Notch and Actin Zones in Drosophila Embryos Figure 15. Large cells in live cl-8+actDelta cell aggregates are products of cell fusions. CellTracker Red labeled cl-8 cells and CellTracker Green labeled actDelta cells were used in the cell aggregation assay. These CellTracker dyes are confined to the treated cells or to their progeny upon division. The two rows represent samples from two independent assays. Arrowheads point to some fused cells. Note that other cells are either red or green and that there is no bleed-through between the red and the green channels. doi:10.1371/journal.pone.0021876.g015 based on Nintra/NICD activity in the nucleus. There is evidence for the existence of Notch activity independent of Nintra/NICD. For example, a Notch function independent of Presenilin (the enzyme that is required for the release of Nintra/NICD [20–22]) has been described in mammals [62]. A similar Notch activity might be functioning in the dorso-lateral regions of the Drosophila embryo. Our data suggest that this non-canonical Notch activity might be situated at the cell surface or in the cytoplasm and is involved in regulating actin levels and cell fusion. This inference is consistent with the finding of others that Notch activity other than the one based on Nintra/NICD is associated with actin accumu- lation in wing discs [31]. The non-canonical Notch activity we appear to have discovered could be the predominant Notch activity in the dorso-lateral regions of the embryos but it cannot be the only Notch activity. It is well known that Nintra/NICD and canonical Notch signaling is required for the peripheral nervous system (PNS) development from the dorso-lateral regions [20–22]. Our studies show that the PNS development is also suppressed in heph03429 and Nnd1-dse embryos, which raises the question of why we do not see depletion of Notch and actin proteins in the dorso-lateral regions as a consequence of constitutive Nintra/NICD and canonical Notch signaling. There are two possible explanations. One, a minority of cells are involved in the PNS development. Two, the canonical Notch signaling activity might precede the non-canonical Notch signaling activity and the latter determines the ultimate phenotype. Regardless of which explanation is correct, it is remarkable that cells in the ventral and the dorso-lateral regions respond so differently to the loss of hephaestus function or to the loss of negative regulation of Notch activity due to the Nnd1-dse mutation. At an earlier stage (stage 8 or 9), these cells were all the same, as they all adopt the default neuronal fate in Notch or Delta embryos. At later stages, the ventral epidermal cells appear to diverge by blocking non-canonical Notch signaling altogether. It appears that this block is not an intrinsic lack of competence because actin and Notch enriched cells are occasionally observed in the ventral region (the heph03429 embryo in Figure 6 was chosen to make this point; please see Fig. 6C). The block is specific to hephaestus or actin-related Notch activity as the ventral epidermal cells participate in other the actin-dependent processes, for example those involved in producing the denticle belts. 3. A clue to Notch function in the dorso-lateral regions of the embryo The Notch pathway is long known to be involved in actin and adhesion processes in Drosophila. Interestingly, many processes that depend on Notch or hephaestus activity undergo cell fusion or block them (e.g., myoblast fusion [63] or spermatid individuali- zation [64], please see [63] for a review of cell fusion). In this regard, Notch functions in myogenesis are quite instructive. During myogenesis, Nintra/NICD and canonical Notch signaling is required to restrict the number of myoblasts. Not as well known is the fact that Notch activity is also required subsequently for myoblast fusion and differentiation [65]. A Notch activity at these stages is also reported to affect the differentiation of the neighboring epidermal cells and this activity is not based on Nintra/NICD [66]. These reports have not been examined in depth so far because nothing is known about the non-canonical Notch signaling mechanism. The dorso-lateral regions of heph03429 and Nnd1-dse embryos represent an excellent model system for exploring non-canonical Notch mechanism with an unusual empirical power: all aspects of this mechanism in the dorso-lateral regions of the embryos can be compared with the canonical Notch signaling pathway mechanism in the ventral region of the same embryo. We know precisely the process that is defective in the ventral region of heph03429 and Nnd1-dse embryos (neuronal cell fate specification) but we do not know anything about the process that is defective in the dorso-lateral regions. Our data contains two clues to the latter process. One clue is in Figure 16, which shows a contrast-enhanced image of wild type and heph03429 embryos probed with the actin antibody. A close examination of this image PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e21876