Figure 10. Protrusion dynamics in the DME cells of pbl mutants. (A and B) Frames of movie sequences showing DME cell protrusion dynamics in embryos expressing mCherry:Moe in engrailed-expressing cell stripes. Scale bars are 5 mm. (A) en-Gal4, UAS-mCherry:Moe control embryo. (B) Homozygous en-Gal4, UAS-mCherry:Moe; pbl3 embryo. doi:10.1371/journal.pone.0022229.g010 genes (Kr, ptc, pbl, Arf51F) does not prevent closure but affect its dynamics. Identification of these genes demonstrates the power of the high-throughput time-lapse microscopy approach. We per- formed a detailed cell biological analysis of one of these genes, the multifunctional GEF, pbl. In this study we demonstrate its direct involvement in cytoskeletal dynamics of the dorsal epithelium and show that pbl indirectly affects dorsal closure dynamics by regulating head involution. The active state of the small GTPases is controlled by guanine nucleotide exchange factors (GEFs), GTPase activator proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). The pbl gene encodes a GEF, which function as the activator of small GTPases. A remarkable feature of GEFs is that several GEFs can activate the same GTPase and several GTPases can be activated by the same GEF [52]. In addition to this obvious redundancy, most of the GEFs and target GTPases are broadly expressed and their expression patterns widely overlap. This complexity of GTPase regulation by GEFs enables a plethora of possible interactions which makes the functional analysis of the individual GEF at the organism level very complicated. Consistently, pbl is a pleiotropic gene required in a wide range of developmental processes [41–42]. Pbl protein has been shown to be able to activate several GTPases in Drosophila in a tissue and develop- mental stage-specific manner [41]. The essential role of pbl in cytokinesis and mesoderm development has been studied exten- sively in Drosophila. Since both processes require cell shape changes, pbl has been suggested as a component of the intracellular signaling pathway mediating cytoskeletal dynamics. Pbl activates Rho1 in the contractile ring during cytokinesis in blastodermal embryos and the Rac GTPase pathway during mesoderm migration, suggesting two separate functions for pbl in these processes. The mesodermal target of pbl, Rac, has been shown to be essential in the DME cells for normal cytoskeletal dynamics [45]. Unlike pbl, however, loss of Rac activity results in the absence of Identification of Genes Involved in Dorsal Closure protrusions of the DME cells. Since the pbl mutant phenotype presented here is different from the Rac mutant phenotype, it is very unlikely that pbl activates Rac in the epithelial cells during dorsal closure. We suggest that during dorsal closure, pbl might activate the Rho GTPase pathway. Two lines of evidence support this hypothesis. The loss-of-function pbl phenotype of the DME cells very closely resembles to that of the Rho1 mutants, both at the cellular and cuticle levels. Reduced Rho1 or pbl function in the DME cells results in weak actin accumulation and excessive filopodia activity at the dorsal surface. In addition, similar to pbl mutants, zygotic loss of Rho1 activity results in abnormal dorsal cuticle morphology [44][53]. The similarity of the loss of function Rho1 and pbl phenotypes in the dorsal ectoderm suggests that these genes act in the same pathway. According to our model, pbl activates Rho1 in the DME cells, which in turn regulates actin accumulation and protrusion dynamics at the dorsal surface. However, the pbl and the Rho1 mutant phenotypes are not completely identical [54]. This discrepancy could be explained by the presence of maternally-provided proteins or the hypomorphic nature of the mutant alleles used. A further explanation could be that Rho1 is activated by additional GEFs or pbl activates additional GTPases in the DME cells. Mutations in many genes involved in dorsal closure also result in head involution defects [7]. How loss of pbl activity leads to head involution defects is not completely clear. One possible function for pbl during head involution could be the regulation of actin dynamics in the translocating tissues through the activation of one or more GTPase pathways. This hypothesis is less attractive, since no specific actin accumulation or protrusion formation in cells of the head region has been reported so far. However, as our knowledge of the details of head involution is very poor, we can not exclude this possibility. An alternative scenario for the role of pbl in head involution could be that it regulates cell adhesion dynamics in the head region. Our results demonstrate that cell-cell contacts are weak in pbl mutants which eventually leads to ripping of the head epithelium. Activity of several GTPases of the Rho family has consistently been shown to be required for cell adhesion in a wide range of organisms and cell lines [54–55]. A further support for this hypothesis is that mutations of Rho1 and RacGTPases abolish head involution [45][53]. Further experi- ments are required to precisely determine the pbl targets in this developmental process. Analysis of the pbl mutant phenotype suggests a tight mechanical connection between head involution and dorsal closure. Biophysical studies revealed the presence of a force acting on the dorsally-migrating epithelial sheets and exerted by tissues undergoing head involution [56]. This force pulls the dorsal epithelial sheets towards the anterior and provides a mechanical factor which forces the two opposing dorsal epithelial edges towards the dorsal midline thereby tightening the dorsal hole. Since pbl mutations abolish head involution, this force might be lost in these mutant embryos which, as a consequence, would affect closure dynamics indirectly. Moreover, additional forces generated by zippering and actomyosin contraction at the leading edge are also perturbed in pbl mutant DME cells. Efficient zippering requires the coordinated activity of cell extensions whereas actomyosin contraction depends on actin accumulation at the leading edge of the DME cells. Both processes are perturbed in pbl mutants, directly affecting closure dynamics. Thus, the only force serving dorsal closure in the pbl mutants is the force generated by amnioserosa contraction. We consistently detected a normal reduction of apical surface area of amnioserosa cells in pbl mutants as compared to wild type. Since dorsal closure is a robust process, loss of the various forces can be compensated by other PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e22229