Table 1. Summary of RNAi phenotypes of the identified genes. Identification of Genes Involved in Dorsal Closure Penetrance of the RNAi phenotype (%) Biological function Reference Group I Group II Gene name Bx42 CG6700 canoe Notch scab shotgun Kru¨ppel patched pebble ADP ribosylation factor 51F 77.9615.5 73.6629.6 85.4610.8 89.269.4 83.3616.8 92.968.4 42.8619.0 62.4612.9 61.5620.0 70.5611.9 doi:10.1371/journal.pone.0022229.t001 To increase the reliability of our screen, multiple independent tests were performed. Silencing was repeated at least two times with the dsRNAs targeting the same region of the gene product. When the penetrance of the mutant phenocopy of the injected embryos reproducibly exceeded 30% in all of the independent experiments, the gene was selected as a candidate for further analysis. By using this strategy, silencing of 12 genes resulted in abnormal dorsal closure. To avoid a potential effect of the ZCL0423 gene trap- marker in the closure process, these 12 genes were again silenced in embryos constitutively expressing the GFP-tagged actin-binding domain of Moesin (sGMCA) [25]. Live imaging of the RNAi- treated sGMCA embryos revealed that silencing of the majority of the selected genes (11/12) reproducibly caused a dorsal closure defect. To avoid false-positive results caused by off-target effects, 11 candidate genes were repeatedly silenced with dsRNAs targeting a different region of these genes. Therefore, new dsRNAs were designed, synthesized and microinjected into sGMCA embryos. Live imaging of the embryos treated with the new dsRNAs revealed that silencing of 10 out of the 11 candidate genes reproduced dorsal closure defects confirming their role in dorsal closure (Table 1). Dorsal closure defects that have not been described previously were found for six genes, whereas four genes have been previously implicated in dorsal closure. In summary, in these series of experiments dsRNAs covering more than a third of the embryonic transcriptome were injected and a large data set of ,60,000 time-lapse movies were produced and analyzed For the candidate genes, a large number (,100) of embryos were injected with each dsRNA in several independent experiments and very stringent screening criteria were used. Our multiple independent RNAi-screening strategies, combined with a sensitive in vivo phenotyping method, uncovered a novel role for six genes in dorsal closure (Table 1). Group I genes are required for closure of the dorsal hole The identified genes were grouped into two phenotypic categories. In the first phenotypic group, dorsal closure is incomplete and the dorsal hole is not closed (Group I). Silencing of Notch (N), Bx42, shotgun (shg), scab (scb), canoe (cno), and CG6700 genes resulted in this phenotype (Figure 2, Movie S2). Loss-of- function mutations in N, shg, scb and cno have previously been shown to affect dorsal closure [26–29]. Silencing of these genes by signal transduction unknown cell adhesion signal transduction cell adhesion cell adhesion vesicle trafficking pattern formation pattern formation cytoskeleton regulation this study this study [28], [59] [27] [26] [29] this study this study this study this study RNAi phenocopies the previously-described abnormalities of the loss-of-function mutants indicating the specificity of our screening approach. In this phenotypic category, in addition to the four known genes, two novel genes, CG6700 and Bx42, were found to be involved in dorsal closure. Microinjection of dsRNA specific to CG6700 resulted in a severe closure defect (Figure 2, Movie S2). Closure was initiated, the straight movement front of the epithelium was formed, the opposing sheets approached the dorsal midline but some time later closure became arrested. CG6700 is a gene of unknown function and encodes a conserved protein containing a SAC3/GANP domain at the C-terminus. This domain has been shown to be present in proteins with diverse functions such as nuclear export factors (SAC3 of the budding yeast or mammalian GANP/MCM3-associated proteins), eukary- otic translation initiation factor 3 (eIF-3 p25), or regulators of the 26 S proteasome (Nin-1). None of these biological processes has previously been implicated in dorsal closure, thus detailed investigation of CG6700 may reveal additional mechanisms involved in this morphogenetic event. A similar phenotype was observed in embryos treated with Bx42-specific dsRNA (Figure 2, Movie S2). In these embryos, convergence of the epithelial sheets was slow and although the hole started to zipper, closure was not completed. Bx42 encodes for a highly-conserved transcriptional regulator protein involved in various signal transduction pathways [30]. In Drosophila, only its involvement in Notch signaling has been demonstrated, but its vertebrate homologs interact with and modulate the activity of several other transcription factors such as Smad and steroid receptors [30–31]. As all of the Notch, steroid hormone and TGF-b/dpp signaling pathways are required for dorsal closure, further studies are required to determine the exact role of Bx42 in this process [8][32]. Since regulation of biological processes can be considered to be a combination of complex gene regulatory networks, it is tempting to speculate that Bx42 plays a role in dorsal closure by simultaneously participating in several signaling cascades. Unfortunately, there are no loss-of-function alleles of Bx42 available, which makes the functional analysis of this gene complicated. Group II genes affect dorsal closure dynamics Since phenotyping of the silenced embryos was performed in living embryos, we were able to identify not only genes essential for PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22229