Identification of Genes Involved in Dorsal Closure Figure 11. Head involution defects of pbl mutants. (A and B) Immunofluorescence staining of embryos after head involution stage with anti- Fas3 antibody. (A) Wild-type embryo. (B) pbl3/pbl3 mutant embryo. (C–F) Frames from movies of arm:GFP-expressing embryos. (C) Head region of a homozygous arm:GFP embryo. (D) Enlargement of the boxed region in (C). (E) Head region of a homozygous arm:GFP; pbl3 mutant embryo. Asterisk labels a rip in the head epithelium. (F) Enlargement of the boxed region in (E). (A–F) Dorsal view is shown, scale bars represent 50 mm. doi:10.1371/journal.pone.0022229.g011 tissues: the pulling force provided by the amnioserosa is sufficient to close the hole, but the dynamics of closure is abnormal. We provide evidence that pbl is also required for thorax closure during metamorphosis, indicating its general role in epithelial closure processes. The requirement of several GTPases (Rac1, cdc42, Rab11, Rab5, Rab30) has been demonstrated in thorax closure but no function for Rho1 has been reported so far [10][57]. Activation of Rac occurs through the Crk–Mbc–ELMO GEF- complex, but cdc42 or Rab activation during thorax closure is still obscure. Further studies are required to determine whether additional GTPases function during thorax closure and which of these GTPases are activated by pbl. Materials and Methods Drosophila stocks We used the ZCL0423 protein trap line and sGMCA:GFP to visualize the DME cells. The fly stocks en-Gal4, 69B-Gal4, pnr- GAL4, UAS-dicer2, pbl3 and arm:GFP were obtained from the Bloomington Stock Center. For inducible silencing of the selected genes, UAS-RNAi lines were obtained from the Vienna Stock Center. The UAS-Moe:mCherry fly stock was provided by T. Millard [33]. To analyze the actin dynamics of the epithelial cells, homozygous en-Gal4, UAS-Moe:mCherry flies were used. Embryo injection and RNAi screening A commercially available dsRNA library was used for the large scale screen (Open Biosystems, [58]). To select genes expressed in the embryo, microarray data were used [24], (GEO accession number: GSE3955). For the microinjections, freshly laid homo- zygous ZCL0423 or sGMCA:GFP embryos were collected for 30 minutes at 25uC on juice-agar plates, washed with water and dechorionated in 50% Chlorox bleach for 2 minutes. Embryos were oriented on a juice-agar plate and transferred to a coverslip covered with glue. Embryos were desiccated and covered with Voltalef H10S halocarbon oil (VWR). Syncytial blastoderm embryos were injected laterally with dsRNAs at ca. 50% egg length. The concentration of injected dsRNA solution was <0.5 mg/ml in TE buffer. Microinjections were performed with glass capillaries using Transjector 5246 (Eppendorf). Capillaries were prepared with a Flaming/Brown micropipette puller P-97 (Sutter Instrument Co.). After injection, coverslips were transferred PLoS ONE | www.plosone.org 11 July 2011 | Volume 6 | Issue 7 | e22229