For determining the effect of Notch and Delta interaction on F- actin in cultured cells, cell aggregations were performed using S2 cells expressing heat shock inducible Notch or Delta and procedures described in [26–28]. Cells were fixed with parafor- maldehyde and processed for labeling using antibodies against Notch and Delta, and Phalloidin as the probe for F-actin (AlexaFluor 568-Phalloidin). In vitro cell fusion assays were performed with clone-8 cells that endogenously express Notch (but not Delta), Schneider (S2) cells that express neither Notch nor Delta [23,24], and S2 cells that constitutively express Delta under the control of the Drosophila actin5C promoter (S2-actDelta). For generating the S2-actDelta cell line, actin5C promoter and the Delta cDNA were cloned the into pUAST vector (please see [18,19]) and a stable S2 line established by co-transfecting with it with pCopHygro plasmid [43]. Near confluent cells were washed and resuspended at a concentration of 3–56106 cells per ml in amine- free buffer (IP buffer in [23,24]) with CMTPX (CellTracker Red) or CMFDA (CellTracker Green) (see Molecular Probes manual MP 02925 for details of the dyes). Clone-8 cells were treated with 1 mM CMTPX (CellTracker Red) and S2-actDelta and S2 cells were treated with 10 mM CMFDA (CellTracker Green), incubated in the dark for 45 minutes, washed once with 1 ml IP buffer, resuspended in 2 ml of cell culture medium (with FBS), and incubated in the dark for 2 hours to complete dye activation and removal of un-reacted dye molecules. The cells were pelleted and re-suspended in cell culture medium (with FBS) at a concentration of 36106/ml. Varying cell concentration controlled the size of cell aggregates. 200 ml of Red-clone 8 cells were mixed with 200 ml of Green-S2-actDelta cells or Green-S2-Cells in a microfuge tube and immediately transferred to a 12-well tissue culture-treated microplate. The plates were gently rotated for 5–10 minutes until Notch and Delta driven cell aggregates became apparent. The plates were then covered in aluminum foil and incubated without shaking in the dark for various time periods (2 hours to 2 days). DeltaVision imaging was done at the Neuroscience Core Imaging facility, University of Vermont College of Medicine. All other imaging was done using an upright Nikon SMZ1500 stereoscope or a Nikon 2500 inverted fluorescence microscope fitted with a Spot RT Slider CCD camera. Wild type and mutant embryos were imaged together or with identical settings. Extra Long Working Distance (ELWD) 406objective was used to image live cells through the bottom of the microplate (total magnification was 400X). Images were processed using Photoshop (Adobe) and Canvas (Deneba) programs. Any adjustment made to brightness/ contrast was applied to the whole image and the same settings were used for all compared samples. Results 1. Neurogenesis is suppressed in hephaestus embryos To determine if the loss of hephaestus function affected Notch function during embryogenesis, we examined the CNS develop- ment in mutant hephaestus embryos as this process is under the control of canonical Notch signaling and is the most widely accepted assay for this signaling. The neuronal precursor cells and many of their progeny express the neuronal marker protein Hunchback. When there is loss of canonical Notch signaling, as in Notch embryos, an excess of Hunchback signal is observed. When there is an excess of this signaling, as in transgenic embryos over-expressing Nintra/NICD from an exogenous promoter, a suppression of Hunchback signal is observed (Fig. S2). Examina- tion of heph03429 and heph2 embryos showed that Hunchback expression is suppressed (Fig. 1). Suppression of neurogenesis was Notch and Actin Zones in Drosophila Embryos apparent as early as stage 9 and became progressively severe with age. As expected from the difference in the strengths of the alleles, the phenotype in heph03429 embryos was much stronger than in heph2 embryos. While almost all heph03429 embryos showed defective CNS developments, only about 20% of heph2 embryos showed defective CNS. Thus, two independently isolated alleles manifested similar phenotypes indicating that these phenotypes are linked to the hephaestus gene. We next examined if Notch mRNA expression is affected in heph mutant embryos. For this purpose we used total RNA extracted from 3 to 6 hour-old heph03429 embryos which would be most active for canonical Notch signaling related to neurogenesis (specification of the epidermal precursor cells). Results showed that heph03429 embryos express a high level of Notch mRNA as well as high levels of E(spl)C mRNA, the target of canonical Notch signaling during neurogenesis (Fig. 2). Based on these studies we conclude that the loss of hephaestus function results in increased Notch mRNA expression and possibly as a conse- quence increased Notch activity that suppresses the CNS development. This conclusion is consistent with studies of wing development showing that hephaestus is a negative regulator of Notch activity [14,15,17]. The increased level of Notch mRNA in heph03429 embryos suggests that the Notch gene is a target of hephaestus function related to negative regulation of the Notch pathway. 2. Mutant hephaestus embryos are defective in dorsal closure Our studies also revealed that the dorsal closure process is severely disrupted in hephaestus mutant embryos. For much of wild type embryogenesis the extra-embryonic aminoserosa occupies the dorsal region of the embryo. Aminoserosal cells help the dorso- lateral epidermal cells move to the dorsal midline for fusion and closure of the embryo. They also undergo apoptosis in a progressi- ve manner to accommodate the advancing lateral epidermis [40]. In heph03429 embryos the dorsal closure process was blocked and Figure 1. Mutant heph embryos manifest suppression of the CNS development in the ventral region, a phenotype linked to excess canonical Notch signaling phenotype. Expression of Hunchback, a well-known neuronal marker, was used to assess the CNS development. Hunchback expression in Notch and gain-of- canonical Notch signaling (Nintra/NICD) embryos is shown in Figure S2. Suppressed neurogenesis phenotype became apparent as early as stage 9 (about four hours of embryogenesis) and was severe at stage 14–15 (about 14–16 hours of embryogenesis). Variation in the suppression of neurogenesis in heph embryos was observed, which is presumably due to variable maternal contribution. All embryos shown were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g001 PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e21876