Many of these functions, particularly those related to actin, cytoskeletion, or extracellular matrix appear to be based on a mechanism that does not involve Suppressor of Hairless or Nintra/ NICD that are required for canonical Notch signaling [31–38]. The mechanism(s) underlying these non-canonical Notch func- tions is(are) poorly understood (e.g. [33]). As a consequence, we do not know much about the molecular features associated with these functions. For example, we do not know whether Nintra/NICD production is a part of the known non-canonical Notch functions. We also do not know whether the canonical and the non-canonical Notch activities function everywhere in the developing animal (together or in tandem) or are confined to distinct regions of the developing animal. The ventral region of the Drosophila embryo was the region where the role of Notch in cell fate specification was first discovered about 70 years ago [39]. Ever since then it has served as an excellent model for the study of the function and mechanism of canonical Notch signaling. Features first identified here were subsequently found in all animals. The development of the Central Nervous System (CNS) is initiated in the ventral region, within clusters of proneural cells that have acquired the potential to become neuronal cells. Most proneural cells activate canonical Notch signaling that promotes the expression of Enhancer of split Complex (E(spl)C) genes and become the epidermal precursor cells. The remaining cells block this signaling and the expression of E(spl)C genes and become the neuronal precursor cells. The epidermal precursor cells remain in the periphery of the embryo and differentiate the epidermis that includes a series of actin-rich denticle belts. The neuronal precursor cells migrate interiorly and differentiate into the CNS with elaborate actin-based processes (e.g., axons). The dorso-lateral regions of the developing Drosophila embryo support many processes that require the cell fate specification functions of Notch, for example the specification of neuronal precursor cells that differentiate the peripheral nervous system or the pericardial cells that differentiate the dorsal vessel (heart). These regions are also involved in actin-based processes such as gastrulation and dorsal closure processes. There are no published reports of the role of Notch in these actin-based processes. It is possibly because of epistasis: loss of Notch function suppresses the production of epidermal cells that are required for many aspects of gastrulation and dorsal closure [40,41]. In any case, both the ventral and the dorso-lateral regions of the Drosophila embryo are involved in cell fate specification and actin-based processes. Consequently, both the regions were expected to have the same potential for canonical and non-canonical Notch signaling and manifest similar molecular features associated with Notch protein activities. If hephaestus negatively regulated Notch functions in embryos (as it does during wing development), its loss of function was expected to have a similar effect on Notch features in the ventral and the dorso-lateral regions. Results reported here show that in mutant hephaestus embryos the level of Notch mRNA and protein activity is increased indicating that Notch mRNA might be targeted by the Hephaestus protein for negative regulation. Remarkably, in hephaestus embryos the Notch protein accumulates at or near the cell surface in the dorso-lateral regions but not in the ventral region. Notch accumu- lation in the dorso-lateral regions is associated with actin accumu- lation, cell fusions, and disruption in dorsal closure and cardioge- nesis processes. The same phenomenon is observed in mutant Notch embryos that have lost negative regulation at the level of mRNA 39 processing suggesting that the hephaestus phenotypes are possibly consequences of increased Notch activities. Over- expression of Nintra, thereby canonical Notch signaling, recapit- Notch and Actin Zones in Drosophila Embryos ulates the molecular and morphological phenotypes in the ventral regions of mutant hephaestus or Notch embryos but not the phenotypes in the dorso-lateral regions. These data suggest that the Drosophila embryo is zoned based on whether or not PTB and Notch activities are directly linked to actin-based processes. Thus, cells that appear to be the same (epidermal cells) and part of the same epithelial layer might differ in developmental potential depending on their place of origin in the embryo (ventral or the dorso-lateral regions). Materials and Methods hephaestus alleles used in our studies are heph03429 and heph2. They are loss of function alleles due to P element insertions. heph03429 appears to be the stronger allele as its homozygotes do not survive to adulthood but a few heph2 homozygotes do ([13,17], FlyBase). heph stocks were obtained from Bloomington Stock Center. They contain a TM3 Sb1 balancer and not a TM3 Sb1 Ser1 balancer as stated in the description sheet. Our studies also indicate that the expressivity of zygotic phenotypes of heph03429 is drastically reduced if the mother is heterozygous for Notch or Serrate alleles. For example, in the background of the TM3actGFPSer1 balancer the accumulation of actin in the dorso-lateral regions of heph03429 embryos that manifests in embryonic stage 14 is delayed until embryonic stage 17 (Fig. S1). The yellow white (yw) fly stock served as the wild type (WT) control. The Notch mutants used are N55e11 or N264-47; the Notch gain-of-function allele used was Nnd1-dse [18,19]. Zygotic heph03429; N- embryos were obtained using standard genetic crosses and stocks with green (GFP) and blue (lacZ) balancers. Stocks were maintained at 18uC and experimental embryos were collected at room temperature (23uC to 27 uC) or 29–30uC (yw control and Nnd1-dse) over a 0–24-hour period. They were processed immediately for immuno-labeling studies or aged for an additional day or two before processing (periods for stock raised at 18uC or 29–30uC were corrected for the difference in developmental rate). Embryos were immuno-labeled with antibodies against Notch [25], Hunchback (gift from Paul MacDonald), Kruppel (gift from John Reinitz), Pericardin (EC11, DSHB), or Actin (Abcam, ab49846), following standard protocols [42,43]. The nuclear stain DAPI was included where required. Embryos were devitellinized by hand for phalloidin labeling. Acridine orange hydrochloride hydrate (Acros Organics, 423340010) was used to detect apoptosis in embryos using the protocol described in [44]. Embryos were sorted using green/blue balancers and/or morphological phenotypes. RNA expression was analyzed by northern blotting [24,27]. For exogenous expression of Nintra/NICD, we used a UAS-Nintra/ NICD transgene [45] and daughterless Gal4 (daGal4) or heat shock protein 70 Gal4 (hsGal4) drivers provided through the males. hsGal4 flies and embryos were reared at 18uC and transferred to 30uC at different stages. All offspring embryos would express Nintra/NICD, as homozygous parents were used. Although similar results were obtained with imuno-fluorescence, detailed studies were done with immuno-cytochemical procedures (based on Horse Radish Peroxidase or Alkaline Phosphatase activity) because they were efficient and cost-effective, enabling us to examine simultaneously thousands of embryos and determine stage-specific expressivity and penetrance of mutant phenotypes. Although Phalloidin and actin antibody gave similar results, we relied on the latter for double labeling studies because it was less tedious. Wild type and mutant embryos were processed identically and mounted in Phosphate Buffered Saline with 0.5% triton6100 (PBT) using strips of glass cover-slip as props so that embryos could be rolled to desired position for imaging. PLoS ONE | www.plosone.org 2 July 2011 | Volume 6 | Issue 7 | e21876