onto a home-made coverslip holder suitable for simultaneously carrying 14 coverslips. Time-lapse analysis For the large-scale screen, after injection, embryos were allowed to develop to stage 13 under oil and were subsequently imaged at 25uC on an Olympus CellR fluorescent microscope equipped with a disc-scanning unit. A 10X objective and an F-View II camera (Soft Imaging System, M u¨nster) were used for time-lapse imaging. Stage positions for each embryo were adjusted manually. Unfertilized eggs or embryos leaking cytoplasm were not imaged. Each embryo was imaged for 13 hours, and images were acquired every 12–15 minutes. Time-lapse movies for each injected embryo were stored as multi-dimensional tiff files and analyzed using ImageJ software. Publication quality images of dsRNA treated embryos were made with Leica TCS SP5 confocal microscope. DsRNA samples were coded, injections and analysis of the movies were performed blind. For the time-lapse movies of pbl mutants, embryos expressing arm:GFP or Moe:mCherry were imaged with an Olympus FW1000 confocal microscope. Geometric parameters of the closure were measured with ImageJ and analyzed with Microsoft Excel and DataFit. Velocity of the epithelial sheet translocation (v), the rate constant of zippering (kz) and the fractional contribution of zippering (fz) to the velocity of the closure were calculated as described previously [22]. Immunohistochemistry Immonostainings were performed as described earlier [49]. Primary antibodies used were anti-Tubulin (1:100, Sigma) and anti Fas3 (1:50, DSHB). To stain actin, embryos were incubated for 2 hrs in rhodamin-phalloidin (2 unit/ml in PBT, Molecular Probes). Specimens were mounted in 50% glycerol/PBS and examined with an Olympus FW1000 confocal microscope. Z- stacks of optical sections were recorded, maximum intensity projections of the optical sections were made with ImageJ, intensity values and color balance were adjusted with GIMP software. Supporting Information Movie S1 Distribution of the EGFP signal in the ZCL0423 protein trap line. Movie shows dorsal closure of an embryo simultaneously expressing ZCL0423-EGFP and mCherry-tagged actin binding domain of Moesin (mCherry:Moe). Moe is shown at the top, ZCL0423 in the middle and the overlay at the bottom with mCherry:Moe in red and ZCL0423-EGFP in green. The mCherry:Moe highlights actin and the protein trap EGFP fusion labels the leading edge of the DME cells. (MOV) Movie S2 Dorsal open phenotypes generated by RNAi. Movies show the absence of dorsal closure of dsRNA injected embryos expressing the ZCL0423-EGFP protein trap fusion protein. Scale bar represents 50 mm. (MOV) Movie S3 Abnormal dorsal closure dynamics generated by RNAi. Movies show abnormal dorsal closure dynamics of dsRNA-injected embryos expressing the ZCL0423-EGFP protein trap fusion protein. Scale bar represents 50 mm. (MOV) Movie S4 Dorsal closure in pbl mutant embryos. Movies show a dorsal view of convergence and zippering of the two opposite epithelial cell sheets. The leading edge of the DME cells is Identification of Genes Involved in Dorsal Closure highlighted by the ZCL0423 protein trap. Scale bar is 50 mm. The movie on the left shows normal closure in a wild type control embryo and the movie on the right shows dorsal closure of a pbl3/ pbl3 mutant embryo. (MOV) Movie S5 Pulsative behavior of the amnioserosa cells in pbl mutants. Pulsation of the amnioserosa cells in wild-type control and in pbl mutant embryos is shown. The cells are outlined by arm:GFP. Scale bars are 10 mm. The movie on the left shows amnioserosa cells in a wild type control embryo and the movie on the right shows amnioserosa cells of a pbl3/pbl3 mutant embryo. (MOV) Movie S6 Cell shape changes in pbl mutant embryos. Epithelial cells expressing arm:GFP are shown in embryos undergoing dorsal closure. Scale bars are 10 mm. The movie on the left shows elongation of epithelial cells in a wild type control embryo and the movie on the right shows elongation of epithelial cells in a pbl3/pbl3 mutant embryo. (MOV) Movie S7 Protrusions of DME cells in pbl mutant embryos. Movie sequences of protrusions forming at the leading edge of the DME cells are shown. Only engrailed expressing epithelial cells are visible due to en-Gal4 driven mCherry:Moe expression. Scale bars are 5 mm. The movie on the left shows cell protrusions in a wild type control embryo and the movie on the right shows protrusions in a pbl3/pbl3 mutant embryo. (MOV) Movie S8 Head involution defects of pbl mutants. Movies show a dorsal view of head involution of embryos expressing arm:GFP. The movie on the left shows head involution of a wild type control embryo and the movie on the right shows a pbl3/pbl3 mutant embryo unable to undergo head involution. Enlargements of the boxed regions show cells shape changes of the wild type and the pbl mutant embryo. (MOV) Table S1 List of genes tested by dsRNA microinjection. Table shows the genes targeted by RNAi, position in the dsRNA library and dsRNA concentration. (XLS) Table S2 Quantitative parameters of dorsal closure of individual embryos. Table shows quantitative parameters of closure dynamics in buffer-injected control embryos, in homozy- gous pbl3 mutant embryos and embryos silenced for pbl and Arf51F. Velocity of the epithelial sheet translocation (v), the rate constant of zippering (kz) and the fractional contribution of zippering (fz) to the velocity of the closure are shown. (XLS) Acknowledgments We thank T. Millard, W. Chia for fly stocks; M. Boutros, K. Spirohn for reagents; and J. Kriston-Vizi, Cs Koncz for helpful discussions on bioinformatics analyses. We also thank Barry J. Irvine for critical reading of the manuscript; Margit Szathma´ri, Ildiko´ Velkeyne´ Krausz for valuable technical assistance; and the members of the Cellular Imaging Facility and J. Szabad for help with microscopy. Author Contributions Conceived and designed the experiments: FJ ME. Performed the experiments: FJ LH AB NK PV. Analyzed the data: FJ LH. Wrote the paper: FJ. PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e22229