Identification of Genes Involved in Dorsal Closure Figure 2. Dorsal open phenotypes generated by RNAi. RNAi phenotypes of genes in the phenotypic category I. Movie sequences show the absence of dorsal closure of dsRNA injected embryos expressing the ZCL0423 protein trap fusion protein. All embryos are shown in dorsal view with anterior to the left. Scale bar represents 50 mm. doi:10.1371/journal.pone.0022229.g002 closure but also genes affecting the dynamics of closure (Group II). Accordingly, in the second phenotypic group closure took place, but with abnormal dynamics. Kru¨ppel (Kr), patched (ptc), ADP ribosylation factor 51F (Arf51F) and pbl genes belong to this phenotypic category. Since silencing does not result in a dorsal hole in the larval cuticle, these genes have not previously been implicated in dorsal closure (Figure 3, Movie S3). However, application of our in vivo screening approach revealed the requirement of these novel genes in dorsal closure. RNAi for Kr and ptc caused similar closure phenotypes. During wild-type closure the dorsal hole retains an ellipsoidal teardrop- shape throughout the entire closure process. In the embryos silenced for Kr and ptc, however, the dorsal hole is asymmetric. (Figure 3, Movie S3). In these embryos the dorsal hole is closed but a misalignment of the epithelial sheets can be detected. Kr is a gap gene functioning as a transcription factor, whereas ptc is a segment polarity gene and encodes for the Hedgehog-receptor. Both Kr and ptc are required for the patterning of the embryonic epithelium. Proper alignment of the segmented epithelium along the dorsal fusion seam requires the accurate contact of each cell with its matching cell in the opposing epithelial sheet. This remarkable accuracy of cell matching ensures the maintenance of the segmented pattern during dorsal closure [33–34]. Silencing of Kr and ptc disturbs segmentation which in turn, consistent with the observed phenotype, results in misalignment of the epithelial sheets. Silencing of Arf51F also induced abnormal closure dynamics. Arf51F encodes for a conserved member of the Arf family of small GTPases regulating membrane trafficking. However, the mamma- lian homolog of Arf51F (Arf6) has been implicated in the regulation of subcortical actin remodeling, cell adhesion dynamics and cell migration [35–36]. Null mutants of Arf51F are viable but they exhibit defects in cytokinesis in the male germ line and in the brain [37]. In addition to these phenotypes, live imaging of the embryonic morphogenesis also revealed a requirement for Arf51F in dorsal closure. In Arf51F-silenced embryos, the convergence of the lateral epithelial sheets took place normally, while zippering was inefficient in both anterior and posterior ends of the dorsal hole (Figure 3, Movie S3). As a consequence, the dorsal opening became abnormally narrow. The abnormal dynamics phenotype was characterized in a quantitative manner using a mathematical model of dorsal closure [22]. In the movies, quantitative features (height and width) of the dorsal opening were measured and the velocity of the epithelial sheet translocation (v), as well as the fractional contribution of zippering (fz) to the velocity of the closure were calculated. Silencing of Arf51F resulted in a decrease of fz suggesting that Arf51F function is essential for efficient zippering (Figure 4, Table S2). Suppression of pbl expression by RNAi also disturbed closure dynamics. The pbl gene encodes a guanine nucleotide exchange factor involved in the regulation of several members of the Rho GTPase family. In embryos injected with pbl dsRNA, the epithelial gap displayed a circular shape instead of the wild type ellipsoidal shape (Figure 3, Movie S3). The epithelial sheets converged towards the midline and the hole became sealed, however, the outline of the dorsal hole retained its roundish shape during the entire closure process. Surprisingly, by quantitative analysis of the closure dynamics in the pbl- silenced embryos, v and fz values were PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e22229