fluor 568 phalloidin (Invitrogen) was used to visualize F-actin. Drosophila E-Cadherin (DCad2) was recognized using rat anti-DCad2 (Developmental Studies Hybridoma Bank) at a dilution of 1:50. Snail and neurotactin double stainings were performed in heat-fixed embryos using mouse anti-neurotactin (BP106; Developmental Studies Hybridoma Bank) and rabbit anti-Snail (a gift from M. Biggin, Lawrence Berkeley National Laboratory, Berkeley, CA) at dilutions of 1:100 and 1:1,000, respectively.
Embryos were dechorionated with 50% bleach and fixed for 25 min at room temperature (23°C) with a 1:1 mixture of 25% glutaraldehyde in 0.1 M cacodylate buffer and heptane. The vitelline membrane was then manually removed with a needle, and embryos were dehydrated by gradually stepping up the concentration of ethanol (25%, 50%, 75%, 95%, and 100%). Embryos were then incubated for 10 min with a 1:1 mixture of ethanol and tetramethylsilane (TMS), and then with 100% TMS. The TMS was allowed to evaporate, and we transferred the embryos to the microscope stand and performed metal coating using a Desk II Sputterer (Denton Vacuum). Samples were imaged using a tabletop scanning EM (TM-1000; Hitachi).
Primers for dsRNA were designed with E-RNAi (Arziman et al., 2005
). Primers included the sequence of the T7 promoter (5′-TAATACGACTCACTATAGGGAGACCAC-3′) followed by the following recognition sequences: Arm-F, 5′-CCTGGTTACCATAGGCCAGA-3′; Arm-R, 5′-TGCCATCTCTAACAGCAACG-3′; ECad2-F, 5′-GAGAGGAGGCAACAGAAACG-3′; ECad2-R, 5′-GGACATACTCTCTAGCGGCG-3′; α-catenin-F, 5′-AAGCTGCAAAATCGGGTAATGAAAA-3′, α-catenin-R, 5′-TCTAAGACTCGTTTGGTGTAAATAC-3′; Fog-F, 5′-TGGTGACCAGTTCTCTTTCC-3′; Fog-R, 5′-TGTTGCAGTTGCCGAAGT-3′; T48-F, 5′-CCGCCGGCTACTTGGA-3′; and T48-R, 5′-GAAAGAAGTCGATAAGCTGG-3′. For twistRNAi
, and control CG3651RNAi
, we used the Twi01, Sna01, and control primer pairs (Martin et al., 2009
). Primer pairs were used to amplify a PCR product from genomic DNA. PCR products were used in a transcription reaction with T7 polymerase using the MEGAscript transcription kit (Applied Biosystems). Annealing was performed by adding 10 mM EDTA, 0.1% SDS, and 0.1M NaCl to the reaction and incubating this mixture in a water bath heated to >90°C, which was allowed to cool for several hours. The dsRNA was purified by phenol/chloroform extraction and resuspended in injection buffer (5 mM KCl and 0.1 mM sodium phosphate, pH 7.0). Newly laid embryos were injected laterally and incubated for 2.5–3 h at room temperature (23°C) before imaging gastrulation. We injected AJ dsRNA at a concentration of ~1 mg/ml. However, tears in the myosin II network could be observed even after diluting dsRNA to a lower concentration. For most experiments, Arm dsRNA was used at 0.2 mg/ml. For double RNAi experiments, Arm dsRNA was used at 0.2 mg/ml, and Twist and Snail dsRNA was used at 2 mg/ml. For triple RNAi experiments, Arm dsRNA was used at 0.2 mg/ml, and Fog and T48 were used at 0.8 mg/ml and 0.65 mg/ml, respectively.
Online supplemental material
Fig. S1 shows that knockdown of E-cadherin, Arm, or α-catenin disrupts the myosin II network and results in membrane tethers. Fig. S2 shows myosin-GFP displacement as a function of time for the two types of laser ablation experiments shown in . Video 1 shows myosin-GFP in wild-type and armM/Z
mutant embryos. Video 2 shows laser ablations in myosin-GFP embryos. Video 3 shows myosin-mCherry and membrane-GFP in an armRNAi
embryo. Video 4 shows the contraction of a myosin II fiber in an armM/Z
mutant embryo. Video 5 shows the unrestrained myosin II contraction that occurs after tearing in an armRNAi
embryo. Video 6 shows membrane tether formation in a membrane-GFP embryo. Video 7 shows myosin-GFP in a double RNAi control arm-CG3651RNAi
embryo. Video 8 shows the suppression of tears and loss of cell–cell adhesion in a myosin-GFP arm-snaRNAi
embryo. Video 9 shows that contraction pulses continue despite loss of cell–cell adhesion in a myosin-GFP arm-twiRNAi
embryo. Video 10 shows the suppression of epithelial tears in a membrane-GFP arm-twiRNAi
embryo. Online supplemental materials is available at http://www.jcb.org/cgi/content/full/jcb.200910099/DC1