Notch and Actin Zones in Drosophila Embryos Figure 12. Enrichment of Notch receptor near the cell surface results in F-actin enrichment also near the cell surface. A. F-actin levels in S2 cells expressing Notch (S2-Notch cells) and S2 cells expressing ligand Delta (S2-Delta cells) that were not mixed together. B. F-actin levels in S2- Notch and S2-Delta cells brought together by Notch and Delta binding. Notch and Delta binding results in Notch receptor enrichment (clustering) at contact points between the two cell types (Delta does not show this response [27]). Notch enrichment diminishes over time, possibly due to production of Nintra/NICD or reduction in Notch synthesis. doi:10.1371/journal.pone.0021876.g012 development of the CNS is suppressed and there was a discernible depletion in the level of the Notch protein. Suppression of the CNS development is consistent with the known role of hephaestus as a negative regulator of canonical Notch signaling during wing and bristle development in the larval and pupal stages [14,15,17]. Excess canonical Notch signaling is well known to suppress neurogenesis in embryos [58,59]. Depletion of the Notch protein in the ventral region that could be explained using data from mammalian systems showing that Nintra/NICD is turned over by a proteolysis process linked to the activity of the transcription factor Mastermind [29,30]. Thus, excess canonical Notch signaling could result in Notch protein depletion if the rate of Nintra/NICD production and degradation is higher than Notch synthesis. If that were the case, it would suggest that the mechanism responsible for down regulating Notch activity targets not Nintra/NICD produc- tion or degradation but Notch synthesis. Combining the data from heph alleles and the Nnd1-dse allele (presented here and in [18,19]), it appears that most of Notch mRNA transcribed following Notch activation is targeted for degradation by a mechanism that requires the Notch 39 UTR and the dse. It is possible that the Hephaestus protein is part of the RNP complex that regulates this mechanism. In its absence, Notch protein synthesis continues instead of being suppressed. There is growing evidence that ligand-independent canonical Notch signaling in involved in development [60,61]. It would be interesting to know if this signaling is also affected by the Hephaestus-based down-regulation mechanism. Thus, understanding how exactly hephaestus negatively regulates canonical Notch activity might provide insights into an important aspect of Notch pathway regulation that was hitherto obscure: down-regulation after activation of Notch by a ligand. As many human diseases are linked to gain of canonical Notch signaling, a better understanding of hephaestus and Notch 39 UTR and dse functions might lead to novel mechanistic insights into these diseases. 2. Revelations about a non-canonical Notch activity The surprising finding in our study is the different response of the dorso-lateral regions of the embryo to the loss of hephaestus function or the loss of negative regulation of Notch mRNA 39 processing (due to the Nnd1-dse mutation). The simplest explanation is that Notch function is not required in these regions and de- repression of Notch protein synthesis results in the accumulation of Notch protein in these regions (as there is no signaling dependent depletion). This explanation, however, does not account for Pericardin accumulation, actin accumulation, or the block in dorsal closure. Pericardin level during cardiogenesis is well PLoS ONE | www.plosone.org 10 July 2011 | Volume 6 | Issue 7 | e21876