(A) Sequence alignment of Sano with that of D. virillis, A. gambiae, T. castaneum, N. vitripennis, and D. pulex. Cyan, completely conserved residues in all species; Blue, identical residues; Yellow, similar residues. (B) GFP-tagged Sano protein was overexpressed in the trachea using btl-Gal4. GFP signal (green) is observed in the cytoplasm and is enriched in the apical domain where Crb, an apical membrane marker, localizes (red). (C) α-Sano antibody (green) recognized overexpressed Sano protein in the cytoplasm with apical enrichment in the trachea. Red, SAS. (D) GFP-tagged Sano protein was overexpressed in the salivary gland using sage-Gal4. Sano-GFP (green) was observed in the cytoplasm and was enriched in the apical domain where Crb localizes (red). Scale bars: 20 µm. Embryos in (B–D) are st.14.

(A, B) Adult thorax of WT (A) and of MS1096-Gal4>UAS-Sano (B). Sano overexpression causes misorientation of thoracic bristles. (C, D) Adult wing of WT (C) and of ptc-Gal4>UAS-Sano (D). WT wing shows distal orientation of hairs, whereas overexpression of Sano causes a swirling hair pattern. (E–G) Adult ommatidia of WT (E), sev-Gal4>UAS-Sano (F) near the dorsal/ventral boundary, the equator, and sano mutant clones in the dorsal compartment (G). sano mutant cells have w^+/+ marker and they are distinguishable from the neighboring WT w^−/− cells by the pigment around the cells. Schematic drawings are shown in the panels below the actual images with black and red shapes indicating the orientation of ommatidia normally found in the dorsal and ventral hemisphere of the eye, respectively, and blue shapes indicating a loss of ommatidial asymmetry. sano mutant cells are marked by the light green color in G and H. Green circles in H indicate ommatidia with abnormal photoreceptor number. (H, I) BarH1 expression in WT (H) and in sano mutant clones in the eye discs (I). BarH1 (blue), an R1 and R6 photoreceptor marker reveals ommatidial misrotation in the sano clones (arrows). Red, Elav. The absence of GFP signal indicates sano^KO2 mutant clones. Scale bar: 5 µm. (J, K) WT and sano mutant clones in the eye discs. mδ0.5-lacZ (red), a R4 photoreceptor marker is absent or significantly reduced in the sano clones (arrows). Blue, BarH1. The absence of GFP signal indicates sano^KO2 mutant clones. Scale bar: 5 µm.

(A–F) 2A12 staining in dsh¹ (A), ds^33K (B), rhoA^72F (C), fmi^E59/stan¹⁹² (D), ds^33K/Df(2L)Exel8003 (E), rhoA^72F/rhoA^72O (F) mutant embryos. (G, G′) btl-Gal4-driven overexpression of Dsh in the trachea results in a shortened DT phenotype similar to the Sano overexpression phenotype. Higher magnification of the boxed region is shown in G′. Red, 2A12; blue, SAS. (H) A nuclear fusion marker is normally expressed in Dsh-overexpressing trachea. Blue, Tgo; red, Dys. (I) Quantification of the length of DT of st. 16 PCP mutants. Error bars, SD (*, longer DT length, p<0.05, t-test; **, shorter DT length, p<0.05, t-test). (J) Yeast-two-hybrid assay using the full length and several fragments of the Dsh protein shows that the small fragment (∼100 a.a.) of Dsh between the PDZ and DEP domains binds to Sano. (K) co-IP experiment using embryo extracts confirms the interaction between Sano-HA and Dsh-GFP. Scale bars: 20 µm for (A–G), 10 µm for (G′, H). All embryos shown are st. 16.

(A–D) rp395 β-gal is expressed in the salivary gland (SG), salivary duct (SD), trachea (TR), CNS midline (ML), foregut (FG) and hindgut (HG). (E–H) sano RNA is expressed in the same pattern as rp395 β-gal, except that expression is more transient. Note that expression in the SG (E) disappears by embryonic stage 13 (G, H). (I–L) sano is regulated by Scr (I, J), Trh (K), and Sim (L). Note the loss of sano in the salivary glands in the Scr mutant (arrow in I), the ectopic rp395 expression in HS-Scr embryos (arrowheads in J), the loss of sano RNA in the trachea in the trh mutant (arrow in K) and the loss of sano RNA in the midline cells in the sim mutant (arrowhead in L). (M) There are five alternative sano splice forms, RA-RE, which encode the same ORF. The rp395 P-element is inserted in the first exon of sano-RC and sano-RD. sano^KO1, sano^KO2 and sano^KO3 are null alleles created by a targeted homologous recombination event in which the entire sano ORF was replaced with white⁺. (N, O) sano mRNA expression in st. 10 WT (N) and transheterozygous embryos of sano^KO3 over deficiency (O). sano patterns of expression were undetectable in sano^KO3/Df.

(A–D) st.15 and st.16 WT (A, C) and sano mutant (B, D) embryos stained for 2A12. (E) Quantification of tracheal DT length in WT, sano^KO3 mutant and rescued embryos by btl-Gal4-driven overexpression of Sano in sano^KO3 mutant background. Error bars indicate standard deviation (SD) (*p<10⁻³, t-test). (F, F′) Overexpression of Sano results in shortened DTs (arrows) that fail to connect. Higher magnification of the boxed region is shown in F′. Green, sano-GFP; red, 2A12; blue, SAS. (G) A fusion marker is normally expressed in Sano-overexpressing trachea. Green, sano-GFP; blue, Tgo; red, Dys. (H, I) Sano overexpression causes shortened salivary gland lumen. Asterisks indicate salivary gland of WT (H) and sage-Gal4>UAS-Sano-GFP salivary gland (I). Scale bars: 50 µm in (A–D) and F, 10 µm for (F′, G), 20 µm for (H, I). Embryos in (F–H) are st.16.

(A) Prehair formation is delayed in Sano-overexpressing cells in the pupal wing (asterisks). (B) Sano overexpression causes nonautonomous misorientation of prehairs in specific regions of the pupal wing (arrows). (C, D) Fmi staining in WT and Sano-overexpressing pupal wing cells. Typical zigzag localization of Fmi (blue) and distal orientation of prehairs (actin, red) are shown in WT (C). In Sano-overexpressing cells, Fmi is observed all around the apical membrane and many hairs are misoriented (D). (E) fz-GFP (green) shows a fuzzy distribution throughout the apical margin in Sano-overexpressing clones (red). Scale bars: 10 µm.

(A) Sano-overexpressing pupal wing cells (green) are smaller than neighboring wild-type cells and often have a pentagonal rather than hexagonal shape (arrows). Red, E-Cad. (B) Quantification of the perimeter of three different clones of Sano-overexpressing single pupal wing cell and corresponding wild-type neighbors. Error bars, SD. *, p<0.05, t-test. (C–E) Adult wings. Sano overexpression at the anterior/posterior boundary with ptc-Gal4 results in a narrower region between veins 3 and 4 (arrows in C and D). Sano overexpression in the entire wing with MS1096-Gal4 results in a dramatic decrease in overall wing size. (F–H) E-Cad staining of tracheal cells (metamere 4). WT (F). btl-Gal4-driven Sano-overexpressing trachea (G) reveal a significant decrease in apical domain size. The tracheal cells in rhoA mutant embryos have larger apical domains (H). Arrowheads indicate apical domain of single cells in each allele. (I, J) E-Cad staining of tracheal cells (metamere 1). WT (I) and esg mutant trachea that have fusion defects (J) have comparable apical domain size. All of the embryos shown are st.16. Scale bars: 5 µm in (A), 1 µm in the inset, 10 µm in (F–J).