Notch and Actin Zones in Drosophila Embryos Figure 2. RNA of Notch and E(spl) genes m5 and m8, the latter targets of canonical Notch signaling, were over- expressed in heph03429 embryos. RNAs used on northern blots were extracted from 3 to 6 hours old embryos that manifest peak canonical Notch signaling activity related to the CNS development. rp49 is the loading control. doi:10.1371/journal.pone.0021876.g002 the cuticle poorly developed even a day after embryogenesis had ended in the wild type embryos (Fig. 3). The wild type embryo shown has just completed embryogenesis (,22 hours, end of stage 17). Its dorsal closure process is complete, internal organs are conspicuous (e.g. the gut), and cuticle is fully developed into the rubbery exoskeleton that resists immuno-staining (as antibodies cannot penetrate the tough cuticle). The mutant heph03429 and heph2 embryos shown are more than a day older (,48 hours). In these embryos the amnioserosa (AS) persists in the dorsal region and most of other embryogenesis events are arrested. Although the amnioserosa in heph mutant embryos retains the morphology (and can be easily identified using it), its physiology appeared to be altered as expression of markers such as Kruppel [46] was drastically reduced. While most heph03429 embryos appeared to cease development at about stage 14 to 16 (12–16 hours of embryogenesis), only about 20% of heph2 embryos appeared to cease development at these stages. These determinations are gross approximations as not all parts of the dead embryos ceased development at the same stage (i.e. the embryos were essentially developmental mosaics). Embryos that had ceased development died of necrosis between 48–72 hours after egg laying. 3. Mutant hephaestus embryos are defective in cardiogenesis To determine whether the defect in the dorso-lateral lateral region of heph mutant embryos is specific to the dorsal closure process, we examined development of the dorsal vessel. Its development is initiated shortly after gastrulation and well before the initiation of the dorsal closure process. Pairs of cardioblasts develop on either side of the embryo, from the lateral myoblast cells along the anterior-posterior axis. Notch activity in one cell of each pair suppresses the cardioblast fate and specifies the alternate Figure 3. Dorsal closure process is blocked in mutant heph embryos. While in the wild type embryo the dorsal region was closed and the extra-embryonic amnioserosa (AS) was completely eliminated by about 22 hours after egg laying (left), in heph03429 and heph2 embryos the dorsal closure process was incomplete and AS persisted even after 48 hours. Embryos were labeled using the actin antibody. All embryos/larva shown were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g003 pericardial cell fate. While cardioblasts are responsible for producing the dorsal vessel proper, pericardial cells are responsible for producing support structures and for secreting the extracellular matrix protein Pericardin that is required for attaching the cardio- blasts to the dorso-lateral epidermal cells. The two longitudinal rows of pericardial cell-cardioblast pairs that form on either side of the embryo migrate to the dorsal midline by hitchhiking on the dorso-lateral epidermal cells involved in dorsal closure. After reaching the dorsal midline, they fuse and differentiate the dorsal vessel. Notch activity is also required for this differentiation [47– 51]. In embryos lacking Notch function (Notch embryos), Pericardin is not expressed, as pericardial cells are not formed (Fig S3). In heph03429 embryos both the pericardial cell specification process (prior to dorsal closure) as well as migration of these cells to the dorsal mid-line (that is dependent on dorsal closure) were affected (Fig. 4). Figure 4A and 4B show two views of the same set of embryos; embryos in 4C are from another set. Pericardin expression initiated prematurely, at stage 9, at which stage the wild type (WT) embryos have not even begun to express Pericardin (these embryos begin expression at stage 13). Pericardial cells were in high numbers and studies of numerous independently processed samples indicated that the pericardial cell specification process proceeded in a haphazard manner. For example, Pericardin was found in regions that do not normally express it, such as the head and the tail regions (see heph03429 embryo in 4D). Embryos in 4E show the ventral region of the embryos in 4D to point out that the ventral epidermis is developed, which is consistent with canonical Notch signaling activity in the region. Abnormal phenotypes in heph03429 embryo persisted to the time when dorsal vessel is fully PLoS ONE | www.plosone.org 4 July 2011 | Volume 6 | Issue 7 | e21876