Identification of Genes Involved in Dorsal Closure Figure 9. Dorsoventral polarity of the DME cells in pbl mutants. (A and B) Frames from movies of arm:GFP-expressing embryos. Dorsal view is shown, scale bars are 10 mm. (A) Wild-type embryo. (B) pbl3/pbl3 mutant embryo. Asterisk labels the same cell, progressively elongating along the D/V axis. (C–F) Immunofluorescence staining of DME cells in embryos at dorsal closure stage with anti-Fas3 (green in C and D) and anti-tubulin antibody (green in E and F). Phalloidin staining of actin in DME cells (red in C–F). White arrow indicates the intrusions of the basolateral membrane. Maximum intensity projections of optical sections encompassing the whole cell volume are shown. Scale bars represent 10 mm in C and D and 5 mm in E, F. (C and E) Wild-type embryos. (D and F) pbl3/pbl3 mutant embryos. doi:10.1371/journal.pone.0022229.g009 applied in this study provides temporal information on the gene function and enables the identification of genes required for the effective closure of the dorsal hole. Since the large-scale screening strategy combined with live video microscopy presented here is easily adaptable to the analysis of various embryonic developmental processes, future studies could apply it to uncover additional genes involved in morphogenesis. Large scale automated RNAi screens tend to have low reliability. Screens using invasive dsRNA treatment methods such as microinjection of the embryo, and application of high amounts of dsRNAs run the risk of identifying many false positive hits, impeding the efficiency of further functional studies on the identified genes [20]. In order to considerably increase the specificity of our screen we applied very stringent screening criteria by performing four independent experiments and using low dsRNA concentrations. In addition, we considered only those candidates true positives that reproducibly displayed the specific phenotypes with a high penetrance in all technical repeats performed with two different gene-specific dsRNAs. As a result, ten genes have been identified as being positive hits and silencing of 26 genes known to affect dorsal closure did not result in a reproducible defect in the closure process. In our screen, beyond the four known genes (scb, N, shg, cno), six novel genes were shown to influence dorsal closure. Only two of the novel genes (CG6700 and bx42) have the classic dorsal-open phenotype, the complete absence of closure, while silencing of four PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22229