Figure 8. Amnioserosa dynamics in pbl mutants. (A and B) Frames from movies of arm:GFP-expressing embryos. Dorsal view is shown, scale bars are 20 mm. (A) Wild-type embryo. (B) pbl3/pbl3 mutant embryo. (C) Quantification of amnioserosa cell contraction in a wild- type control embryo (n = 22 cells in 7 embryos) and a pbl3/pbl3 mutant embryo (n = 24 cells in 6 embryos). Bars indicate standard deviation. doi:10.1371/journal.pone.0022229.g008 abnormal head cuticles. The embryos died showing holes in the head region of the cuticle, suggesting a role of pbl in head involution as well (Figure 6). Immunostaining of Fas3 in the mutant embryos revealed that although the epithelial sheets met at the dorsal midline and covered the dorsal hole, the head segments of the embryo did not involute, and the head region of the embryo was not covered by epithelium (Figure 11). In vivo imaging of head morphogenesis in wild-type and pbl mutant embryos expressing arm:GFP revealed a role for pbl in coordinating cell shape changes in the dorsal epithelium and in the involuting tissue (Figure 11, Movie S8). In wild-type embryos, during head involution the epithelium migrates anteriorly and covers the involuting head segments. Live imaging revealed that this requires coordinated cell shape changes both in the dorsal epithelium and in the so-called acron region, the unsegmented anterodorsal part of the head. In the wild-type embryos, the epithelial cells, which became elongated along the D/V axis during dorsal closure, adopted a more cubical shape during their anterior displacement in head involution. In pbl mutant embryos, however, the epithelium failed to migrate anteriorly. The epithelial cells were stretched by the Identification of Genes Involved in Dorsal Closure amnioserosa contraction pointing towards the region of the dorsal midline where closure took place, but after completion of the closure most of the epithelial cells remained elongated and did not move anteriorly (Figure 11, Movie S8). Cells in the acron region also displayed characteristic shape changes during head involution. In the wild-type embryos, the cells at the dorsal midline became elongated along the A/P axis, whereas lateral cells elongated medio-posteriorly. As head involution proceeded, cells reduced their apical surface size and were occasionally extruded from the tissue. In pbl mutant embryos, a disorganized acron structure was detected. The cells were larger than in the wild type embryos and had abnormal shapes. Live imaging of the pbl mutant embryos revealed that, despite their morphological abnormalities, cells in the acron region were able to contract and reduce their apical surface but in an uncoordinated manner (Figure 11, Movie S8). Although some cells were stretched medio-posteriorly by the contraction of the amnioserosa, involu- tion did not take place. Contraction and stretching of the acron cells often caused ripping of the continuous head tissue indicating defects in cell adhesion in pbl mutants (Figure 11, Movie S8). In summary, our observations suggest that there are at least two causes underlying abnormal dorsal closure in pbl mutants. Firstly, dorsal closure defects are a direct consequence of abnormal cytoskeletal dynamics in DME cells. Secondly, dorsal closure might be indirectly affected by the abnormal head involution in pbl mutants suggesting a tight genetic and mechanic coupling between these two morphogenetic processes. Discussion The goal of the present study was to investigate the genetic network regulating dorsal closure of the embryonic epithelium. To achieve this goal, we aimed to identify genes not previously implicated in dorsal closure. A high-throughput functional genomic screen was designed for this purpose and performed on a large scale. Our screening strategy was based on the systematic reduction of gene function by RNAi and the subsequent automated in vivo time-lapse imaging. This high-content assay provided both spatial and temporal information on gene activity and enabled a more comprehensive analysis of gene function. Using this approach we were able to identify not only genes essential for sealing of the epithelial sheets, but also genes which regulate the dynamics of the closure process. In the post-genomic era, application of high-throughput RNAi has enabled the identification of gene functions at the genomic scale. Several dozen high throughput-screens have been performed on Drosophila and human, but these screens were typically based on cell cultures [17]. Although assay systems using cell cultures may provide valuable insights on the processes investigated, they lack the complexity of an intact developing animal and have, therefore, limited adaptability at the organism level. For example, morpho- genetic movements typically require the coordinated effort of many supracellular activities, i.e., the rearrangement of the cells or the interactions of various tissues. Application of high-throughput RNAi in intact Drosophila embryos enabled the functional genomic analysis of such a complex developmental process as the dorsal closure of the embryonic epithelium. It has been suggested previously that the dorsal hole has to be closed in a well-defined, efficient manner [22][48]. Defects of closure dynamics, although they do not necessarily cause morphological abnormalities, might be detrimental on an evolutionary scale. Therefore, to gain a complete overview of the genetics of dorsal closure, the presence or absence of the larval cuticle hole can not be used as the sole screening criterion. Combination of RNAi screening with time-lapse microscopy PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22229