A Functional Genomic Screen Combined with Time- Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos Ferenc Jankovics*, La´ szlo´ Henn, A´ gnes Bujna, Pe´ ter Vilmos, No´ ra Kiss, Miklo´ s Erde´ lyi Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary Abstract Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes. Citation: Jankovics F, Henn L, Bujna A´ , Vilmos P, Kiss N, et al. (2011) A Functional Genomic Screen Combined with Time-Lapse Microscopy Uncovers a Novel Set of Genes Involved in Dorsal Closure of Drosophila Embryos. PLoS ONE 6(7): e22229. doi:10.1371/journal.pone.0022229 Editor: Francois Schweisguth, Institut Pasteur, France Received March 16, 2011; Accepted June 17, 2011; Published July 20, 2011 Copyright: 2011 Jankovics et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Hungarian National Science Foundation (OTKA-H07-B-74357 to F.J.) (http://www.otka.hu) and the Howard Hughes Medical Institutes (HHMI, 55005606 to M.E.) (http://www.hhmi.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jankovic@brc.hu Introduction Dorsal closure of the embryonic epithelium takes place during mid-embryogenesis, when two epithelial sheets migrate towards the dorsal midline where they meet and fuse [1]. The migrating epithelium is pulled by rhythmic contractions of cells in the neighboring tissue called amnioserosa. Cells of the amnioserosa progressively die by apoptosis during closure and the dorsal hole becomes sealed, generating a continuous dorsal epidermis. Other epithelial closure processes such as embryonic wound healing or closure of the adult thorax during metamorphosis, involve a coordinated series of cellular activities that are very similar to those required for dorsal closure [2]. Importantly, there is a surprisingly high degree of evolutionary conservation of mechanisms by which epithelial discontinuities are repaired, making dorsal closure of Drosophila an excellent model for wound healing [3]. Over the last few decades, several large-scale mutant screens have been performed to identify genes affecting embryonic morphogenesis [4–6]. These classical genetic screens also uncov- ered the roles of many genes in dorsal closure. Mutations of these genes led to the classical dorsal open phenotype: a hole in the larval cuticle. Analysis of the larval cuticle revealed that some mutants with dorsal open phenotype also exhibit defects in other morphogenetic events. Abnormalities in developmental processes such as germ band retraction or head involution, in many cases appear to be coupled with dorsal closure defects indicating close cooperation between genetic and structural elements regulating these events [7]. Genetic and cell biological characterization of the dorsal closure mutants revealed that many complex cytoskeletal rearrangements coordinated by several signaling pathways collab- orate to orchestrate closure of the dorsal hole. The TGF-b/dpp pathway has been demonstrated to be the central element of the regulatory network of dorsal closure but JNK, wingless, Notch and the steroid hormone signaling pathways have also been implicated in this process [8]. In addition to the signal transduction cascades, genes encoding structural elements of the cytoskeleton and the cell adhesion complexes have been identified as being involved in dorsal closure, based on the dorsal open phenotype of their mutations [8]. Genetic and cell biological analysis revealed the involvement of several regulators of the cytoskeleton in various stages of dorsal closure. Members of the Rho, Rab and Ras GTPase families have also been implicated in the regulation of the dorsal closure [9–13]. In addition, three GTPase regulators, the Rap1 activator PDZ-GEF, the Rac1 activator myoblast city and the Rac/cdc42 repressor rotund/racGAP84C, were identified as partic- ipating in the complex regulation of GTPase function in the embryonic epithelium undergoing dorsal closure [14–16]. Although the genetics of the dorsal closure have been well explored, apparently not all components have thus far been identified. Despite its obvious potential as a useful model for epithelial closure processes, no systematic loss-of-function screen has been performed for genes affecting dorsal closure. RNAi has been shown to be a powerful experimental tool to efficiently silence specific genes. RNAi-based screening has been used to PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22229