Identification of Genes Involved in Dorsal Closure Figure 3. Abnormal dorsal closure dynamics generated by RNAi. RNAi phenotypes of genes in the phenotypic category II. Frames from movie sequences show abnormal dorsal closure dynamics of dsRNA-injected embryos expressing the ZCL0423 protein trap fusion protein. Arrows indicate misaligned sites. All embryos are shown in dorsal view with anterior to the left. Scale bar represents 50 mm. doi:10.1371/journal.pone.0022229.g003 found to be normal, suggesting a complex effect of pbl on dorsal closure (Figure 4, Table S2). Pbl and N are involved in both dorsal closure and the closure of the adult thorax Closure of the adult thorax during metamorphosis has been shown to share many signaling and structural elements with the dorsal closure of the embryonic epithelium [38]. To test the conservation of our candidate genes between these closure processes, their involvement was also tested in thorax closure. In a genome- wide screen, specific dsRNAs of all Drosophila genes has been expressed by Mummery-Widmer et al. in a tissue-specific manner at the dorsal midline during metamorphosis using the Pnr-GAL4 driver, and the loss-of-function RNAi phenotypes of the adults have been determined [39]. In this experiment – of our candidate genes – only pbl has been shown to be required for thorax closure. Since coexpression of dicer2 has been demonstrated to enhance the RNAi- phenotype, we simultaneously expressed dicer2 with dsRNAs for our candidate genes in the thorax [40]. Under these conditions, silencing of five of the ten tested genes exhibited a phenotype. Silencing of three genes (scb, Bx42, ptc) led to lethality, while silencing of two genes (N and pbl) caused abnormal thorax closure and resulted in the formation of a thorax cleft, suggesting the general requirement of these genes in epithelial closure processes (Figure 5). Pbl is involved in epithelial morphogenesis Pbl, one of the genes exhibiting thorax and dorsal closure defects, was further characterized. We investigated whether the RNAi phenotype is similar to the phenotypes of the loss-of- function pbl mutants by comparing the cuticles of embryos Figure 4. Quantification of abnormal dorsal closure dynamics. (A and B) Graphs showing closure kinetics of the dorsal hole in a buffer- injected control embryo, in a homozygous pbl3 mutant embryo and embryos silenced for pbl and Arf51F. For each category, data of individual representative embryos are shown. (A) ‘‘Width’’ represents the maximal distance between zippering ends. (B) ‘‘Height’’ represents the maximal distance between the converging epithelial layers. Velocity of the epithelial sheet translocation (v), the rate constant of zippering (kz) and the fractional contribution of zippering (fz) to the velocity of the closure were calculated as described [22]. doi:10.1371/journal.pone.0022229.g004 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22229