reveals that the strong actin signals in the heph03429 embryo are more or less amplified and expanded versions of the above- background actin signals in the wild type embryo. The second clue is in heph03429 and Nnd1-dse embryos probed with phalloidin. It appears that these embryos form enlarged versions of the cable- like actin structures that traverse almost the entire length of the dorso-lateral regions in the body of the wild type embryo (please see arrow heads in Fig. 13). It is quite possible that clusters of cells in the dorso-lateral regions undergo partial or full fusion to form actin scaffolds that maintain epithelia integrity during remodeling and migration. Hypertrophy of these actin scaffolds could be the defect in the dorso-lateral regions of heph03429 and Nnd1-dse embryos. At this juncture, we do not know the mechanism by which actin protein level is altered in these mutant embryos. 4. Clues to a higher level of developmental organization heph03429 and Nnd1-dse embryos appear to reveal a new level of developmental organization: broad zones that are competent or refractory to non-canonical Notch signaling activity. We do not know what factors or mechanisms determine these zones. A diverse array of mechanisms is known to regulate Notch activity at the protein level, such as glycosylation, trafficking, and proteolytic processing [67–81]. It is possible that the ventral and the dorso- lateral regions differ in these mechanisms. Understanding the mechanism underlying the zonation of Notch activity in Droso- phila embryos might also have practical implications since the Figure 16. The pattern of actin accumulation in heph03429 embryos resembles the pattern of normal actin levels in wild type embryos. The image of actin labeled wild type and heph03429 embryos was contrast-enhanced to reveal the faint actin patterns in the wild type embryo. doi:10.1371/journal.pone.0021876.g016 Notch and Actin Zones in Drosophila Embryos Notch pathway is an important regulator of stem cell differenti- ation and cancer development. It might help us understand varia- tions within and among stem cell or cancer populations. It is possible that certain populations are composed of cells with potential for only the canonical Notch signaling while others include cells with potential for both canonical and non-canonical Notch signaling. Such differences in potentials might explain why some stem cells just proliferate while others differentiate or why some cancer cells are begin while others are metastatic. Supporting Information Figure S1 Manifestation of heph03429 phenotypes (actin accumu- lation in the dorso-lateral regions) is delayed to stage 17 (end of embryogenesis) when the mother was heterozygous for the Green TM3 balancer with the Ser1 mutant allele. If the mother was heterozygous for a allele of Notch, heph03429 embryos hatched into larvae (data not shown). TM3actGFPSer1 homozygotes ceased development at about stage 6, were severely deformed, or died in the larval stages. Animals were arranged in a multi-well plate and imaged under brightlight and UV light with filter to detect GFP fluorescence. (TIF) Figure S2 Expression of Hunchback in Notch embryos and in Nintra/NICD-overexpressing embryos. A. Too many neural cells were produced in embryos lacking Notch function (N55e11/Y). B. Too few neural cells were produced in embryos expressing high levels of Nintra/NICD and canonical Notch signaling. All embryos were from the same experiment and were processed identically. (TIF) Figure S3 Cardiogenesis (dorsal vessel formation) requires Notch function. Pericardial cells were not formed in N55e11/Y embryos that lack Notch function. All embryos were from the same experiment and were processed identically. (TIF) Figure S4 wingless embryos that are known to experience increased apoptosis do not accumulate actin in the dorso-lateral regions. wgcx4 is a allele of wingless. Both embryos were from the same experiment and were processed identically. (TIF) Figure S5 The sizes of nuclei are similar inside and outside the regions of high actin accumulation in the dorso-lateral regions of heph03429 embryos. This is the full DeltaVision image that was the source for Figure 8C. The similar nucleus sizes across the whole image indicate that multiple DAPI signals within rings of high actin expression are not due to chromosome fragmentation. The odd numbers of DAPI signals within such actin rings indicate fusion rather than defective cytokinesis. (TIF) Figure S6 Nintra/NICD over-expression does not result in increased Pericardin level. Embryos from stage 9 to the end of embryogenesis were studied but only Stage 12 and 14 embryos are shown. Pericardin expression became apparent in wild type em- bryos only at stage 14. A stage 16 wild type embryo with fully formed dorsal vessel is also shown for comparison. All embryos were from the same experiment and were processed identically. (TIF) Figure S7 Excess canonical Notch signaling does not result in increased actin level. A. Excess canonical signaling due to Nintra/ NICD over-expression did not result in increased actin level. Nintra/NICD over-expression did not affect actin level at early stages (stage 11) although other phenotypic consequences of its PLoS ONE | www.plosone.org 13 July 2011 | Volume 6 | Issue 7 | e21876