and relaxed periodically but more frequently and with lower amplitude than wild type cells. In the pbl mutants, the periodicity of the cell surface pulsations decreased from the wild type 191677 s value to 163671 s (n = 85 pulsations in wild type and n = 106 pulsations in pbl mutants) (Figure 7, Movie S5). As closure progressed, however, the amnioserosa cells decreased their apical surface area normally (Figure 8). These results indicate that loss of pbl function disturbs normal pulsing of the amnioserosa cells but does not severely affects the contraction of the whole amnioserosa tissue. As other forces contributing to dorsal closure are represented by the actomyosin contraction and zippering of the DME cells, the role of pbl was also tested in the epithelium. In vivo time-lapse imaging of epithelial cells was performed in pbl mutants expressing arm:GFP (Figure 9, Movie S6). Consistently with previous studies, we found that epithelial cells in the pbl mutants were larger than in the wild type embryos because of the earlier effect of pbl on cell division [47]. In addition, the shape of the pbl mutant epithelial cells was abnormal: several DME cells were detected which were transiently elongated along the anterior-posterior body axis. However, during dorsal closure progression, these cells elongated along their dorsal-ventral (D/V) axis and adopted an approxi- mately wild-type shape. As polarization of the DME cells along the D/V axis is an essential step in dorsal closure, we tested whether the abnormal cell size and shape was linked to abnormal D/V polarity. It has been shown that Fasciclin3 (Fas3) is excluded from the leading edge, whereas microtubules of the DME cells are arranged in parallel bundles along the D/V axis [48–49]. We found that in the pbl mutants the microtubule distribution was similar to the wild type and Fas3 was excluded from the leading edge indicating that pbl is not required for the D/V polarization (Figure 9). Interestingly, immunostaining of Fas3 revealed that the epithelial cells had an abnormal basolateral cell cortex. We detected long intrusions at the lateral membranes of the pbl mutant DME cells (Figure 9). Dynamics of the actin network at the leading edge of the DME cells is critical in dorsal closure. Epithelial cells accumulate actin Identification of Genes Involved in Dorsal Closure and extend actin-rich protrusions at their dorsal surface which have been shown to be required for normal dorsal closure. Regulation of actin accumulation and the dynamics of the extensions depend on Rho and Rac GTPases, which are targets of pbl in other tissues [41][50–51]. To test the involvement of pbl in these processes, we visualized actin in fixed pbl mutant embryos by phalloidin staining. At the leading edge of the pbl mutant DME cells, a slight reduction of actin accumulation was detected (Figure 9). To test whether pbl is involved in the regulation of protrusion dynamics at the leading edge of the DME cells, in vivo time-lapse imaging of the pbl mutant embryos was performed. The actin-rich structures were visualized by expression of Moe:mCherry in the dorsal epithelium with the en-GAL4 driver. In agreement with the hystochemical observations, a weak accumulation of actin was detected at the leading edge of pbl mutant DME cells (Figure 10, Movie S7). During the zippering stage, both filopodia and lamellipodia were extended but the morphology of these protrusions were abnormal. In the pbl mutants protrusions were more extensive, filopodia were longer (4.861.4 mm in wild type [n = 58] versus 7.161.7 mm in pbl mutant embryos [n = 52]) and lamellipodia covered a larger protrusive area, reaching up to 26.667.3 mm2 (n = 12) compared to 14.564.0 mm2 (n = 13) in wild type. Despite of the abnormal protrusions in pbl mutants, towards the end of the closure process, DME cells engaged with cells from the opposite side and zippered the dorsal hole. These results indicate that reduction of pbl function affects actin accumulation and protrusion dynamics of the DME cells. Pbl function is required for head involution It has been suggested previously that head involution, a complex morphogenetic process occurring simultaneously with dorsal closure, influences dorsal closure [7]. To analyze the correlation of these processes in pbl mutants, the embryonic cuticle was examined. In addition to defects in morphogenesis of the dorsal epithelium, the pbl3 mutant and pbl-silenced embryos had Figure 7. Pulsative behavior of the amnioserosa cells in pbl mutants. (A and D) Frames from movies of arm:GFP-expressing embryos. Scale bars are 10 mm. (B and E) Graphs showing amnioserosa cell surface fluctuations of the cells highlighted in A and D. (C and F) Mean of the apical surface maxima and minima for amnioserosa cells. Dashed lines represent average surface area of the cells (n = 18 cells in 3 embryos for wild type and n = 17 cells in 3 embryos for pbl3/pbl3 mutant). (A,B,C) Wild-type control embryo. (D,E,F) pbl3/pbl3 mutant embryo. doi:10.1371/journal.pone.0022229.g007 PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22229