Notch and Actin Zones in Drosophila Embryos Figure 4. Pericardial cells are in excess and mislocalized in heph03429 embryos indicating that cardiogenesis is disrupted in these embryos. A–C. The same heph03429 and wild type embryos imaged from different perspectives. D. Pericardin was present in unusual places in heph03429 embryos, such as the head and the tail. Note the large amnioserosal region (as) that persisted in heph03429 embryos. E. The ventral epidermis was reasonably developed in heph03429 embryos indicating that these embryos produced sufficient amounts of canonical Notch signaling in the ventral region. F. Pericardial cells continued to be in excess and cardiogenesis blocked in heph03429 embryos at the time when embryogenesis and dorsal vessel formation was complete in wild type embryos (stage 17). All embryos shown were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g004 formed in the wild type embryo (embryos in 4F). We observed similar defects in heph2 embryos but they were milder or in a smaller fraction of embryos. These data indicate that the loss of hephaestus function affects not just the dorsal closure process but also additional processes that are dependent on the dorso-lateral regions of the embryo. 4. Notch expression is differently affected in ventral and dorso-lateral regions of mutant hephaestus embryos To determine whether loss of hephaestus function affected Notch protein levels, we studied heph03429 embryos using an antibody made against the carboxyl terminus of the Notch protein [24]. We found a remarkable difference between the level of the Notch protein in the ventral and dorso-lateral regions of the same mutant embryo. Notch protein accumulated to a high level in the dorso- lateral regions of heph03429 embryo but not in the ventral region. Notch accumulation was very pronounced in stage 15 embryos but could be detected even in stage 9 embryos when the effect of the zygotic loss of hephaestus function begins to manifest (Fig. 5). Notch protein expression in the ventral region of heph03429 embryos was always lower than the level in wild type embryos of the same age. This feature is not clearly discernible in stage 15 embryos in Figure 5 due to the higher expression of Notch in the notochord just beneath the surface of wild type embryos (notochord is suppressed in heph03429 embryos). However, it is clearly discernible in stage 9 embryos in which Notch protein expression is confined to the outer layer of cells. We observed the same pattern of Notch protein expression (higher level in the dorso-lateral regions and a lower level in the ventral region) even in heph2 embryos but it was not as obvious and was observed in a smaller fraction of embryos. 5. Actin level is differently affected in ventral and dorso-lateral regions of mutant hephaestus embryos As the dorsal closure process is primarily dependent on actin- based processes [40] and Notch is known to be involved in actin- based processes [31,36–38], we examined actin level in mutant hephaestus embryos. We found that actin accumulated to a high level and in a disorganized manner in the dorso-lateral regions of heph03429 embryos (Fig. 6). In addition, we observed a depletion of actin in the ventral region of heph03429 embryos compared to the level in wild type embryos (see Fig. 6B). We observed a similar pattern of actin accumulation in heph2 embryos but it was less obvious and in a smaller fraction of embryos. To determine if actin accumulation in the dorso-lateral regions of heph03429 embryos was PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e21876