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.

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IN most multicellular organisms, epithelial cells form highly organized tissues with cells polarized along their apical/basal axis. Epithelia are also often polarized within the plane of the tissue, which is called planar cell polarity (PCP) or tissue polarity. In recent years, substantial progress has been made in discerning the mechanisms that lead to PCP in Drosophila in several body regions, especially in the wings and the eyes (reviewed in SHULMAN et al. 1998; ADLER 2002). In the Drosophila wing, each cell produces a single distally oriented hair. This arises from the polarized assembly of an actin-containing prehair at the distal vertex of each hexagonal wing cell (WONG and ADLER 1993). Mutants in PCP signaling have characteristic defects in both the orientation and number of hairs per wing cell (GUBB and GARCIA-BELLIDO 1982; WONG and ADLER 1993). In the eye, polarity is reflected in the mirror image arrangement of ommatidia of opposite chiral forms across the dorsal/ventral boundary, the equator. The photoreceptors within each ommatidium are arranged in an asymmetric trapezoidal shape, with the R7/R8 photoreceptor pointing toward the equator and R3 toward the polar side. Mutations in genes that regulate PCP result in failure of ommatidia to acquire the correct chirality and/or failure to rotate properly (ZHENG et al. 1995; TOMLINSON and STRUHL 1999; WEBER et al. 2000).

Using a number of approaches, investigators have identified genes required for the specification of planar cell polarity in Drosophila. This has led to the identification of the Frizzled pathway as a key for the development of tissue polarity (GUBB and GARCIA-BELLIDO 1982; VINSON and ADLER 1987; WONG and ADLER 1993; STRUTT and STRUTT 1999; ADLER and LEE 2001). Among the key members of this pathway are frizzled (fz), which encodes a serpentine receptor (VINSON et al. 1989); dishevelled (dsh), which encodes a cytoplasmic protein (KLINGENSMITH et al. 1994; THEISEN et al. 1994; KRASNOW et al. 1995); and starry night/flamingo (stan/fmi), which is a cadherin protein with a seven-pass transmembrane domain (CHAE et al. 1999; USUI et al. 1999).

Dsh and Fz family proteins also participate in the Wingless (Wg)/Wnt signal transduction system that regulate a wide variety of developmental events, including cell proliferation and cell fate specification (reviewed in LOGAN and NUSSE 2004). Recent works also have uncovered some common aspects of the canonical Wg-signaling pathway and PCP signaling: The trimeric G-protein G₀-subunit was reported to be required for direct transduction of Fz signals from the membrane to downstream components in both pathways (KATANAEV et al. 2005), and a genetic approach based on fz alleles revealed that even though the PCP signaling and the canonical Wg signaling are genetically distinct, fz controls these two different signaling activities by a common mechanism (POVELONES et al. 2005). However, Wg and PCP signaling differ in their requirements for more downstream factors such as armadillo/ß-catenin (arm/ß-cat) (NOORDERMEER et al. 1994) and the transcription factor pangolin/Drosophila T cell Factor (pan/dTCF) (BRUNNER et al. 1997; VAN DE WETERING et al. 1997) for the former, and small GTPase RhoA/Rho1 (STRUTT et al. 1997), MAP kinase cascade components (BOUTROS et al. 1998; PARICIO et al. 1999), and the inturned/fuzzy (in/fy) group of genes (PARK et al. 1996; COLLIER and GUBB 1997) for the latter. The pathways appear to diverge downstream of Dsh (AXELROD et al. 1998; BOUTROS et al. 1998).

The PCP signal from Fz/Dsh directs asymmetric cytoskeletal reorganization and polarized cell morphology in part by activating RhoA/Rho1 (STRUTT et al. 1997) and its downstream effector, Drosophila Rho-associated kinase, Drok (WINTER et al. 2001). RhoA acts as a molecular switch that gates signaling to downstream targets, both nuclear and cytoskeletal. In eye development, RhoA signals via Jun N-teminal kinase and Jun to regulate PCP-dependent transcription (STRUTT et al. 1997; FANTO et al. 2000; WEBER et al. 2000). In the wing, RhoA signals via Drok, which functions by regulating myosin II activity via phosphorylating Spaghetti squash (Sqh), a Drosophila homolog of nonmuscle myosin II regulatory light chain (MRLC) (KARESS et al. 1991; WINTER et al. 2001). Mutations in Drok result in wing cells forming multiple hairs of normal orientation. In the eye, mutations in Drok lead to abnormalities in the rotation of ommatidia (WINTER et al. 2001).

Here we show that the expression of a newly identified gene, target of wingless (tow), is negatively regulated by a canonical Wg-signaling pathway and that tow interacts with Drok in PCP signaling. Loss-of-function mutations in tow show neither a PCP phenotype nor wg-signaling-related defects, but its overexpression leads to wing cells forming multiple hairs of normal polarity much like loss-of-function mutations in Drok. We found that Tow is a nuclear protein in pupal wing cells and that spaghetti squash (sqh) mRNA was downregulated when Tow was overexpressed. This downregulation is presumably at least part of the reason for the Tow overexpression multiple-hair-cell phenotype.

Drosophila stocks:

Alleles used in this study were UAS-wg and UAS-dTCF^DN (gifts from R. Nusse); sev-Gal4 UAS-fz (a recombinant of sev-Gal4 and UAS-fz, gift from D. Strutt); UAS-RhoA, UAS-Rho^V14, UAS-Drok-CAT, and UAS-Drok-CAT-KG, sqh^AX3 (gifts from L. Luo); dsh¹, -tub-Drok, Drok², RhoA^72O, zip¹, zip^IIX62, ck¹³, ck⁰⁷¹³⁰, and UAS-RedStinger (Bloomington Stock Center). stan^VC31, a dominant-negative allele of stan, was induced by EMS, and it is a recessive lethal. sha^VB13 is a temperature-sensitive hypomorphic allele of shavenoid (sha; also called kojak) (HE and ADLER 2002).

Enhancer trap screening:

An enhancer trap screen was performed as described by KIM et al. (2006).

X-gal staining of third instar larvae:

Imaginal discs were dissected from third instar larvae in phosphate-buffered saline (PBS), fixed in 1% glutaraldehyde in PBS for 20 min at room temperature, and washed twice for 10 min with PBTr (0.5% Triton X-100 in PBS). The disc complexes were immersed in 200 µl of X-gal staining solution (10.0 mM NaH₂PO₄·H₂O/Na₂HPO₄·2H₂O [pH 7.2], 150 mM NaCl, 1.0 mM MgCl₂, 3.1 mM K₄[Fe^II(CN)₆], 3.1 mM K₃[Fe^III(CN)₆], 0.3% Triton X-100) adding 8 µl of 50 mg/ml X-gal to dimethylformamide for several hours at 37°. Disc complexes were washed twice for 10 min each with PBTr and mounted in 70% glycerol. LacZ expression patterns were viewed on Nikon microscopes and photographed with an AxioCam camera. Larvae derived from 7500 new insertions were stained for lacZ expression, and line 4938 was selected since its expression appeared opposite to that of wg in the wing discs.

Plasmid rescue and flanking sequence analysis:

A total of 1–5 µg of the tow-lacZ genomic DNA was fully digested with EcoRI or SacII, and the DNA fragments were self-ligated. After precipitation, the ligated DNAs were transformed into Escherichia coli strain XL-1 Blue by electroporation. Of the ligated DNAs, the genomic DNA flanking of the P-element insertion was selected by an ampicillin-resistant marker.

The flanking DNA was sequenced using either the IR primer (CGA-CGG-GAC-CAC-CTT-ATG-TTA-TTT-CAT-CAT-G) or the A primer (GAG-TTA-ATT-CAA-ACC-CCA-CGG). The sequence was identified using the BLAST program in the database of genomic clones sequenced by the Berkeley Drosophila Genome Project (http://www.fruitfly.org/).

Clonal analysis:

Flip-out clones were generated by the Flp-mediated recombination technique (GOLIC 1991; XU and RUBIN 1993). Clones were induced at 48–60 hr after egg laying (AEL) by heat shock at 37° for 1 hr, and third instar larvae were dissected and fixed. The genotype of the larvae used to make wg flip-out clones was act>y⁺>Gal4, UAS-CD2/UAS-wg; hs-FLP, MKRS/tow-lacZ.

Immunohistochemistry:

Imaginal discs were dissected from third instar larvae, fixed with fixation buffer (0.1 M PIPES, pH 6.9, 1 mM EDTA, 1.0% Triton X-100, 2 mM MgSO₄, 1% formaldehyde), blocked in a solution [50 mM Tris–Cl (pH 6.8), 150 mM NaCl, 0.5% NP-40, 5 mg/ml bovine serum albumin (BSA)] and stained overnight at 4° with appropriate antibodies in a washing/incubation solution (50 mM Tris–HCl, pH 6.8, 150 mM NaCl, 0.5% NP-40, 1 mg/ml BSA). After washing several times, secondary antibodies were applied for 2–4 hr at room temperature or on ice. The cuticles were mounted in antifade mounting solution (BioMeda). The fluorescence images were obtained with a confocal microscope system (Zeiss LSM510).

Primary antibodies used in these experiments were rabbit anti-ß-gal (Serotec, 1:2000), mouse anti-Wg (4D4, DSHB, 1:10). Secondary antibodies linked to Cy2 or Cy3 were obtained from Amersham Pharmacia Biotech and used in 1:1000.

RNA in situ hybridization:

In situ hybridization of third instar larvae was done as described before (TAUTZ and PFEIFLE 1989). Digoxigenin-labeled antisense RNA probes were synthesized from full-length cDNA of tow.

Generation of tow mutants:

To obtain a deletion mutant of tow, we induced imprecise excision of the P element in the tow-lacZ line. The flanking sequence is occasionally deleted during P-element jumping. The source of the transposase in this dysgenic cross was [2-3]Ki, and it mobilized P[lacW] from the insert site. First, tow-lacZ homozygous females were crossed to male [2-3]Ki, and the male progeny were crossed to balancer virgins (w; TM3/TM6B). Among the progeny, those whose P element had jumped out had white eyes; hence we selected male progeny with white eyes, which do not have the Ki phenotype. The selected males were balanced with TM3 or TM6B by crossing individually to TM3/TM6B virgins. Of the 500 lines, one null mutant, tow⁷⁵⁴, whose deletion range covers the whole open reading frame (ORF) of the tow transcript, was confirmed by genomic PCR.

tow^fz inversion mutant was created by hs-FLP-mediated inversion. The P/Piggy Bac insertion alleles tow^d02991 and fz^f07870 were kindly provided by the Harvard Exelixis Stock Collection. The relevant transposons contain FRT sequences (PARKS et al. 2004). A recombinant chromosome carrying both insertions was obtained by meiotic crossing over. Male flies that were w hs-flp; tow^d02991 fz^f07870/TM6 were generated and heat-shocked several times during larval and pupal life. These were crossed to w; fz^R53 females and the progeny were screened for phenotypically fz flies. Stocks were established from several such flies and salivary gland squashes were performed to confirm the presence of the predicted inversion. The inversion breaks both the tow and the fz transcription units. It results in a phenotypically null fz allele and a tow allele that appears to be a biochemical null.

Generation of transgenic flies:

Sequencing confirmed that the fly EST GH12583 that encodes Tow carries an intact tow gene. We cloned the full-length EST into the pUAST vector (BRAND and PERRIMON 1993) to generate flies carrying the UAS-tow construct. Various Gal4 drivers were used to analyze the consequences of overexpressing Tow.

arm-tow-GFP is a fusion at the C-terminal end of the full-length Tow open reading frame to the N-terminal end of GFP. It was cloned into the pCaSpeR vector with an armadillo (arm) promoter, and germline transformation was performed by standard methods (SPRADLING and RUBIN 1982).

Actin staining:

Pupal wings were stained with rhodamine-labeled phalloidin, as previously described (WONG and ADLER 1993).

Mosaic analysis of Drok² clones:

FLP/FRT-mediated recombination was used to generate clones homozygous for Drok² in the wing via heat-shock-induced recombination 48–72 hr AEL. Homozygous wing clones in newly eclosed adults were visualized using the MARCM system (LEE and LUO 1999) by the presence of the membrane marker mCD8GFP. The genotype used was y w Drok² FRT^19A/tubP-GAL80, hs-FLP, FRT^19A; UAS-mCD8GFP/+; tubP-Gal4/+.

Genetic interaction and sectioning of adult eyes:

For genetic interaction studies with sev-Gal4 UAS-fz (STRUTT et al. 1997), flies heterozygous for both sev-Gal4 UAS-fz and the tow mutation were analyzed. Adult heads were cut and fixed overnight in 4% paraformaldehyde solution at 4°, washed three times for 15 min in 0.1 M phosphate buffer (pH 7.2), and postfixed for 1 hr in 1% OsO₄ until the specimen turn black or dark brown. After washing three times for 10 min in distilled water, the samples were dehydrated with ethanol and propylene oxide. Next, the propylene oxide was exchanged with Spurr and embedded in a mold and polymerized for 24 hr at 65°. The block was sectioned into 200- to 300-nm thick slices with an ultramicrotome through the equatorial region of the eye. The slices were stained with 1% Toluidine Blue O on a hot plate for several minutes, and ommatidia were examined for incorrect rotation and chirality. At least five sections from independent eyes were analyzed for each genotype.

Genetic interaction assays using multiple wing hairs:

Wings were mounted in Euparal (Asco Labs) or Gary's Magic Mount and examined under bright-field microscopy. The multiple-wing-hair phenotype in Figure 6 and Table 2 was induced by heat shock at 24 hr after puparium formation (APF) at 37° in animals carrying one copy of hs-Gal4, UAS-tow and the mutation or transgene of several components in Drok pathway. The multiple-wing-hair phenotype was quantified in the dorsal surface of an area enclosed by the L3 and L4 vein and the first intervein and the imaginary line starting perpendicularly from the tip of second intervein to the L3. Since overexpression of Tow usually causes the wing blade surface to be uneven and wavy, counting the number of multiple hairs was easier in the central region of the wing than in other regions. The number of multiple hair cells was counted and averaged for at least 10 wings/genotype.

View larger version (110K): In this window In a new window Download PPT slide   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 -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/-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.

 

View this table: In this window In a new window   TABLE 2 Genetic interaction with tow gain-of-function phenotype

 

Subcellular localization of Tow:

ap-Gal4/+; UAS-RedStinger/arm-tow-GFP pupae were fixed in 4% paraformaldehyde in PBS at 4° overnight. UAS-RedStinger line expresses a variant DsRed (red fluorescent protein) with a nuclear localization signal under the control of UAS. The GFP signals were seen without staining or after anti-GFP staining (1:4,000; Molecular Probes, Eugene, OR). The wings were mounted in Prolong Gold Mounting Media (Molecular Probes) and examined in a ATTO CARV confocal unit attached to a Nikon camera.

Northern hybridization:

Heat shock was given to pupae of hs-Gal4/+; UAS-tow/+ flies (at 24 hr APF) at 37° for 40 min two times with a recovery period of 40 min. Total RNA was isolated with Trizol^R (Gifco). Thirty micrograms of total RNA/sample was separated by formaldehyde gel, blotted onto nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and hybridized with ³²P-labeled DNA probe specific for sqh. The sqh fragment was amplified by RT–PCR from wild-type flies using the sqh-specific primers sqh-RTP1-5' (AGT-GCA-GCT-GGT-CGA-AAG-TT) and sqh-RTP2-3' (ATT-CGA-GGT-AGT-CGA-ACA-GA).

Western blotting:

hs-Gal4/+; UAS-tow/+ pupae were heat-shocked as described for the Northern blot analyses. Total protein was isolated and analyzed using standard methods. Thirty micrograms of total protein/sample was separated by 4–20% gradient SDS–PAGE gel, blotted onto PVDF membrane, and incubated with anti-Sqh or anti-phospho-Sqh antibody (Cell Signaling).

Identification of Wg-responsive enhancer trap, 4938, inserted at the tow locus:

To identify novel molecules involved in wing development, we performed an enhancer trap screen (KIM et al. 2006). The P[lacW] transposon (BIER et al. 1989) was randomly mobilized, and larvae derived from 7500 P-element insertions were stained for ß-gal expression. Line 4938 was further analyzed because it drove lacZ expression in the wing pouch but not at the dorsal/ventral (D/V) compartment boundary of the wing disc (Figure 1A) where wg is expressed. We show below that this enhancer trap is inserted into the tow gene and that its expression is identical to that of tow.

View larger version (53K): In this window In a new window Download PPT slide   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-dTCFDN derepresses tow-lacZ expression at the A/P boundary of the wing (indicated by arrow). The genotype of the larvae is UAS-dTCFDN/+; dpp-Gal4/tow-lacZ. (D) Ectopic expression of Wg (asterisks in D-I and D-iii) represses tow-lacZ. (D-i) ß-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) towfz mutant (tow and fz double mutant) created by hs-FLP-mediated inversion. Homozygous towfz mutant shows typical fz mutant phenotype. Trans-heterozygote of tow754 and towfz 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.

 
The pattern of 4938 enhancer trap (tow-lacZ hereafter) expression led us to test whether Wg signaling could regulate its expression. First, we induced Wg-expressing flip-out clones using the FLP–FRT system (GOLIC 1991; XU and RUBIN 1993) and found that the ß-gal expression of tow-lacZ was repressed nonautonomously in the vicinity of the clones (Figure 1D). The nonautonomy is expected as Wg is secreted and acts on neighboring cells (ZECCA et al. 1996; NEUMANN and COHEN 1997) and antibody staining is not sensitive enough to detect levels of ligand known to be biologically active in distant cells (NEUMANN and COHEN 1997; STRIGINI and COHEN 2000). We also utilized the UAS/Gal4 system (BRAND and PERRIMON 1993) to express a dominant-negative form of Drosophila T cell factor (dTCF). The wild-type dTCF protein is a transcription factor activated by Wg signaling, while dTCF^DN (VAN DE WETERING et al. 1997) inhibits Wg signaling. Expression of dTCF^DN in a Decapentaplegic (Dpp) domain [i.e., a stripe that runs perpendicular to the D/V wg stripe at the anterior/posterior (A/P) boundary] leads to derepression of ß-gal activity of tow-lacZ within the Dpp domain (Figure 1C). Thus, Wg signaling negatively regulates the expression of tow-lacZ enhancer trap. In other experiments, we found the expression of wg was not affected by overexpression or absence of tow (Figure 1B). Drosophila frizzled 2 (Dfz2) is a receptor for canonical Wg signaling pathway (BHANOT et al. 1996) and is expressed in the wing in a pattern very similar to that of tow, i.e., with the lowest levels found at the D/V boundary (CADIGAN et al. 1998). The interaction between Wg and DFz2 plays a crucial role in shaping the Wg morphogen gradient and in determining the response of cells to the Wg signal. We found that the expression of Dfz2 was also not affected by tow (data not shown). Thus, tow transcription is a downstream target of Wg signaling.

Sequencing of genomic DNA fragments flanking the insertion showed that the P element was inserted into the first intron of a novel gene (CG14821) that we named target of wingless (tow), which encodes a protein of 347 amino acids. Homologs of tow are found in other insects but not in more distantly related organisms. Sequence analysis revealed that there is a highly homologous gene, GA13273, in Drosophila pseudoobscura and that it is also very well conserved in other Drosophila species, such as D. simulans, D. yakuba, D. erecta, D. ananassae, D. mojavensis, and D. virillis (their genome sequences are available at http://species.flybase.net/). In addition, the first 60 amino acids in the N terminus show homology to a gene in Anopheles gambiae. Tow does not contain informative motifs. However, the sequence is notable for containing eight runs of multiple Gln residues and one Pro-rich region and for being unusually rich in Gln (20.2%) and Pro (10.1%) (Figure 1G).

In situ hybridization to wild-type wing discs using a tow-specific probe showed the same expression pattern as 4938 lacZ staining, in that tow mRNA was expressed in the wing pouch except at the D/V boundary (Figure 1F).

tow null mutant is viable:

To obtain a loss-of-function allele of tow, we mobilized the tow-lacZ P element. We obtained an allele, tow⁷⁵⁴, which deletes the entire tow open reading frame and a part of a neighboring gene, CG10077 (Figure 1E-i). By genomic DNA sequencing, we determined that the 7.4-kb deletion removes the entire tow gene except for the first (noncoding) exon and part of the first intron and 160 amino acids of the 3'-end of CG10077-RA, which is the longer of its two transcripts. RT–PCR confirmed that tow⁷⁵⁴ was a null allele as no tow mRNA could be detected in tow⁷⁵⁴ homozygous flies (data not shown). Homozygotes of tow⁷⁵⁴ were viable and showed no obvious phenotype. Similar results were obtained with trans-heterozygotes of this tow mutant and a deficiency for the region.

Since tow⁷⁵⁴ deletes a part of another gene, we made another allele using hs-FLP-mediated inversion. We obtained an inversion mutant allele, tow^fz, in which a genomic DNA fragment between the first intron of tow and the final intron of fz was inverted (Figure 1E-ii). Homozygous tow^fz flies were also viable and showed a typical fz mutant phenotype in the wings and thorax. No tow mRNA was detected in homozygous tow^fz flies in RT–PCR experiments (data not shown). Trans-heterozygotes of tow⁷⁵⁴ and tow^fz flies were normal, showing that Tow is nonessential.

Overexpression of Tow causes planar polarity defects manifested by multiple wing hairs:

Gain-of-function studies can often give a clue as to the function of a gene and are particularly useful in analyzing functionally redundant genes. We generated transgenic flies, which carried a UAS-tow cDNA transgene and crossed them to various Gal4 drivers (BRAND and PERRIMON 1993). Activation of transcription by Gal4 in apterous (ap)-expressing cells results in overexpression in thorax and dorsal wing blade. Interestingly, when Tow was overexpressed by the ap-Gal4 driver, it caused a disorganization of microchaetae and stunted macrochaetae on the thorax as well as defects in the scutellum (Figure 2B). A disorganized microchaetae phenotype was also seen as a consequence of the misexpression of several planar polarity genes, and gain-of-function screening using this bristle defect has been successfully used in finding a novel component of planar polarity (PARICIO et al. 1999; FEIGUIN et al. 2001). Therefore, this phenotype led us to hypothesize that Tow is also involved in PCP. It is intriguing that a gene whose expression is regulated by Wg signaling could have a function in PCP signaling because the canonical Wg signaling and PCP signaling have been known to diverge into distinct pathways downstream of Dsh (AXELROD et al. 1998; BOUTROS et al. 1998).

View larger version (83K): In this window In a new window Download PPT slide   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.

 
We overexpressed Tow in the wing using several wing-specific Gal4 drivers, all of which resulted in multiple hair cells (Figure 2D). In wild-type wings, each cell produces a single, distally oriented wing hair, and multiple-hair-cell phenotypes are associated with PCP defects. Most PCP genes, including fz and dsh, exhibit both stereotypical orientation and multiple-hair-cell defects (GUBB and GARCIA-BELLIDO 1982; WONG and ADLER 1993), but others such as Drok show only the multiple-wing-hair phenotype (WINTER et al. 2001; Figure 2E).

To investigate the cellular basis of the Tow overexpression phenotype, we used phalloidin staining to examine the distribution of F-actin during hair morphogenesis. When we used MS1096-Gal4 to drive Tow expression mainly in the dorsal region of the wing (CAPDEVILA and GUERRERO 1994), a majority of wing cells formed more than one F-actin bundle (Figure 2F). Notably, most prehairs maintained a roughly distal orientation. The similarities between the pupal and adult phenotypes indicate that the multiple-hair-cell phenotype of adult Tow-overexpressing wings is likely the result of a failure to restrict F-actin bundle assembly to a single site during prehair formation. Due to the similarity of their multiple-hair-cell phenotypes, we thought it possible that Tow might be involved in the Drok pathway. In addition, MS1096-Gal4-driven Tow overexpression caused extra bristles in the anterior wing margin and in the wing blade near the most distal region, which suggests that ectopic expression of Tow in the wing margin where it is not normally expressed results in formation of extra bristle sense organs in those regions (data not shown).

Tow acts downstream of Frizzled/Dishevelled in planar cell polarity:

The proper level of Fz/Dsh signaling is critical for the generation of wild-type PCP, and it has been reported that both overexpression and loss of function of many PCP genes result in polarity defects in the eye (ZHENG et al. 1995; STRUTT et al. 1997; BOUTROS et al. 1998) and the wing (KRASNOW and ADLER 1994; AXELROD et al. 1998; GUBB et al. 1999; USUI et al. 1999; TREE et al. 2002). The multiple-hair-cell phenotype of Tow gain of function led us to hypothesize that Tow might also act downstream of Fz/Dsh. To assess the genetic interaction of the Fz pathway and tow, we employed two kinds of assays in the eye and in the wing.

In the Drosophila eye, PCP is reflected in the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline, the equator. This pattern is generated posterior to the morphogenetic furrow when ommatidial preclusters rotate 90° toward the equator, adopting opposite chirality, depending on their dorsal or ventral positions (GUBB 1993; Figure 3A). Polarity defects are manifested in the loss of mirror-image symmetry, with the ommatidia misrotating and adopting random chirality or remaining symmetrical (GUBB 1993; THEISEN et al. 1994; ZHENG et al. 1995; STRUTT et al. 1997; BOUTROS et al. 1998; WOLFF and RUBIN 1998).

View larger version (85K): In this window In a new window Download PPT slide   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.

 
The sevenless (sev)-driven gain-of-function Fz or Dsh phenotype (sev-Gal4 UAS-fz or sev-Gal4 UAS-dsh) has previously been used successfully to identify and study new components of the Fz/Dsh planar polarity pathway (STRUTT et al. 1997; BOUTROS et al. 1998). We employed the same assay to determine if Tow could be a new component of the Fz pathway. sev-driven overexpression of Fz in the eye results in planar polarity defects characterized by abnormal ommatidial chirality and misrotation due to overactivation of planar polarity signaling (STRUTT et al. 1997; Figure 3B). Null tow⁷⁵⁴ flies showed no eye polarity defects and the overexpression of Tow using a sev driver also failed to produce an eye phenotype (Figure 3A). However, we found that a null mutation of tow acted as a strong dominant suppressor of the gain of function of the sev-Gal4 UAS-fz eye phenotype (Figure 3C; Table 1), as is the case for PCP genes such as RhoA (STRUTT et al. 1997).

View this table: In this window In a new window   TABLE 1 Quantification of genetic interactions with the sev-Gal4 UAS-fz phenotype

 
We confirmed these results using another tow allele, tow^fz. Introduction of a tow^fz chromosome could also suppress the sev-Gal4 UAS-fz eye phenotype (Table 1). Since tow^fz is a double mutant for tow and fz, and a fz mutant itself can dominantly suppress the sev-Gal4-driven Fz overexpression phenotype (STRUTT et al. 1997; Table 1), we tried to rescue fz activity in the tow^fz mutant by introduction of an arm-fz-GFP chromosome (STRUTT 2001). The suppression of the sev-Gal4 UAS-fz eye phenotype by tow^fz was still seen when the arm-fz-GFP chromosome was present (Table 1). In addition, co-overexpression of Tow and Fz resulted in enhanced polarity defects; notably, the frequency of misrotation of ommatidia was increased (Figure 3D; Table 1). Thus the sev-Gal4 UAS-fz phenotype is sensitive to both an increase and a decrease in tow dose. These data suggest that Tow could be a downstream target of the Fz pathway or it could function in parallel in eye planar polarity. In addition, two kinds of tow alleles showed the same suppression results. Therefore, the deletion of CG10077 in the tow⁷⁵⁴ mutant does not seem to affect the genetic interaction with fz.

We also examined genetic interactions between tow and dsh, a downstream effector of Fz. We made use of the dsh¹ allele, which is defective for PCP function without affecting canonical Wg signaling (AXELROD et al. 1998; BOUTROS et al. 1998). In a dsh¹ mutant, typical polarity defects such as swirling hair patterns and multiple wing hairs are seen (WONG and ADLER 1993; Figure 4A). To assess the genetic interactions between dsh and tow, we quantified the multiple-hair-cell phenotype in a defined region, the dorsal surface of the central region of the wing (the region is described in detail in MATERIALS AND METHODS).

View larger version (95K): In this window In a new window Download PPT slide   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.

 
In dsh¹ hemizygous males, an average of 4.8 cells with multiple hairs were present in this region (Figure 4, A and E). This multiple-hair-cell phenotype was suppressed by the reduction of tow dosage, leading to an average of only 0.8 multiple hair cells in the defined region, when one copy of the tow⁷⁵⁴ chromosome was introduced (Figure 4, B and E). The polarity of wing hairs in dsh¹ hemizygotes was not affected by tow dosage. ap-Gal4-driven UAS-tow resulted in multiple hairs over the entire dorsal surface of the wing, and in our target region an average of 14.9 cells with multiple hairs were found (Figure 4, C and E). When Tow was overexpressed by ap-Gal4 in a dsh¹ hemizygous background, the average number of cells exhibiting multiple hairs was substantially increased to 44.3/wing region (Figure 4, D and E). ap-Gal4-driven overexpression of Tow caused the wing blade to be very uneven and wavy, so the polarity phenotype in dsh¹ looked enhanced. However, when Tow was overexpressed by a weaker driver such as hs-Gal4, it was obvious that only the number of multiple wing hairs was increased and no apparent enhancement of polarity phenotype was detected (data not shown). In the control wings of dsh¹/Y; ap-Gal4/+ or dsh¹/Y; UAS-tow/+ flies, no remarkable variation of the number of multiple hair cells was observed. We found a similar effect between tow^fz and dsh¹ alleles when fz activity was rescued by the arm-fz-GFP chromosome (data not shown). In a previous study, the overexpression of Drok reduced the average number of multiple hair cells in dsh¹ hemizygotes, and a reduction in Drok dose caused an increased number of multiple hair cells, which led to the suggestion that Drok was activated by Fz signaling (WINTER et al. 2001). We found an opposite dose response between tow and dsh¹. Taken together, these experiments suggest that Tow functions downstream of Fz/Dsh perhaps to inhibit Drok activity.

Genetic interaction of tow with other PCP genes:

To see if tow genetically interacts with other known PCP genes, we crossed the tow⁷⁵⁴ null mutant to several PCP mutants and examined the phenotypes of the trans-heterozygotes or double homozygotes. The dominant allele of stan, stan^VC31, causes a swirling polarity pattern and a small number of multiple hair cells on the wing. The number of multiple hair cells was increased by a reduction in tow dosage (Figure 5, A and B). The introduction of one copy of the tow⁷⁵⁴ null allele also can enhance the phenotype of the sha mutant. Mutations in sha result in a delay in hair morphogenesis and, in some cells, no hair or only several small hairs form (HE and ADLER 2002). The morphogenesis of the hair involves temporal control by sha and spatial control by the genes of the Fz pathway, and there is a strong genetic interaction between mutations in these genes (REN et al. 2006). tow mutation caused an increased number of small hairs and multiple wing hairs in the hypomorphic sha^VB13 background (Figure 5, C and D). We also looked for interactions with other PCP genes such as Van Gogh [Vang; also known as strabismus (stbm)], which encodes a putative membrane protein (TAYLOR et al. 1998; WOLFF and RUBIN 1998); fy, which encodes a novel four-pass transmembrane protein (COLLIER and GUBB 1997); and prickle (pk), which encodes a protein with LIM domains (GUBB et al. 1999). However, there was no distinct interaction between the tow mutant and Vang^TBS42, a dominant allele of Vang (TAYLOR et al. 1998); fy^JN11/fy^JN12, weak alleles of fy (COLLIER and GUBB 1997); and pk^D, a dominant allele of pk (ADLER et al. 2000).

View larger version (96K): In this window In a new window Download PPT slide   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) stanVC31/+ wing in the anterior region. (B) Trans-heterozygotes of tow754 and stanVC31 showing increased multiple wing hairs. (C) shaVB13 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 shaVB13 background.

 

tow genetically interacts with the Drok pathway:

Transient overexpression of Tow driven by hs-Gal4 with heat shock at 24 hr APF caused the formation of multiple hair cells throughout the wing blade. Once again, the multiple hairs had largely normal polarity. In the flies that carried one copy of both hs-Gal4 and UAS-tow, an average of 10.9 cells with multiple hairs was found in the defined region (Figure 6A; Table 2). When Drok was simultaneously overexpressed [using a -tubP-Drok transgene (WINTER et al. 2001)] with Tow, the average number of multiple hair cells was reduced by more than one-half to 4.6/wing region (Figure 6B; Table 2). This suppression was confirmed with co-overexpression of the catalytic domain of Drok (Drok-CAT) (WINTER et al. 2001) and Tow by hs-Gal4, which resulted in 5.3 multiple hair cells/region (Table 2). Complementary dosage responses were seen with loss-of-function mutations in Drok. The presence of a Drok² null allele (Drok²/+) enhanced the multiple-hair-cell phenotype of hs-Gal4 UAS-tow by 2.3-fold to 24.7 multiple hair cells/region (Figure 6C; Table 2). Similarly, when a kinase-dead form of Drok, Drok-CAT-KG (WINTER et al. 2001), which is not a dominant-negative form, was co-overexpressed with Tow, the phenotype was slightly enhanced to 13.5 multiple hair cells/region (Table 2).

Drok is a downstream effector of RhoA/Rho1, which functions as a molecular switch that gates signaling from Fz/Dsh to downstream targets (STRUTT et al. 1997; WINTER et al. 2001). The presence of a RhoA^72O allele (STRUTT et al. 1997) slightly increased the number of multiple hairs caused by hs-Gal4 UAS-tow to 13.3/region (Table 2). On the other hand, co-overexpression of Tow and wild-type RhoA via UAS-RhoA (LEE et al. 2000) also resulted in a reduced number of multiple hair cells (2.1/wing region, Table 2). A constitutively active form of RhoA, UAS-Rho^V14 (LEE et al. 2000), caused lethality when overexpressed by hs-Gal4, so we could not examine the effect.

An important substrate of Drok is sqh, which encodes the Drosophila homolog of the nonmuscle MRLC. Sqh is phosphorylated by Drok in vivo (KARESS et al. 1991; JORDAN and KARESS 1997). Reducing the wild-type sqh gene dosage from two to one by introducing a single copy of the sqh^AX3 null allele (JORDAN and KARESS 1997) also slightly enhanced the multiple-wing-hair phenotype caused by Tow overexpression (Table 2).

Zipper (Zip) also functions in hair morphogenesis and interacts with Fz/Dsh (WINTER et al. 2001). Loss of one copy of the zip gene enhanced the dsh¹ and hs-Fz multiple-hair-cell phenotypes, suggesting that myosin II functions positively downstream of Fz/Dsh in hair development. We found that reducing the dose of zip substantially enhanced the multiple-hair-cell phenotype caused by overexpression of Tow (Figure 6D, Table 2). This was confirmed for two independent zip alleles, zip¹, a null mutant, and zip^IIX62, a hypomorph (Table 2).

We also found an interaction with crinkled (ck), which encodes a myosin VIIA. In the mouse, a mutation in myosin VIIA (shaker-1 mutant) caused stereocilia disorganization and the formation of multiple stereocilia bundles (SELF et al. 1998). ck was previously found to be involved wing-hair morphogenesis and to interact with Fz/Dsh signaling in Drosophila. Mutations in ck cause multiple wing hairs as well as a split-hair phenotype (GUBB et al. 1984; TURNER and ADLER 1998), and a reduction in ck gene dose suppressed the dsh¹ multiple-hair phenotype (WINTER et al. 2001). The genetic interaction of ck with components of the Fz/Dsh pathway has opposite effects to that of zip, and the seemingly antagonistic relationship between myosin II and myosin VIIA suggested a mechanism in which the balance of the activities or stoichiometry of these two myosins is critical for the common process that they regulate (WINTER et al. 2001). We also found a complementary genetic interaction of tow with these two myosins. Contrary to the enhancement of the Tow overexpression phenotype by the introduction of the zip mutant chromosome, reduction in the ck dose suppressed the multiple-wing-hair phenotype due to overexpression of Tow to only one or two multiple hair cells per region (Table 2). This was also confirmed by two independent alleles, ck¹³ and ck⁰⁷¹³⁰ (Table 2).

Tow is localized in the nucleus in pupal wing cells and overexpression of Tow downregulates the level of sqh mRNA:

GFP was fused to the C terminus of Tow to generate a tool for studying the subcellular localization of the Tow protein. The tow-lacZ signal was uniformly distributed in the pupal wing blade, so we expressed Tow-GFP under the control of the ubiquitous promoter of arm (VINCENT et al. 1994) and examined arm-Tow-GFP distribution in pupal wings at various times between 24 and 36 hr APF. The GFP signal in pupal wings was always found in what we suspected to be nuclei. To confirm this, we colocalized arm-Tow-GFP and a nuclear fluorescent protein (using the UAS-RedStinger line, which expresses a variant red fluorescent protein with a nuclear localization signal under the control of UAS). Indeed, the two signals colocalized, consistent with Tow being primarily a nuclear protein in the pupal wing cells (Figure 7A).

View larger version (29K): In this window In a new window Download PPT slide   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.

 
Due to the lack of the mutant phenotype for tow, we could not test the function of Tow-GFP by simply looking at the rescue of a tow mutation. Hence, we expressed Tow-GFP in hs-Gal4/+; UAS-tow/tow⁷⁵⁴ flies. The tow⁷⁵⁴ null mutation can suppress the multiple-hair-cell phenotype caused by the hs-Gal4-driven overexpression of Tow by about two-thirds (Table 2). Introduction of the arm-tow-GFP chromosome slightly but significantly increases the number of multiple hair cells, so we assume that arm-Tow-GFP mimics endogenous Tow function. However, we note that we still cannot rule out the possibility that arm-Tow-GFP also localized to ectopic sites where Tow is not normally present.

The nuclear localization of Tow-GFP led us to test whether the multiple-wing-hair phenotype caused by Tow overexpression could be due to repression of the mRNA level of one or more components of the Drok pathway. Total RNA was isolated from the hs-Gal4 UAS-tow pupae to which heat shock was given 24 hr APF, and Northern hybridization results showed that the mRNA level of sqh, a substrate of Drok, was repressed in the Tow-overexpressing flies (Figure 7B). Western blotting experiments confirmed that Sqh protein and phosphorylated Sqh level were also repressed in the Tow-overexpressing flies (data not shown). On the other hand, no difference in mRNA level was detected in homozygous tow⁷⁵⁴ or tow^fz mutants (data not shown), or trans-heterozygotes of these (Figure 7B), consistent with the normal phenotypes of those flies.

A novel gene whose expression is regulated by the canonical Wg-signaling pathway has a function in PCP signaling:

tow was identified in an enhancer trap screen, and its expression pattern in the wing imaginal disc was the opposite to that of wg. The expression of tow was negatively regulated by Wg signaling: When Wg was expressed ectopically, tow was downregulated nonautonomously, and when Wg signaling was blocked by misexpression of a dominant-negative form of dTCF (VAN DE WETERING et al. 1997), the expression of tow was derepressed in the Gal4-expressing domain. The expression of wg or its receptor Dfz2 was not affected by tow, which suggested that tow is a downstream target of Wg signaling. From its expression pattern and the regulation by Wg signaling, we expected that tow might function in the Wg-signaling pathway. Our data, however, showed that Tow has little, if any, function in Wg signaling. The only evidence supporting a role for tow in Wg signaling was that the overexpression of Tow in the wing resulted in several extra bristles at the anterior wing margin and in the wing blade near the distal margin. The specification of sensory bristle cells is dependent on wg activity (PHILLIPS and WHITTLE 1993; COUSO et al. 1994). Since the expression of wg or Dfz2 was not affected by the overexpression of Tow, this extra bristle phenotype might be caused by Tow in some way regulating the expression of one or more downstream genes. On the other hand, surprisingly, we obtained evidence for tow acting in Fz-dependent planar cell polarity signaling.

Studies of the canonical Wg-signaling pathway and Fz-dependent polarity pathway have indicated that divergent signaling mechanisms are involved (AXELROD et al. 1998; BOUTROS et al. 1998). Although both the Wg- and Fz-signaling pathways require the function of Dsh, they demonstrate differential requirements for more downstream factors. Our study, however, showed that tow expression was negatively regulated by Wg signaling in wing discs and that it functioned in the regulation of the actin cytoskeleton and hair morphogenesis during PCP signaling. In both the wing and the eye, the interactions suggested that Tow functioned as a downstream regulator/component of PCP signaling and the Fz/Dsh pathway. The significance of the regulation of tow expression by Wg in wing discs for the function of tow during hair morphogenesis is unclear as these two events are temporally separated. Further studies will be required to determine this.

Interactions between tow and other PCP genes:

We tested for genetic interactions between tow and other PCP genes and found that the reduction in tow dose enhanced the multiple-hair-cell phenotype of a dominant stan allele. This interaction is the opposite of what we saw between tow and dsh. The basis for this difference is unclear but might be due to Tow affecting the expression of different target genes for these two phenotypes. That tow mutations could interact with PCP genes by altering gene expression would not be surprising. The PCP system is sensitive to the relative level of components, and interactions were seen previously between PCP genes and mutations in the grainy head transcription factor (LEE and ADLER 2004). Our experiments provided evidence for tow regulating sgh expression and this could explain at least part of the interaction with dsh.

The sha gene encodes a putative actin-binding protein whose expression increases dramatically just prior to hair initiation (REN et al. 2006). Consistent with the expression of sha playing a key role in regulating hair initiation, sha mutant wing cells either fail to form a hair or have delayed hair formation. Those cells that form delayed hairs produce multiple short ones. Previously, it was found that decreases in PCP gene function enhanced the sha mutant phenotype. We found that reduction in tow dose also enhanced a sha mutant phenotype. This could be due to an indirect effect of tow on the PCP pathway and/or to tow regulating sha expression directly or by an alternative mechanism.

Proper level of Tow is important in regulation of the actin cytoskeleton:

Mutations in a number of genes involved in PCP signaling result in multiple-wing-hair phenotypes as well as a swirling pattern of hairs on the Drosophila wing. Drok is thought to act as an effector of small GTPase RhoA and to link Fz/Dsh signaling to the actin cytoskeleton (WINTER et al. 2001). Drok mutations alter only the number of hairs produced and not the hair orientation; thus these two processes likely diverge upstream of Drok.

The gain-of-function phenotype of Tow (distally oriented multiple hairs) suggested that it functioned in the Drok pathway. Genetic interactions revealed that Drok and tow have an antagonistic relationship. Increasing Drok expression suppresses and decreasing Drok expression enhances the multiple-wing-hair phenotype caused by Tow overexpression. RhoA is upstream of Drok and functions to activate Drok. Thus, it is not surprising that overexpression of RhoA also suppresses the Tow multiple-hair-cell phenotype while mutation of RhoA enhances it. The phosphorylation of Sqh/MRLC by Drok leads to Sqh activation and, consistent with this, a reduction in sqh dose enhances the Tow multiple-hair-cell phenotype.The two cellular myosin genes zip (myosin II) and ck (myosin VIIA) also interact with Tow in a manner consistent with the interactions of these genes with Drok. These data suggest that the balance between Drok and Tow is important for the regulation of the actin cytoskeleton.

The arm-tow-GFP transgene revealed that Tow is localized in the nucleus in pupal wings. From the subcellular localization of Tow, we assumed that it might influence the expression of components involved in the Drok pathway. The overexpression of Tow downregulates sqh, a substrate of Drok. This leads to a decrease in the level of Sqh and phosphorylated Sqh protein. This provides a putative mechanism that can explain the multiple-hair-cell phenotype caused by Tow overexpression, which is similar to the Drok mutant phenotype. In a Drok mutant, the level of phosphorylated Sqh was reduced, indicating that Drok is required for maintaining the proper level of Sqh phosphorylation (WINTER et al. 2001). Genetic interactions of tow with Drok pathway components also fit such a model. The mutation of genes in the Drok pathway enhanced the multiple-hair-cell phenotype caused by Tow overexpression, and the overexpression or the introduction of transgenes of Drok pathway components suppressed the Tow overexpression phenotype. We conclude that the proper level of Tow (and/or the relative level of Tow and Drok) is important in the regulation of the actin cytoskeleton. However, it remains to be resolved whether the downregulation of sqh by overexpression of Tow is direct or not. The absence of a tow mutant phenotype suggests that there could be other genes whose function in the regulation of sqh is redundant with tow.

We thank L. Luo, R. Nusse, D. Strutt, the Bloomington Stock Center, and the Harvard Exelixis Stock Collection for providing the fly stocks and the Developmental Studies Hybridoma Bank for the antibody. This research was supported by a grant (M103KV010002 04K2201 00230) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, the Republic of Korea, to J.Y. and a grant from the National Institute of General Medical Science (GM-37136) to P.N.A.

¹ Present address: Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, MD 21205-2185.

³ These authors contributed equally to this work.

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