accumulation. We performed high-resolution analysis using the DeltaVision restoration microscopy, which like confocal micros- copy provides expression information in a single optical plane. We chose to focus on the dorso-lateral cells in late stage 15 heph03429 embryos, as they show the highest level of Notch and actin accumulation. We used Alexa Fluor 488 conjugated and highly cross-adsorbed secondary antibody against the Notch antibody made in hamster, Alexa Fluor 647 conjugated and highly cross- adsorbed secondary antibody against actin antibody made in mouse, and DAPI for nuclear labeling. These fluorophores have no overlap in emission spectra. In all embryos we examined (n = 15), there was significant overlap in Notch and actin accumulation. Low magnification images of dorso-lateral regions of embryos are shown in Figure 8A–B. In many areas there was significant co-localization as shown by yellow color signals (inset in Fig. 8A) but in others there was none. Analysis at a higher magnification showed that Notch and actin accumulated at or near the cell surface, with little or no Notch in the nucleus (Fig. 8C–D). Interestingly, Notch and actin accumulation were across surfaces of many cells that appeared to have fused (see multiple DAPI signals in C and D). Cell fusion rather than defective cytokinesis appeared to be the explanation because the number of nuclei ranged from 2 to 11 nuclei (odd numbers are not expected with defects in cytokinesis). Furthermore, the sizes of nuclei were approximately the same within fusions and outside fusions indicating that multiple DAPI signals are not due to chromosomal fragmentation (Fig. S5). Thus, the loss of hephaestus function resulted in high levels of Notch, actin, and cell fusions. 7. Nintra/NICD over-expression does not lead to abnormal Pericardin or actin levels Accumulation of Notch could imply excess canonical Notch signaling. As shown in Figure S2, exogenous Nintra/NICD over- expression mimics the classic gain-of-canonical-Notch-signaling phenotype: suppression of neurogenesis in the ventral region of heph03429 embryos. Therefore, we examined if exogenous Nintra/ NICD expression also mimicked the high levels of Pericardin and actin in the dorso-lateral region of the embryo. Results of experiments examining pericardin level in UAS-Nintra/NICD- Notch and Actin Zones in Drosophila Embryos daGal4 embryos are shown in Figure S6. We did not observe high levels of pericardin at any stage. In fact, pericardin level was suppressed. Comparable stages of wild type embryos showed normal Pericardin levels (embryos D–E). We obtained similar results with both daGal4 and hsGal4 drivers. Results of our experiments examining actin level in UAS-Nintra/ NICD-hsGal4 embryos showed no effect at earlier stages (stages 11–12) but showed suppression of actin level at later stages (stages 13–14) (Fig. S7A). The latter result is the opposite of what we observed in mutant heph03429 embryos. We also observed the loss of aminoserosa (AS) in Nintra/NICD expressing embryos, which is the opposite of what we observed in heph03429 embryos. We then examined embryos of the hyperactive Notch allele l(1)NB. This allele contains a mutation in the extracellular region that is a negative regulator of Nintra/NICD production. As a consequence, Nintra/NICD and canonical Notch signaling is produced at a high level in l(1)NB/Y embryos and these embryos die near the end of embryogenesis (stage 17) or in early larval stages ([28,53], FlyBase). Results with l(1)NB embryos showed that actin level is not increased (Fig. S7B). We also did not observe increased levels of pericardin in l(1)NB embryos. In fact, we found that the dorsal vessel was missing or partially formed. These observations are in accord with our Gal4/UAS-Nintra/NICD transgene data. Thus, it appears that while Nintra/NICD and canonical Notch signaling are sufficient to explain the mutant phenotypes in the ventral region of heph03429 embryos, they are insufficient to explain the mutant phenotypes in the dorso-lateral regions. 8. Nnd1-dse embryos mimic the phenotypes of mutant hephaestus embryos The block in the dorsal closure process could be due to Notch accumulation in the dorso-lateral region that is disrupting actin metabolism. On the other hand, it could be unrelated to Notch accumulation. The latter was a distinct possibility since Nintra/ NICD over-expression failed to reproduce the dorsal closure or actin phenotypes. To distinguish between the two possibilities, we examined embryos of the temperature-sensitive Nnd1-dse allele that produces constitutive and high levels of endogenous Notch activi- ties at the restrictive temperature. The lesion in Nnd1-dse is a Figure 8. Notch and actin accumulated near the cell surfaces in the dorso-lateral regions of heph03429 embryos. A, B. Low magnification images from DeltaVision restoration microscopy showing that Notch and actin expression overlap. C, D. Two sets of high magnification images from DeltaVision microscopy showing that Notch and actin accumulate near the surfaces of fused cells and not in the nucleus (marked by DAPI). doi:10.1371/journal.pone.0021876.g008 PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e21876