sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. the non-autonomous polarizing activity of and genes randomize the orientation of the SOP division relative to the body axis [4], [6], [7], [8]. The mechanisms whereby these PCP proteins act to position the aPKC-Par6 complex and orient the mitotic spindle in SOPs are not known. Asymmetric localization of Fz and Vang at opposite poles at the apical cortex of epithelial cells is an early read-out for PCP [17], [18]. Asymmetric distribution of Fz and Vang further underlies the local coordination of planar polarization by contributing to the cell-cell propagation of polarity (reviewed in [9], [10]). Additionally, asymmetric localization Masitinib manufacturer of Fz and Vang provides subcellular cues Masitinib manufacturer for the polarization of the cytoskeleton. The mechanisms whereby Fz and Vang act intracellularly to polarize epithelial cells are partly understood in the context of wing epidermal cells [19], [20]. The extent to which similar mechanisms operate in asymmetrically dividing SOPs remains to be determined. Previous studies have shown that Fz localizes at the posterior cortex of SOPs prior to division whereas Vang accumulates at the anterior apical cortex [8]. Additionally, while Fmi localizes at the apical cortex with no sign of asymmetry in SOPs [7], recent studies have suggested that Fmi associate with either Vang or Fz to form distinct complexes at opposite poles of the cell [21], [22]. The asymmetric distribution Masitinib manufacturer of Fz and Vang in dividing SOPs therefore suggests that Fz and/or Vang may act locally to regulate the activity of downstream effectors. However, the relative roles of Vang and Fz in positioning the aPKC-Par6 complex and in orienting the mitotic spindle are, however, still elusive. Here, we have studied the relative contributions of Vang and Fz in orienting the SOP polarity axis at mitosis. We have used a live imaging assay to show that Dsh acts in part by inhibiting Vang to restrict the localization of a centrosome-attracting activity. Using clonal analysis, we have shown that both and act redundantly to orient the SOP polarity axis in response to PCP. These observations indicate that both Vang, at the anterior cortex, and Fz, at the posterior cortex, contribute to the a-p orientation of dividing SOPs. Materials and Methods Flies The following genotypes were studied: Figure 1: Open in a separate window Figure 1 Dsh acts via Vang to inhibit a centrosome-attracting activity at anaphase.(A,B) Time-lapse recording of SOP division in wild-type (A) and mutant pupae (B) using RFP-PonLD (red), Histone2B-RFP (red) AurA-GFP (green). RFP-PonLD is mis-segregated in the mutant SOP. The two centrosomes, marked by AurA-GFP, appear to interact with the cortical domain marked by RFP-PonLD and moved off-center towards this domain at anaphase. (C) Quantification of the defects in RFP-PonLD segregation in wild-type, and double mutant SOPs. The severity of the phenotype is significantly different from those associated with all other genotypes in a two-by-two comparison using a Fischer exact test. No other difference in pairwise comparisons was statistically significant. 1. FRT42D/FRT42D; neurP72GAL4 UAS-AurA-GFP/+; 4. FRT42D/FRT42D; neurP72GAL4 UAS-AurA-GFP/(B,B), (C,C) and double mutant (D,D) SOPs. In D, nls-GFP (in blue) was used as clonal marker. The extent of the Pins-positive cortical domain in late prophase SOPs was measured as an angle value. The results of this quantification are shown in the bottom panels. Masitinib manufacturer 6. FRT42D/FRT 42D 7. FRT42D/FRT42D Figure 3: Open in a separate window Figure 3 Fz and Vang are individually dispensable to orient the SOP polarity axis.The orientation of SOP division was studied along the border of wild-type control (ACB), mutant (CCD) and (ECF) clones. SOPs were identified using Sens (red in A, B, E and F) in fixed tissues and GFP-PonLD (green in C and D) in living tissues. Orientation of the division was determined using Pins (blue in A, B, E and F) or GFP-PonLD (green in TRADD C and D). We used nls-GFP (in green) as a clone marker. (ACB) wild-type SOPs inside (GFP? in A) Masitinib manufacturer and outside (GFP+ in B) control clones. (CCD) mutant (GFP? in C) and wild-type (GFP+ in D) SOPs. (ECF) (GFP? in E) and wild-type (GFP+ in F) SOPs. The orientation of SOP division was measured as an angle between the axis of SOP polarity oriented towards Pins and Pon and a line corresponding to the clone border at the position of the dividing SOPs (see A for a graphic representation). Angle values corresponding to the genotypes studied in top panels are plotted in the bottom panels. The orientation of the asymmetry axis, relative to the clone margin, was divided in four categories,.