Notch and Actin Zones in Drosophila Embryos Figure 5. Notch protein level is different in different regions of heph03429 embryos. A. Notch protein accumulated in the dorso-lateral region of stage 15 heph03429 embryos but not in the ventral region. B. Accumulation of Notch protein in the dorso-lateral regions, and depletion in the ventral region, of heph03429 embryos became apparent as early as Stage 9. All embryos shown were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g005 due of increased apoptosis, we performed Acridine Orange labeling assay that reveals dying cells during Drosophila embryogenesis [44]. Results showed that heph03429 embryos undergo apoptosis equal to or less than the wild type embryos of comparable stages (Fig. 7). It is well established that embryos homozygous for a allele of the wingless gene experience Figure 7. Apoptosis is not increased in heph03429 and Nnd1-dse mutant embryos. Normal apoptosis observed in the CNS of wild type embryo was not observed in either heph03429 or Nnd1-dse embryos as the CNS development in the latter embryos was suppressed. Embryos are at stage 14. The brightness of yolk is due to autofluorescence. doi:10.1371/journal.pone.0021876.g007 increased cell death [52]. Therefore, we examined wingless embryos and found that they do not accumulate actin in the dorso- lateral regions (Fig. S4). These results rule out cell death (apoptosis) as the cause of actin accumulation in mutant hephaestus embryos. 6. Notch and actin accumulation patterns overlap in mutant heph03429 embryos As Notch and actin protein accumulation patterns were comparable in the dorso-lateral regions of heph03429 embryos, we examined whether there is overlap between Notch and actin Figure 6. Actin level was high and disorganized in the dorso-lateral regions of heph03429 embryos. A, B, and C: the same set of embryos shown from different perspectives. Absence of CNS labeling in the ventral region of the heph03429 embryo is due to the absence of neuronal cells. Embryos are at stage 15. All embryos shown were from the same experiment and were processed identically. doi:10.1371/journal.pone.0021876.g006 PLoS ONE | www.plosone.org 6 July 2011 | Volume 6 | Issue 7 | e21876