The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing

Abstract

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.

Figure 1.—

Expression of the tow enhancer trap is negatively regulated by Wg signaling in the wing disc. (A) Wild-type tow-lacZ expression (X-gal) is found in the whole-wing pouch except the D/V boundary (indicated by white dashed line). (B) Wg expression (red) is not affected by ptc-Gal4-driven overexpression of Tow (green). The genotype of the larvae used is ptc-Gal4/UAS-GFP; UAS-tow/+. (C) dpp-Gal4-driven UAS-dTCF^DN derepresses tow-lacZ expression at the A/P boundary of the wing (indicated by arrow). The genotype of the larvae is UAS-dTCF^DN/+; dpp-Gal4/tow-lacZ. (D) Ectopic expression of Wg (asterisks in D-I and D-iii) represses tow-lacZ. (D-i) β-Galactosidase activity of tow-lacZ (red). (D-ii) Wg flip-out clones marked by Wg antibody (green). (D-iii) Merged image of i and ii. (E-i) Structure of tow gene, enhancer trap P-element insertion, and deletion created by imprecise excision. tow transcript has a very long 5′-UTR, and 7.4-kb genomic DNA is deleted in the tow⁷⁵⁴ mutant, which removes the whole ORF of the tow gene and 160 amino acids of 3′ of the neighboring gene, CG10077. Homozygous tow⁷⁵⁴ mutant flies are normal. (E-ii) tow^fz mutant (tow and fz double mutant) created by hs-FLP-mediated inversion. Homozygous tow^fz mutant shows typical fz mutant phenotype. Trans-heterozygote of tow⁷⁵⁴ and tow^fz shows a normal phenotype. (F) In situ hybridization to wild-type wing disc using a tow-derived RNA probe shows a pattern very similar to that of tow-lacZ. (G) Sequence of Tow protein. The residues in red are Gln repeats, and in blue are Pro repeats. Bars in A–D and E, 50 μm.

Figure 2.—

Overexpression of Tow causes disorganization of microchaetae on the thorax and multiple hairs in the wing. (A and B) Microchaetae phenotype in adult thorax. Anterior is left. (A) Thorax of ap-Gal4/CyO. Microchaetae are regularly oriented and point posteriorly. (B) Thorax of ap-Gal4/+; UAS-tow/+. Misexpression of Tow results in disorganization of microchaetae and defects in the scutellum (arrow). (C–F) Adult (C–E) and pupal (F) wing phenotypes. In all of the wings, proximal is to the left. (C) Wild-type adult wing. (D) Multiple-hair-cell phenotype of MS1096-Gal4-driven UAS-tow adult wing. A few multiple hairs are indicated by red arrows. (E) Multiple hairs on the adult wing in the Drok² mutant clone. The clonal border is marked by a white dashed line. (F) Phalloidin staining of the MS1096-Gal4-driven UAS-tow pupal wing reveals multiple F-actin prehairs. White arrows represent several multiple F-actin bundles.

Figure 3.—

tow null mutation dominantly suppresses a polarity-specific Fz gain-of-function phenotype. (A–D) Tangential sections of the equatorial region of adult eyes. Anterior is left, and dorsal is up. Each panel contains eye sections (top) and a schematic of the same area with arrows reflecting ommatidial polarity (bottom). (A) sev-Gal4/UAS-tow. It has completely normal eyes. Equator is indicated by gray lines, and it shows ommatidia of normal dorsal and ventral chirality with black and blue arrows, respectively. (B) sev-Gal4 UAS-fz/+. Transient sevenless-driven overexpression of Fz in the eye results in planar polarity defects. Red arrows represent misrotated ommatidia; green arrows without flags represent symmetrical nonchiral ommatidia. Circles mark unscorable ommatidia, usually due to missing photoreceptors or abnormal morphology. (C) sev-Gal4 UAS-fz/tow⁷⁵⁴. The mutation in tow is able to dominantly suppress the gain-of-function sev-Gal4 UAS-fz eye phenotype. It is almost completely suppressed. (D) sev-Gal4 UAS-fz/UAS-tow. Co-overexpression of Fz and Tow by sev-Gal4 results in the enhanced polarity defects. Ommatidia look different in the different genotypes due to fixation or staining variation.

Figure 4.—

tow mutant suppresses dsh¹ multiple-hair-cell phenotype, and its gain-of-function phenotype is enhanced in a dsh¹ background. (A–D) Representative multiple-hair-cell phenotypes (circled in red) on a dorsal surface of the central region of the adult wing. The genotypes are (A) dsh¹/Y, (B) dsh¹/Y; tow⁷⁵⁴/+, (C) ap-Gal4/+; UAS-tow/+, and (D) dsh¹/Y; ap-Gal4/+; UAS-tow/+. The dsh¹ polarity phenotype looked like it was enhanced because ap-driven overexpression of Tow caused the wing blade to be very uneven. (E) The number of multiple hair cells. Error bars represent standard errors. In all of the adult wings, proximal is to the left.

Figure 5.—

Genetic interaction of tow and other PCP components (A–D) Representative wing-hair phenotype of the dorsal compartment of the adult wing. (A) stan^VC31/+ wing in the anterior region. (B) Trans-heterozygotes of tow⁷⁵⁴ and stan^VC31 showing increased multiple wing hairs. (C) sha^VB13 homozygous adult wing in the center of the most posterior region. (D) Reduction of tow dosage increased number of small hairs and multiple wing hairs in a sha^VB13 background.

Figure 6.—

Evidence that Tow genetically interacts with Drok pathway. (A–D) Representative multiple-hair-cellphenotype (circled in red) of various genotypes in a defined region of the adult wing. In A–D, heat shock was given at 37° 24 hr APF. (A) hs-Gal4/+; UAS-tow/+ wing. (B and C) Overexpression of Drok via a α-tubP-Drok transgene results in a reduced number of multiple hairs, and reduction of Drok dosage causes an increase in the number of multiple hair cells. (B) hs-Gal4/+; UAS-tow/α-tub-Drok. (C) Drok²/+; hs-Gal4/+; UAS-tow/+. (D) hs-Gal4/zip¹; UAS-tow/+. zipper mutation results in a substantial increase of multiple hairs. In all of the wings, proximal is to the left.

Figure 7.—

Tow protein is localized in the nucleus, and overexpression of Tow represses sqh mRNA. (A) Subcellular localization of Tow in pupal wing cells of arm-tow-GFP. (A-i) GFP signal (green) showing subcellular localization of Tow protein. (A-ii) ap-Gal4-driven UAS-RedStinger wing, which results in the red fluorescent protein in the nucleus. A-i and A-ii are merged in A-iii. GFP and nuclear signal show the same localization patterns. (B) hs-Gal4-driven overexpression of Tow represses the level of sqh mRNA.