Figure 10. Nnd1-dse embryos show Notch protein accumulation pattern that is similar to that observed in heph03429 embryos. While the Notch protein accumulated in the dorso-lateral region, it was depleted in the ventral region. Depletion was more clearly discernible in stage 9 embryos that have not yet produced the notochord (that tends to obscure the depletion in images of embryos at later stages). Please compare Nnd1-dse images in this figure with those of heph03429 embryos in Figure 5. All embryos shown were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g010 Figure 11. Reduction of Notch activity in heph03429 embryos suppresses actin accumulation in the dorso-lateral regions. N55e11 is a allele of Notch. heph03429/+; N55e11/Y and heph03429 ; N55e11/Y embryos were obtained from the same cross. +; N55e11/Y embryos are mostly composed of neuronal cells (with very few if any epidermal cells) and therefore show a higher level of actin compared to wild type embryos of similar stages. Suppression of actin level in the ventral region of heph03429 ; N55e11/Y was due to rescue of epidermal tissue (a consequence of increased canonical Notch signaling). Actin accumulation was not observed in the dorso-lateral regions of these embryos. doi:10.1371/journal.pone.0021876.g011 Notch and Actin Zones in Drosophila Embryos indeed F-actin (Fig. 13A). They also revealed a feature that was not apparent from actin antibody labeling: F-actin accumulates in longitudinal strings, as if the embryos are forming multiple longitudinal scaffolds (Fig. 13B). Such scaffolds are faintly visible even in the wild type embryos (see arrow heads in Fig. 13B) suggesting that normal scaffolds are enlarged in heph03429 and Nnd1- dse embryos. These results suggest that Notch accumulation at the cell surface has the potential to lead to F-actin accumulation in nearby locations, in the same pattern as in wild type embryos. 10. Persistent Notch-ligand interaction results in cell fusions in vitro A glaring phenotype in the dorso-lateral regions of heph03429 and Nnd1-dse embryos is cell fusions. Although Notch is involved in processes that include cell fusions (for example, muscle develop- ment or cardiogenesis), whether it is directly involved in cell fu- sions as our data suggest is unknown. Therefore, we examined this issue in cultured cells that are simpler than embryos and have served as an excellent in vitro model system for the analysis of Notch and/or Delta activities for more than 20 years (for example [23,24,26–28,54–57]). We used clone 8 cells, that constitutively express Notch from the endogenous gene in order to retain as much of the natural regulations as possible, and S2-actDelta cells that constitutively express Delta from the acting5C promoter. Untransfected S2 cells (S2 cells) served as control cells. We treated cl-8 cells with either S2-actDelta cells or S2 cells for varying periods from 2 hours to 20 hours. After about 4 hours, we started to notice large cells. Samples from a high-cell-density experiment and a low-cell- density experiment are shown in Figure 14. These large cells became apparent only when cells were grown under natural conditions (in tissue culture treated plates or flasks) and were easily lost when the cell aggregates were washed or centrifuged. To find out whether the large cells were products of cell fusion, we rendered clone 8 cells red and S2-actDelta cells or S2 cells green with cytoplasmic dyes that are confined to the treated cells for several cell generations. We mixed red clone 8 cells with either green S2-actDelta cells or green S2 cells, and processed them as described above but in the dark (to prevent fluorescence quenching). Double colored large cells indicating cell fusions became apparent in cl-8/S2-actDelta cell samples at about six hours and progressively increased over time. Eighty to ninety percent of cell aggregates (of 25–100 cells each) showed one or few fused cells (it is difficult to rule out fusion in the remaining aggregates as fused cells were easily dislodged). Two examples of cell fusion after 12 hours of incubation are shown in Figure 15. Mixtures of cl-8 cells and S2 cells that were processed identically do not aggregate and did not show evidence of cell fusion (Fig. S8). We have also not observed cell fusion in cells expressing Nintra/NICD. These results suggest that persistent interaction between Notch and ligand (Delta) expressing cells (made possible only by binding between Notch and ligand {Delta} at the interface between cells) has the potential to lead to cell fusions and supports our observation of cell fusions in heph03429 and Nnd1-dse embryos. The low level of fusions observed in vitro could be due to lack of proper conditions or additional factors (fusogens) that promote cell fusions in embryos. Discussion 1. Novel aspects of canonical Notch signaling Data presented in this report show that the loss of hephaestus (dmPTB) function affects the ventral and the dorso-lateral regions of Drosophila embryos very differently. In the ventral region, PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e21876