mutation in the Down Stream Element (dse) that is critical for the negative regulation of Notch mRNA 39 processing and protein production. At the restrictive temperature, more than two-thirds of the embryos die at various stages [18,19]. If constitutive Notch activity is the cause of actin-related phenotypes in heph03429 embryos, we expected to observe similar phenotypes in Nnd1-dse embryos. We found that actin protein accumulated in the dorso- lateral regions of more than 50% of un-hatched Nnd1-dse embryos that had developed past embryonic stage 14 (Fig. 9A). The same embryos also manifested suppression of CNS development in the ventral region and blocked dorsal closure process (Fig. 9B, C). Immuno-labeling with the Notch antibody showed that Notch protein also accumulated in the dorso-lateral regions of Nnd1-dse embryos (Fig. 10). We checked for increased apoptosis in Nnd1-dse embryos and found no evidence for it (please see Fig. 7). The similarities between Nnd1-dse and heph03429 phenotypes strongly suggested that the defects in the dorso-lateral regions of mutant heph embryos are caused by increased and/or constitutive Notch activity. Since Notch activity is required for producing epidermal cells, including those in the dorso-lateral regions, loss of Notch function can be expected to be epistatic to the loss of hephaestus function. However, since hephaestus is a negative regulator and the effect of the loss of Notch function in Notch zygotic embryos manifests progressively from stages 9 to 13 (possibly due to variation in maternal contribution), we expected a fraction of Notch; hephaestus zygotic double mutant embryos to develop beyond stage 13. These embryos would enable us to determine if the actin accumulation in hephaestus mutant embryos is suppressed in the absence of Notch. Therefore, we generated double zygotic mutant embryos of heph03429 and one of the Notch alleles (N55e11 and N264-47). About 70% of these embryos manifested the Notch phenotype of excess neuronal cell types at the expense of epidermal cells. In other words, Notch is indeed epistatic to hephaestus, which is also consistent with data from studies in wing development indicating that hephaestus functions after Notch activation [17]. The remaining 30% showed varying degrees of suppression of Notch pheno- types. A sample of embryos probed with the actin antibody is Notch and Actin Zones in Drosophila Embryos shown in Figure 11. Actin level was higher everywhere in N55e11/ Y embryos than in comparable wild type embryos, possibly because they are composed of mostly neuronal cells. That high level of actin was drastically reduced when the embryos were also homozygous for the heph03429. Reduction in the ventral region was likely to be due to partial suppression of the neurogenic phenotype (possibly as a consequence of persistent canonical Notch signaling generated by maternal Notch protein). In the dorso-lateral regions of the same embryos, actin accumulation was not observed. We conclude from these observations that (1) actin accumulation in heph03429 is dependent on Notch activity and (2) this Notch activity occurs later than the canonical Notch signaling activity in the ventral region. While these results are not conclusive (due to the complexity of hephaestus and Notch interactions), they are consistent with our hypothesis that hephasestus negatively regulates Notch that in turn positively regulates actin levels in the dorso-lateral regions of the Drosophila embryo. 9. Notch enrichment at the cell surface results in F-actin accumulation at the site Since Notch and actin accumulated near the cell surface in heph03429 and Nnd1-dse embryos, we wondered if Notch accumula- tion at the cell surface results in actin accumulation. To find out we relied on the phenomenon of Notch receptor clustering, as it is the most natural way to enrich for Notch at the cell surface. When cultured Drosophila cells expressing Notch are treated with cells expressing a Notch ligand (e.g., Delta), the first and most rapid response is Notch receptor clustering at the sites of contact with ligand expressing cells. In the absence of the ligand expressing cells, Notch expression is distributed all around the cell membrane and in the cytoplasm. Delta does not cluster in response to Notch binding [27,54]. We generated Notch clusters and examined actin levels using using Phalloidin that detects F-actin. Results showed clear enrichment of F-actin near Notch receptor clusters (Fig. 12). To determine if actin that accumulates in heph03429 and Nnd1-dse embryos is also F-actin, we probed these embryos with fluores- cently labeled Phalloidin. These experiments confirmed that most of actin that accumulated in heph03429 and Nnd1-dse embryos is Figure 9. Embryos of the gain-of-Function Notch allele, Nnd1-dse, manifest heph03429-like phenotypes. A. Actin accumulated in the dorso- lateral regions of Nnd1-dse embryos. B. The CNS development is suppressed in the ventral region of Nnd1-dse embryos. C. Dorsal closure was blocked in Nnd1-dse embryos. Please compare Nnd1-dse images in this figure with those of heph03429 embryos in Figures 3 and 6. Embryos in A and B are at stage 14; the embryos in C are at stage 17 when embryogenesis ends in wild type embryos. All embryos were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g009 PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e21876