identify gene function systematically and rapidly in Drosophila and in many other organisms [17–21]. Therefore, we carried out a large-scale RNAi-based genetic screen to identify genes regulating embryonic dorsal closure. It has been shown that several forces provided by various tissues contribute to dorsal closure, and loss of one of these forces can be compensated by the others [22]. In these cases the opening is closed completely, but the dynamics of the closure is abnormal. A description of these abnormalities requires a quantitative analysis of the phenotype using a mathematical model. Dynamic parameters such as length and width of the dorsal hole have to be measured and displacement velocity of the epithelium or fractional contribution of the various forces can be determined [22]. Since the previous studies used solely a dorsal opening in the larval cuticle as a phenotypic output, mutations with defects in the closure dynamics were not revealed. To overcome this limitation, we have applied a high-content screening strategy providing detailed temporal information about the dynamics of the phenotype. We combined large-scale RNAi screening with automated time-lapse video microscopy and monitored the dynamics of the closure process in living dsRNA-treated embryos. Here we describe a genomic-scale RNAi-based loss-of-function screen for genes involved in embryonic dorsal closure. The application of automated in vivo video microscopy to detect phenocopies enabled us to identify genes not only essential for the closure but also genes that affect closure dynamics. We have identified novel dorsal closure genes involved in various biological processes, including small GTPase regulation, signal transduction, vesicle trafficking and embryonic patterning. Furthermore, we present a detailed cell biological analysis of the multifunctional guanine nucleotide exchange factor (GEF) pbl. Pbl affects dorsal closure dynamics both directly by regulating actin dynamics of the closing epithelium and indirectly as an essential regulator of head involution. Identification of Genes Involved in Dorsal Closure Results RNAi screen revealed the role of six novel genes in dorsal closure To identify novel genes involved in embryonic dorsal closure, we used RNAi-based genetic screening coupled with in vivo fluorescent video microscopy. Early Drosophila embryos were microinjected with dsRNAs and allowed to develop until stage 13, when germ band retraction begins. Treated embryos were then subjected to live cell imaging and the whole closure process was recorded. To visualize the leading edge, the protein trap line ZCL0423 was used which specifically labels the first row of cells in the dorsally-migrating epithelial sheets enabling easy and quick screening of the closure process [23]. In the ZCL0423 homozy- gous embryos, the GFP signal appeared after completion of germ band retraction in the dorsal-most epithelial (DME) cells and co- localized with the actin cables. After dorsal closure was complete, the GFP signal disappeared from the epithelial cells (Figure 1, Movie S1). For the large-scale screening, we individually silenced a large number of genes and tested their involvement in dorsal closure by in vivo fluorescent confocal video microscopy. To increase the efficiency of the screening, genes were preselected which have been shown to be expressed during embryogenesis [24]. Specific dsRNAs for 2,520 genes were microinjected into embryos and time-lapse images were collected (Table S1). Initially, 32 embryos were injected with each dsRNA and on average 21 embryos were imaged in vivo. Image sequences were collected into movies and analyzed by visual inspection. For a more detailed analysis, in some cases the length and width of the dorsal holes were also measured. Phenotypic abnormalities were recorded into a database, the identified genes were classified by phenotypic category and the penetrance of the morphological defects was determined (Table 1). Figure 1. Distribution of the GFP signal in the ZCL0423 protein trap line. (A and B) Frames from movie sequences of ZCL0423/+; 69B-Gal4/ UAS-mCherry:Moe embryos undergoing dorsal closure. Embryos coexpress the ZCL0423 protein trap EGFP fusion and the mCherry-tagged actin binding domain of Moesin (mCherry:Moe). (A) Expression of mCherry:Moe driven by the 69B-Gal4 driver in the epithelium highlights actin. (B) The protein trap EGFP fusion is specifically expressed in the DME cells, where it labels the leading edge. (C) Merged images, GFP in green, mCherry in red. doi:10.1371/journal.pone.0022229.g001 PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e22229