Identification of Genes Involved in Dorsal Closure Figure 5. Abnormal thorax closure generated by RNAi. (A) Control thorax with pnr-Gal4/+ genotype. (B–C) Thoracic cleft phenotypes induced by pnr-Gal4-driven expression of UAS-RNAi constructs. (B) Silencing of N. (C) Silencing of pbl. The transformant ID of the UAS-RNAi constructs are KK100002 for N and GD35350 for pbl. Figures show dorsal views of adult thoraxes with anterior to the left. doi:10.1371/journal.pone.0022229.g005 homozygous for the strong hypomorphic pbl3 allele and the cuticles of pbl dsRNA-treated embryos. Cuticle abnormalities were detected in the pbl mutant embryos identical to the embryos silenced for pbl confirming the accuracy of our screening method. In most of the embryos (88%, n = 24) injected with pbl dsRNA, a serious disturbance of the epithelial matching was observed. Instead of the segmentally repeated rows of cuticle hairs present in wild-type embryos, the pbl-silenced embryos displayed disorga- nized rows of hairs meeting at one point around the dorsal midline (Figure 6A–C). We detected identical segmental misalignments in all of the homozygous pbl3 mutant embryos. In addition, live imaging of pbl mutants expressing EGFP specifically in the DME cells revealed defects of closure dynamics identical to the pbl- silenced embryos (Figure 6D,E). In pbl mutants, quantitative parameters of the closure were similar to that of pbl-silenced embryos indicating further that the RNAi phenotype precisely phenocopies the loss-of-function phenotype of the pbl gene (Figure 4, Movie S4, Table S2). Pbl is involved in actin dynamics of the DME cells Pbl functions as a multifunctional RhoGEF involved in the regulation of Rho and Rac GTPases [41–42]. Rho1 and Rac genes have been shown to play a role in several aspects of dorsal closure involving amnioserosa cell contraction and dynamic cytoskeletal rearrangements in DME cells [43–45]. It has been shown that several forces exerted by the epithelium and the amnioserosa contribute to dorsal closure [22]. Therefore, the function of pbl was tested both in epithelial and in amnioserosa cells. The closure phenotype in pbl-silenced and pbl mutant embryos is reminiscent of cases where the amnioserosa contraction is disturbed either by genetic methods or by laser ablation [22]. This similarity suggests an important role for pbl in the coordination of amnioserosa contraction. The apical surface of the wild type amnioserosa cells pulsate rhythmically contributing to dorsal-ward displacement of the epithelial sheets [46]. In vivo examination of amnioserosa cell activity of the pbl3 homozygous embryos revealed that the mutant amnioserosa cells contracted Figure 6. Pbl is required for the morphogenesis of the dorsal epithelium. (A–C) Cuticle preparations of embryos. (A) Wild-type cuticle. (B) Cuticle of an embryo injected with dsRNA for pbl. (C) Cuticle of a homozygous pbl3 mutant embryo. (D and E) Frames from movies of embryos expressing ZCL0423 protein trap- EGFP fusion protein. Embryos are shown in dorsal view, scale bars represent 50 mm. (D) Wild type embryo. (E) pbl3/ pbl3 mutant embryo. doi:10.1371/journal.pone.0022229.g006 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e22229