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doi: 10.1242/10.1242/dev.00588

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The Drosophila bZIP transcription factor Vrille is involved in hair and cell growth

Sébastien Szuplewski^*, Benjamin Kottler and Régine Terracol

Laboratoire de Génétique du Développement et Evolution, Institut Jacques Monod, 2 Place Jussieu Tour 43, 75251 Paris Cedex 05, France

Author for correspondence (e-mail: terracol{at}ijm.jussieu.fr)

Accepted 9 May 2003

	SUMMARY

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Vri is closely related to bZIP transcription factors involved in growth or cell death. vri clonal and overexpression analyses revealed defects at the cellular level. vri clones in the adult cuticle contain smaller cells with atrophic bristles. The phenotypes are strictly cell autonomous. Clones induced in the eye precursor cells lead to individuals with smaller eyes and reduced number of ommatidia with an abnormal morphology and shorter photoreceptor cell stalks. Overexpression of vri is anti-proliferative in embryonic dorsal epidermis and in imaginal discs, and induces apoptosis. On the wing surface, larger cells with multiple trichomes are observed, suggesting cytoskeletal defects. In salivary glands, vri overexpression leads to smaller cells and organs. We also show that vri is involved in locomotion and flight and interacts genetically with genes encoding actin-binding proteins. The phenotypes observed are consistent with the hypothesis that vri is required for normal cell growth and proliferation via the regulation of the actin cytoskeleton.

Key words: vrille, Growth, Proliferation, Differentiation, Apoptosis, Cytoskeleton, Drosophila melanogaster

	INTRODUCTION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The vrille (vri) gene was previously identified as an enhancer of decapentaplegic (dpp) embryonic and adult phenotypes (George and Terracol, 1997). vri RNA expression is dynamic throughout development. In the embryo, vri transcripts are maternally provided and later expressed in morphogenetically active epithelia: in foregut, pharynx, hindgut, in stripes along dorsal and lateral epidermis and in tracheal track. At third larval instar, vri is expressed in gut, brain and in imaginal discs. vri was later characterized as a clock-controlled gene expressed in circadian pacemaker cells (Blau and Young, 1999) and acting as a direct repressor of the Clock and cryptochrome genes (Glossop et al., 2003; Cyran et al., 2003).

vri encodes a bZIP transcription factor (George and Terracol, 1997) with a typical basic DNA-binding domain and a leucine zipper involved in homo- or heterodimerization (Vinson et al., 1989). Vri is closely related, in the bZIP extended domain, to proteins implicated in cell death or growth. The more closely related factors are, with 60% identity and 93% similarity with Vri, gene 9 of Xenopus (Brown et al., 1996) (which is induced by thyroid hormone during the tadpole tail resorption program) and E4BP4 (a transcription factor of the human placenta that acts as a repressor of the protein E4 of adenovirus, which is involved in apoptosis) (Cowell et al., 1992; Cowell and Hurst, 1994). This factor was later isolated as NFIL3A a transactivator of the interleukin 3 (IL3) promotor in human T cells (Zhang et al., 1995). In pro-B lymphocytes, the murine homolog of NFIL3A (100% identity in the bZIP domain) is a delayed anti-apoptotic early transcription factor induced by IL3 stimulation acting through the Ras/MAPK (mitogen-activated protein kinase) and PI3K (phosphatidylinositol 3-kinase) pathways. The alteration of these pathways is likely to contribute to human B-lineage leukemia (Ikushima et al., 1997; Kuribara et al., 1999). Vri and NFIL3A are also related to the segmentation gap gene product of Drosophila Giant (Capovilla et al., 1992), to CES-2, the product of a cell death specification gene of C. elegans (Metzstein et al., 1996), and to the sub-family of proteins (PAR) (Haas et al., 1995). In mammals, the PAR bZIP proteins include DBP (Mueller et al., 1990), TEF (Drolet et al., 1991), VBP (Iyer et al., 1991) and HLF (hepatic leukemia factor) (Hunger et al., 1992). It has been postulated that members of the CES-2/PAR family are evolutionarily conserved regulators of programmed cell death (Metzstein et al., 1996; Seidel and Look, 2001). In other respects, it is noteworthy that the PAR family genes present, like vri and the chicken and mouse homologs of NFIL3A/E4BP4, circadian oscillations (Wuarin and Schibler, 1990; Falvey et al., 1995; Fonjallaz et al., 1996; Blau and Young, 1999; Mitsui et al., 2001; Doi et al., 2001).

We show that vri partial loss of function induces flight and other locomotory defects associated with a downward bending wing phenotype and hair defects. Furthermore, vri interacts genetically with genes encoding actin-binding proteins: bent encoding a myosin light chain kinase (Ayme-Southgate et al., 1991; Daley et al., 1998), karst encoding a ßHeavy-spectrin (Thomas and Kiehart, 1994; Thomas et al., 1998; Zarnescu and Thomas, 1999), and actinin encoding an actin crosslinking spectrin superfamily member (Fyrberg et al., 1990; Fyrberg et al., 1998; Roulier et al., 1992; Dubreuil and Wang, 2000). Clonal analysis shows that vri is cell autonomous and that the absence of vri reduces cell and hair size in wing and to smaller eyes with a reduced number of ommatidia of abnormal morphology with shorter photoreceptor cell stalks. The overexpression of vri reduces salivary glands growth and nuclei size, a phenotype typical of an inhibition of endoreplication. Overexpression of vri in the embryo and in imaginal discs has an anti-proliferative effect. In the wing disc, increased apoptosis is observed and at the wing surface cells with multiple trichomes are formed, indicative of cell cycle arrest and defects in regulation of actin cytoskeleton. We propose that vri may control cell growth and proliferation via the regulation of the actin cytoskeleton.

	MATERIALS AND METHODS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fly stocks
Flies were raised on standard media and all crosses were performed at 25°C unless stated otherwise. vri alleles are described by George and Terracol (George and Terracol, 1997) and new 25D P insertions lines at the Berkeley Drosophila Genome Project (BDGP: http://flypush.imgen.bcm.tcm.edu/pscreen). The following stocks were used: a, Actn, bt and kst alleles (FlyBase, 2003); w¹¹¹⁸, FLP/FRT (Xu and Rubin, 1993); EGUF/hid (Stowers and Schwartz, 1999); hs FLP; Sp/SM6-TM6B (Tb), GFP FRT40A, Act5C>CD2>GAL4, UASGFP [III] (Neufeld et al., 1998a); pnr-GAL4[III] (Calleja et al., 1996); vg-GAL4[II] (Simmonds et al., 1995); ey-GAL4[III] (Hazelett et al., 1998); en-GAL4[II] and hs-GAL4[III] (Brand and Perrimon, 1993); C765-GAL4[X] (Guillén et al., 1995); MS1096-GAL4[X] (Capdevila and Guerrero, 1994); A9-GAL4[X] (Haerry et al., 1998); F4-GAL4[II] (Weiss et al., 1998); UAS-rbf[III] (Du et al., 1996); rbf¹¹, rbf¹⁴ (Du and Dyson, 1999); UAS-dap[III], UAS-E2F-Dp[III] and UASCycE[III] (Lane et al., 1996); UAS-string[II] (Neufeld et al., 1998a); and UAS-dPTEN (Gao et al., 2000). Balancer chromosomes and other stocks are described elsewhere (Lindsley and Zimm, 1992; FlyBase, 2003).

DNA preparation, vri deficiency mapping
Standard molecular biology experiments were performed as described by Sambrook et al. (Sambrook et al., 1989). Genomic DNA preparations and PCR amplifications were performed as described by Szuplewski and Terracol (Szuplewski and Terracol, 2001). Positions are from cDNA1 (3.8) start in the genomic region: primer 1 (forward) 5'-CGATTGTCTGCACGCTGG-3' (322-339), primer 2 (reverse) 5'-GTTCCTTCTCCGGCGATC-3' (2570-2553), primer 3 (reverse) 5'-CAAGGCAAGGCTGGAGAG-3' (6083-6066) and primer 4 (reverse) 5'-AGTATCTGCAGCGCACGG-3' (7379-7352). P-lacW (Bier et al., 1989) primers: 5'P (reverse) 5'-CCTCTCAACAGCAAACGTGTACTG-3' (90-67), 3'P (forward) 5'-TCTCTTGCCGACGGGACC-3' (10653-10670), lacZ (forward) 5'-GATCATCTGGTCGCTGGG-3' (1935-1952). vri⁵ (Török et al., 1993) was mapped by plasmid rescue (George and Terracol, 1997) and upstream sequence from the 5'P primer is: (1218) 5'-GATTCTTGCATCATTCGGCG-3'. The insertion site is identical to that determined from the P-lacW 3' end (AG034151) by the BDGP (http://www.fruitfly.org/p_disrupt/) (Spradling et al., 1995; Spradling et al., 1999). PCR products were cloned into pGEM plasmid (Promega) and sequenced with the SP6 or T7 primers. PCR fragments and breakpoint sequences were: vri^5R7.2, 2.2 kb from primers lacZ-3; P-lacW/genomic, 5'-GCAGTGCACGGCAGATACAC/TTGCCGCTTCGGTCACCCGT-3'; vri^5R1.5, 3.3 kb from primers lacZ-4; P-lacW/genomic, 5'-CAACATCAAATTGTCTGCGG/CGATGATGGTGAAGTTAACG-3'; vri^5R5.24, cloned by plasmid rescue; and genomic/P-lacW, 5'-TGATTTAAGCAGAGTATTTC/GCTAAATACTGGCAGGCGTT-3'. DNA was sequenced by the dideoxy-chain termination method (Sanger et al., 1977) using the US Biochemicals sequencing kit (Pharmacia) or performed on a ABI Prism^TM 377 DNA sequencer (Applied Systems). DNA sequences were compiled using the Genetics Computer Group software (GCG) (Devereux et al., 1984) and compared with the Drosophila database using the BDGP (http://www.fruitfly.org/blast/) Blast Searches program (Altschul et al., 1990).

Clonal analysis
Mitotic clones were generated using the FLP/FRT technique (Golic and Lindquist, 1989; Xu and Rubin, 1993). vri alleles were recombined onto a P[ry⁺ hs-neo FRT]40A second chromosome. To generate somatic clones in adult y w/y w; vri P[ry⁺ hs-neo-FRT]40A/CyO females were crossed to y P[ry⁺ hs FLP]1/Y; P[y+] P[ry⁺ hs-neo FRT]40A/Bc males and yellow clones analyzed in y P[ry⁺ hs FLP]1/y w; P[y⁺] P[ry⁺ hs-neo FRT]40A/vri P[ry⁺ hs-neo FRT]40A female progeny. Clones were heat-shock induced at the third larval instar by 1 hour exposure at 38°C. To generate mitotic clones in the eye, we used the EGUF/hid method (Stowers and Schwarz, 1999). y w/y w; P[ry⁺ hs-neo FRT]40A GMR-hid l(2)CL-L1/CyO; ey-GAL4 UAS-FLP/ey-GAL4 UAS-FLP females were crossed to y w/Y; vri P[ry⁺ hs-neo FRT]40A/CyO males at 29°C. GAL4-expressing clones were induced by the FRT `flip-out' method (Struhl and Basler, 1993) by crossing P[ry⁺ hs FLP]/Y; UAS-vri/SM6-TM6B (Tb) males to Act5C>CD2>GAL4, UAS-GFP/Act5C>CD2>GAL4, UAS-GFP females. In both types of experiments, clones were heat-shock induced in the progeny 10-48 hours after egg deposition by 30 minute exposure at 37°C. Tb⁺ female larvae were dissected at mid third larval instar.

Scanning electron microscopy, retina sections, embryonic and wing phenotypic analyses
Flies were fixed in 3% glutaraldehyde, 0.1 M PBS (2 hours at room temperature and then 24 hours at 4°C), dehydrated in ethanol series and then in amyl acetate series. Electron microscopy was performed on a JEOL JSM 6100 scanning electron microscope. To observe adult retinas, flies were fixed in carnoy, embedded in paraffin wax and 7 µm sections were cut on a Leica RM 2145 microtome. Embryonic cuticles were prepared as described by Wieschaus and Nüsslein-Volhard (Wieschaus and Nüsslein-Volhard, 1986). Wings were dissected, collected in 70% ethanol and mounted in Euparal (Labosi).

Histology
Larval tissues were dissected in 1xPBS, fixed in PBT [1xPBS, 0.1% Tween 20 (Sigma) plus 4% paraformaldehyde] for 25 minutes, washed three times for 5 minutes in PBT, stained for 10 minutes in PBT, 0.1 µg/ml DAPI (Sigma), washed overnight in PBT, incubated 24 hours in 80% glycerol and mounted in Citifluor (Kent Scientific Industry Project). Acridine Orange (Sigma) staining was performed as described in Gaumer et al. (Gaumer et al., 2000). Preparations were observed on a Leica DMR fluorescence microscope using a Micro Max (Princeton Instrument) camera and collected using the Metaview Imaging System software (Universal Imaging Corporation).

UAS constructs and transformation experiments
vri cDNAs are described by George and Terracol (George and Terracol, 1997). The 3.8 kb XbaI fragment from pBScDNA3.8 was cloned into the XbaI site of the pUAST transformation plasmid (Brand and Perrimon, 1993) and the 3.3 kb SalI-XbaI fragment from pBScDNA3.3 was cloned into the XhoI and XbaI sites of pUAST. pUAST constructs were co-injected with the pUCh2-3 helper plasmid in the pole cell region of w¹¹¹⁸ preblastoderm embryos (Spradling and Rubin, 1982). 20 UAS-vri1 (3.8 kb) and 27 UAS-vri2 (3.3 kb) independent transformed lines were recovered. The following lines were used in this study: UAS-vri1⁶[III], UAS-vri1⁹[III], UAS-vri1¹³ [III], UAS-vri1¹⁴[X] and UAS-vri2⁸[II].

	RESULTS

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Characterization of vri 5' region and new alleles
The 5' extension of vri was characterized by the recovery of new cDNAs and P-insertion alleles. From the nine cDNAs vri identified by the BDGP (http://www.fruitfly.org/EST/) (Rubin et al., 2000), six isolated from embryos (0-24h) had the same 5' end as cDNA1 (3.8 kb) (Fig. 1). One cDNA from adult heads starts at +15 bp and two, from embryos, start at -6.061 kb from cDNA1 start. The complete sequence provided for one of them, RE29005, shows that the cDNA is 3,073 kb long with a 7814 bp first intron and is 380 bp shorter at the 3' end (Fig. 1). This cDNA encodes the same 610 amino acid putative protein as cDNA2 (3.3 kb). Four new lethal and one viable P-insertion lines were mapped (BDGP, http://flypush.imgen.bcm.tcm.edu/ pscreen) in the vri region (Fig. 1) that we identified as new vri alleles. BG01569-3 (vri⁹) maps at -75 bp, upstream of vri⁴, vri⁶ (-66) and vri⁸ (-57), and KG03762 (vri¹⁰) maps in intron 2 near vri⁵ and vri⁷. They do not complement vri alleles. KG01220 (vri¹¹) maps at -16.7 kb and KG10174 (vri¹³) at -8.6 kb. They complement partially vri alleles. EY01490 (vri¹²) is a PUAS viable line and maps at -12.6 kb.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Mapping of vri 5' end and generation of deletion alleles by P-element mutagenesis. The position of the vri alleles and the extent of the deletions generated are shown. The localization of the vri cDNAs, the other described loci, the putative GadFly genes and the BDGP cDNAs are presented. The positions of the genomic (1, 2, 3, 4) and PlacW primers used for PCR amplification are shown (arrows). vri1 (cDNA3.8) and vri2 (cDNA3.3) are the two types of cDNAs used for UAS constructs. DS00714 distal end of a P1 phage isolated by the BDGP, DM194B8, T7 end and DM77B6, SP6 end of two cosmids isolated by the European Drosophila Mapping Consortium.

vri affects wing shape and size, hair morphology and flight and interacts with genes encoding actin-binding proteins
vri¹¹ and vri¹³ over vri lethal alleles and Df(2L)tkv^Sz2 (25D2-4; 25D6-E1) lead to flies with pleiotropic phenotypes (100% penetrance). The wings are shorter and downward (or more rarely upward) bending, and posterior scutellar macrochaete are shorter and upturned (Fig. 2A,B). They are poorly viable and present locomotory and flight defects. vri¹¹/vri¹³ flies present the same phenotypes. Wings are notably smaller and regions of atrophic or missing hairs are present at the margin and on the surface of the wing (Fig. 2C,D). The downward bending phenotype has been described for other mutants. This is the case for mutants in the arc gene, which encodes an adherens junction-associated PDZ domain protein (Liu and Lengyel, 2000) and in the karst gene encoding ß-heavy-spectrin involved in cell integrity, polarity and adhesion (Thomas and Kiehart, 1994; Thomas et al., 1998; Zarnescu and Thomas, 1999). This phenotype is also observed for mutations in bent encoding a myosin light chain kinase expressed in indirect flight muscle and tubular muscle (Ayme-Southgate et al., 1991; Daley et al., 1998). Viable mutants in bent are flightless, this is also the case for mutants in muscle actin, and in actin-binding proteins such as flightless I encoding a gelsolin family homolog (Davy et al., 2001) and actinin encoding an actin crosslinking protein of the spectrin superfamily (Fyrberg et al., 1990; Fyrberg et al., 1998; Roulier et al., 1992; Dubreuil and Wang, 2000). We have tested for interactions in double heterozygotes between vri and arc, karst, bent and actn alleles. No significant interaction was observed with arc, but only the hypomorphic a¹ allele was tested. With the three other genes, reduced viability and wing hair phenotypes similar to those of vri are observed (Fig. 2E,F). With bent (b¹) and Actn⁸ (null), Actn¹⁴ and Actn^G0077, flies also present progressive locomotory defects affecting rear legs.

View larger version (93K): [in this window] [in a new window]   Fig. 2. vri affects wing shape and hair morphology and interacts with actin-binding protein encoding genes. (A) Wild-type adult female fly. (B) vri11/vri5 adult female fly with shorter downward bending wings and shorter and upturned posterior scutellar bristles (arrow). (C) Wild-type wing. (D) vri11/vri5R5.24 wing, smaller than wild type with notching at the anterior margin (right-hand arrow) and multiple regions with atrophic or missing hairs appearing as non pigmented zones on the wing surface (arrows). (E) vri5R7.2/kst01318 wing showing missing hairs at the anterior margin (top arrow) and notching at the posterior margin (bottom arrow). (F) vri5R7.2/ActnG0077 wing with missing hairs at the anterior margin (top arrow) and on the wing surface (bottom arrow).

View larger version (67K): [in this window] [in a new window]   Fig. 3. Mutations in vri decrease hair and cell size in adult cuticle. y/y; vri/vri somatic clones were induced at third larval instar by the FLP/FRT technique and observed in females. (A) y/y; vri2/vri2 clonal left posterior scutellar macrochaete (arrow) with a reduced size compared with the y+/y; vri+/vri right one. (B) vri1 clone showing a socket with no macrochaete on the head (arrow). (C) vri5 clones with pigmented cuticle. (D) y/y; vri5/vri5 clones at the wing anterior margin in the triple row region with smaller and thinner stout mechanosensory bristles. The space between bristles is reduced (vertical arrows), which is characteristic of smaller cells, and the margin is concave (double-headed arrow). (E) At the anterior wing margin, the average distance between stout bristles of the triple row is significantly reduced in y/y; vri/vri clones (Student's test: P<0.001) compared with y/y; vri+/vri+ clones. The values are percentages of wild type±s.e. Clones, analyzed from 12 to 31 wings, contained from two to 33 bristles.

View larger version (104K): [in this window] [in a new window]   Fig. 4. vri loss of function allele reduces eye size, alters morphology and size of ommatidia and photoreceptors. Scanning electron micrographs of vri clonal eye were generated by the EGUF/hid method. Photoreceptor cells bearing the dominant photoreceptor cell lethal transgene GMR-hid die during metamorphosis. The l(2)CL-L1 recessive cell lethal mutation leads to lethality at an earlier stage of eye development. The crosses were performed at 29°C and the eyes were observed in males. (A,C,F) vri+ FRT40A/vri+ FRT40A control eyes from [ry+ hs-neo FRT]40A/[ry hs-neo FRT]40A GMR-hid l(2)CL-L1; ey-GAL4 UAS-FLP/+ flies. (B,D,G) vri5R7.2/vri5R7.2 clonal eyes from vri5R7.2[ry+ hs-neo FRT]40A/[ry hs-neo FRT]40A GMR-hid l(2)CL-L1; ey-GAL4 UAS-FLP/+ flies. (B) Clonal eye smaller than control (A) and with a rough aspect. (C,D) The ommatidia are disorganized and present an abnormal morphology. They are spherical rather than hexagonal and bristles are missing or atrophic. (E) The number of ommatidia is reduced in vri clonal eyes. Values are mean numbers of ommatidia (±s.e.) observed in male eyes. The mean number of ommatidia is reduced by 10% in vri5, 17% in vri1 and 19% in vri5R7.2 eyes compared with the vri+ control eyes (Student's test: P<0.001). (F,G) Longitudinal sections through adult retinas. (G) Clonal retina with photoreceptor stalks twisted and shorter than control (F).

Overexpression of vri using the driver pannier, the expression of which is restricted to dorsal tissues throughout development (Calleja et al., 1996; Heitzler et al., 1996), leads to embryonic lethality due to defects in the dorsal epidermis and dorsal closure (Fig. 5B). Use of the vg-GAL4 driver (Simmonds et al., 1995), which directs GAL4 expression in the wing pouch, leads to notching at the wing margin (Fig. 5E). Overexpressing vri with the leaky hsp70-GAL4 (Brand and Perrimon, 1993) driver results in atrophic or missing bristles on the adult cuticle (Fig. 5H). Similar phenotypes are observed by overexpressing with the same drivers the inhibitor of proliferation Rbf (Datar et al., 2000) (Fig. 5C,F,I) or Rho1, a small GTPase involved in actin cytoskeleton regulation (Hariharan et al., 1995). Overexpression of vri driven with ey-GAL4 (Hazelett et al., 1998), the expression of which is specific to the eye disc and starts in the embryonic eye disc primordia, results in atrophy of the eye (Fig. 6). We observed similar phenotypes (not shown) by overexpressing Rbf (Datar et al., 2000) or Rho1 (Hariharan et al., 1995). According to the strength of the UAS-vri transgene and the temperature, a progressive reduction in the size of the eye and a rough aspect are observed (Fig. 6A-D). The ommatidia are disorganized and bristles are either duplicated or missing. In some flies, the eyes are totally absent (Fig. 6E) and dissection of third instar larvae revealed no eye disc and atrophic brain (not shown). The stronger phenotype observed at 29°C leads to an absence of head (Fig. 6F). These phenotypes are compatible with an inhibition of proliferation.

View larger version (107K): [in this window] [in a new window]   Fig. 5. vri overexpression induces atrophy of embryonic epidermis, adult wing and thoracic hairs. (A) Wild-type embryo. (B) UAS-vri16/pnr-GAL4 dead embryo showing atrophic dorsal epidermis and anterior hole and head defects. (C) pnr-GAL4/UAS-rbf dead embryo with a similar phenotype. (D) Wild-type wing. (E) vg-GAL4/+; UAS-vri113/+ wing at 29°C showing notching at the margin. (F) vg-GAL4/+; UAS-rbf/+ wing at 25°C with notching of the posterior margin. (G) Scanning electron micrographs of wild-type thorax. (H) hsp70-GAL4/UAS-vri19 thorax at 29°C showing atrophic macrochaete. (I) hsp70-GAL4/UAS-rbf thorax at 29°C with a similar phenotype.

View larger version (114K): [in this window] [in a new window]   Fig. 6. vri overexpression induces atrophy of the eye. Scanning electron micrographs of ey-GAL4/UAS-vri19 eye series with progressive reduction of the eye at 25°C (A,E), and extreme phenotype with UAS-vri114/+; ey-GAL4/UAS-vri113 at 29°C (F). (A) Rough eye with a moderate reduction in size. (B,C) Ommatidia are disorganized with a globular aspect, bristles are duplicated or missing and regions of apparent irregular growth are observed (B). (D) Very reduced eye. (E) Eye totally absent. (F) At 29°C the UAS-vri114/+; ey-GAL4/UAS-vri113 flies lack all head structures derived from eye-antennal disc. The proboscis, which is derived from the labial disc, is present.

View larger version (93K): [in this window] [in a new window]   Fig. 7. vri overexpression alters trichome number, size and morphology in wing. All wings are from females. (A) A9-GAL4/+ control wing. (B) Magnified view of A9-GAL4/+ control wing around L4 vein. (C) A9-GAL4/UAS-vri114; UAS-vri113/+ wing with a slightly reduced size, enlarged cells and reduced L5 vein. Long ectopic bristles are observed (magnification in the top right-hand corner). (D) Magnified A9-GAL4/UAS-vri114; UAS-vri113/+ wing surface, around L4 vein. Larger cells are observed with multiple trichomes. (E) MS1096-GAL4/+ control wing. (F) Magnified view of MS1096-GAL4/+ control wing around L3 vein. (G) MS1096-GAL4/+; UAS-vri28/+ wing. (H) Magnified MS1096-GAL4/+; UAS-vri28/+ wing showing enlarged L3 vein, enlarged cell size with multiple trichomes, some of them with an abnormal morphology. (I) MS1096-GAL4/+; UAS-vri28/UAS-vri28 wing with a greatly reduced size and an abnormal morphology. (J) Magnified MS1096-GAL4/+; UAS-vri28/UAS-vri28 vein around L3 vein showing multiple trichomes with a reduced size and an abnormal differentiation.

vri overexpression induces apoptosis and reduces cell size and endoreplication
To observe the effect of vri overexpression at earlier stages, larval tissues were studied. With the strong UAS-vri transgenes such as UAS-vri1⁹, their combination with MS1096-GAL4 is pupal lethal and leads to increased apoptosis in the undifferentiated proliferating imaginal wing disc (Fig. 8A,B). Overexpression of vri was driven in the salivary gland using the F4-GAL4 transgene whose expression starts in late stage 13 of embryogenesis, once cell proliferation in the salivary primordium is complete, between the first and second rounds of endoreplication. The driver stays active throughout larval stages. F4-GAL4/+; UAS-vri1⁹/+ glands grow to about one half the size of the control gland (Fig. 8C,D). Nuclei are smaller indicating an inhibition of endoreplication and often present a degenerative aspect characterized by a condensation of the chromatin (pyknosis), suggestive of high levels of apoptosis. Overexpression in the fat body, another polytenic tissue, using the `flip out' technique, results in small clones with smaller cells, indicating an inhibition of endoreplication (Fig. 8E,F).

View larger version (85K): [in this window] [in a new window]   Fig. 8. vri overexpression induces apoptosis and inhibits cell growth in larval tissues. Tissues were dissected at third larval instar. (A,B) Wing imaginal discs stained with Acridine Orange. (A) MS1096-GAL4/+ control disc with low level of apoptosis. (B) MS1096-GAL4/+; MS1096-GAL4/+; UAS-vri19/+ with increased number of apoptotic cells (arrow) in the wing pouch region. (C,D) DAPI staining of male salivary gland. (C) F4-GAL4/+ control gland. (D) F4-GAL4/+; UAS-vri19/+ gland (same magnification as control) with a size reduced by about one half and with pyknotic nuclei (arrows). (E,F) Fat body clones co-expressing vri and GFP induced by the `flip out' technique in hsp FLP; UAS-vri19/Act5C<CD2<GAL4, UAS-GFP females. (E) GFP detection (green) with vri overexpressing clones of greater green intensity. (F) GFP and DAPI (red) stainings showing clones (yellow) with smaller cells.

	DISCUSSION

TOP SUMMARY INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
vri is required for normal hair and cell growth
The functional analysis of vri performed by mutant clone induction observed in adult indicates that vri is cell autonomous and involved in hair and cell growth. The fact that vri acts in a strict cell-autonomous manner suggests that it does not regulate the expression of a diffusive molecule such as a growth factor or a hormone. Smaller cells are recovered on the whole cuticle with shorter thinner or atrophic bristles. In the wing, clones have an abnormal shape and degenerative tissues are observed. These defects could be due to cytoskeleton defects. Clones induced in the eye precursor cells result in smaller eyes with a significantly reduced number of ommatidia with an atrophic morphology and a reduced size with the stronger allele. The photoreceptor cell stalks are shorter and atrophic. These results suggest that vri cells grow more slowly and are less viable than vri⁺ cells, even when they are not surrounded by wild-type cells

Smaller cells and bristles can result from metabolic defects, for example, mutations in tRNAs, ribosomal proteins (Minute) or rRNAs. This is the case for mutations in the S6 kinase gene, a regulator of ribosomal protein production (Montagne et al., 1999) and in Drosophila myc, diminutive, which is involved in growth and metabolism (Johnston et al., 1999). They are also found with genes regulating the cell cycle, such as rbf (Datar et al., 2000), and with genes of the Ras/MAPK and Insulin/PI3K pathways controlling growth (Stocker and Hafen, 2000; Oldham et al., 2000; Prober and Edgar, 2001). Hypomorphic mutations in the rbf gene, which encodes a homologue of the retinoblastoma protein, Rb, an important regulator of cell proliferation and differentiation leads to atrophic bristles and rough eyes (Du, 2000). Null-clone cells in wing discs are smaller than wild-type cells and exhibit many pyknotic nuclei, suggesting elevated levels of apoptosis. rbf-null clones induced in the eye exhibit slight to moderate hypoplasia, missing or duplicated bristles and fused ommatidia. However, RBF inhibits cell cycle progression, rather than cellular growth directly (Datar et al., 2000). Cells homozygous for partial loss-of-function mutations in components of the Ras/MAPK pathway such as Ras1 grow slowly and remain small. Ras1 controls growth, survival and differentiation in the eye (Wassarman and Therrien, 1997; Halfar et al., 2001) and coordinates cellular growth and cell cycle progression in the wing (Diaz-Benjumea and Hafen, 1994; Karim and Rubin, 1998; Prober and Edgar, 2000). Cells devoid of the lipid kinase PI3K (Leevers et al., 1996; Weinkove et al., 1999) have a reduced growth and size, while loss of its antagonist dPTEN results in bigger cells than wild type (Goberdhan et al., 1999; Huang et al., 1999; Gao et al., 2000).

Smaller cells and bristles can also result from cytoskeletal defects. For example, the miniature and dusky genes encoding ZP proteins required for cytoskeletal reorganization are involved in growth and morphogenesis of cells and hairs in the wing (Roch et al., 2003). Mutant wings are smaller, phenotype also observed in vri viable combinations. Drosophila bristles are single cells with very long extensions supported by actin bundles of crosslinked actin filaments (Tilney et al., 2000a; Tilney et al., 2000b). The assembly of actin filaments is under the control of the Rho GTPases, including Rho1, Drac1 and DCdc42. Rho GTPases act as molecular switches involved in many processes such as morphogenesis, chemotaxis, axonal guidance and cell cycle progression (Hall, 1998; Van Aelst and Symons, 2002). They regulate actin cytoskeleton and are involved in planar polarization, hair morphology and outgrowth processes (Eaton et al., 1995; Eaton et al., 1996; Guichard et al., 1997; Hall, 1998; Adler et al., 2000), and photoreceptor morphogenesis. The dominant-negative form of Drac1, N17Drac1, leads to reduced and disordered rhabdomeres (Chang and Ready, 2000; Colley, 2000), while overexpression of Rho1 induces rough eyes and causes atrophy of the retina (Hariharan et al., 1995). Regulation of actin cytoskeleton is coupled with other pathways, including MAPK and PI3K pathways (Hall, 1998). For example Drac1 acts upstream of JNK cascade and DCdc42 downstream of Dpp pathway in dorsal and thorax closure processes (Ricos et al., 1999). lilli acts in cytoskeleton regulation, control of cell identity and cell growth, in parallel with the Ras/MAPK and PI3K/PKB pathways. It is noteworthy that retinal cells and wing margin bristles lacking lilly are significantly smaller than wild type (Wittwer et al., 2001; Tang et al., 2001). The flightless I-mediated cytoskeletal regulation involves PI3K, and the small GTPases Ras, RhoA and Cdc42 (Davy et al., 2001). Viable mutations in flightless I cause also ultrastructural defects in the indirect flight muscles.

vri overexpression inhibits proliferation and alters cytoskeleton regulation and endoreplication
Overexpression of vri in the embryo and imaginal discs induces an atrophy of the resulting tissues. With strong transgenes, the discs do not proliferate and the adult structures are totally absent. Similar phenotypes are observed when the inhibitor of proliferation Rbf or the regulator of actin cytoskeleton Rho1 are overexpressed. In wing discs, strong transgenes lead to lethality and increased apoptosis is observed. On the wing surface larger cells with multiple trichomes are observed. With strong viable transgenes, the wing is reduced and trichomes are disoriented with abnormal morphology. This phenotype has been attributed to cell cycle arrest in G1 phase. Inactivation of Cdc2 kinase which regulates entry into mitosis or of the myb gene required for G2/M transition and maintenance of diploidy leads to a similar phenotype (Weigmann et al., 1997; Katzen et al., 1998). This phenotype is observed when the human cyclin-dependant kinase inhibitor P21CIP/WAF1 is overexpressed with UASP21 driven by the dpp-GAL4 transgene (Karim and Rubin, 1998). We observed the same phenotype when overexpressing dacapo, the Drosophila P21 homolog (De Nooij et al., 1996), under the control of MS1096-GAL4. DAP binds to Cyclin E/Cdk2 complexes inducing cell cycle arrest in G1 phase and epidermal cell proliferation inhibition (Lane et al., 1996). The same phenotype is also observed when disrupting cytoskeleton regulation by overexpressing the activated form of Drac1, DRac1N17 (Eaton et al., 1995) or a dominant-negative form of DRacGAP, DRacGAP^EIE. DRacGAP is a negative regulator of the Rho-family GTPases, Drac1 and DCdc42, regulating actin cytoskeleton via EGF/Ras signaling pathway in the developing wing. The P21 overexpression phenotype is suppressed by UAS-Ras1^V12, an activated form of Ras1, or UAS-DRacGAP (Sotillos and Campuzano, 2000).

In salivary glands, vri overexpression leads to glands whose size is reduced by about one half with numerous pyknotic nuclei, suggesting inhibition of endoreplication and elevated levels of apoptosis. A reduction in the size of the gland and an inhibition of the level of endoreplication is observed when the genes encoding cell cycle regulators cyclin E, dacapo (Follette et al., 1998; Weiss et al., 1998) and rbf (Datar et al., 2000) are overexpressed. Pulses of Cyclin E are required to drive endocycle S phase, and continuous expression inhibits endoreplication cycles. In the fat body (polytenic tissue), vri-overexpressing clones are small with smaller cells often presenting a degenerative aspect.

Possible level of vri function
vri was previously identified as an enhancer of dpp phenotypes both in embryo and adult (George and Terracol, 1997). dpp encodes a TGFß homolog closely related to BMP4 (bone morphogenetic protein 4), which acts as a morphogen at different stages of development (reviewed by Podos and Ferguson, 1999). Dpp plays a proliferative role in all imaginal discs at larval stages but induces a cell cycle arrest in G1 phase in the eye-antennal disc during third larval instar (Horsfield et al., 1998). Dpp might promote cell growth and/or proliferation directly, in a cell-autonomous manner (Burke and Basler, 1996; Martin-Castellanos and Edgar, 2002). Other genes have been identified or described as enhancers of dpp phenotypes. Some of them are integral members of the dpp pathway, which is the case for tkv (Terracol and Lengyel, 1994), Mad and Med (Raftery et al., 1995), but others [such as lilliputian (Su et al., 2001) and cyclope (Szuplewski and Terracol, 2001)] have been shown to act in different pathways. Interestingly, lilli has been also identified in other screens as a dominant suppressor of activated MAP kinase pathway phenotypes (Dickson et al., 1996; Neufeld et al., 1998b; Rebay et al., 2000). lilli acts in parallel with the Ras/MAPK and PI3K/PKB pathways in the control of cell identity, cell growth and/or cytoskeletal arrangement (Wittwer et al., 2001; Tang et al., 2001). vri mutations do not alter dpp pathway target gene expression and probably act in a parallel pathway.

vri overexpression phenotypes suggest a role in cell cycle and proliferation. However, these phenotypes are not rescued by simultaneous overexpression of the genes encoding activators of proliferation, Drosophila E2F, cyclin E or string. Therefore, it is unlikely that Vri is either a direct repressor of genes that activate proliferation or an activator of those acting as inhibitors of proliferation like rbf or dacapo. It could act upstream in the Ras/MAPK or PI3K pathways regulating growth and involved in the regulation of the mammalian homolog of Vri, NFIL3A, acting mostly as a repressor. Genetic interactions have been tested in double-heterozygotes with available members of these pathways and vri, but no reduction in viability nor any strong phenotypes was recovered. This could result from genes with non-limiting products and/or be due to the functional redundancy of vri. Alternatively, vri may control cell size independently of growth signals.

vri loss-of-function and overexpression phenotypes, more probably, could result from primary defects in cytoskeletal actin network. Although cytoskeletal integrity and adhesion are altered in mutants of regulators of cell growth and proliferation, these effects are indirect. Wing hair atrophy phenotypes were observed in interaction with the karst gene involved in cytoskeleton arrangement. The downward-bending wing and the reduction of the photoreceptor stalk size (Pellikka et al., 2002) are two other phenotypes observed in vri and kst. We observed new vri phenotypes affecting wing shape flight and locomotion. Locomotory defects could result from neurological or muscular alteration (or both). We found interaction with the actn and bent genes involved in muscle actin function, which suggests that the effect is rather at the muscular level, although we have not observed gross defect in indirect flight muscle. However these defects appear degenerative and must be studied in more detail. We also observe hair atrophic phenotypes in interaction with these two genes, suggesting an effect in other cell types. Although the locomotory and hair defects are not necessarily related, it is notable that the genes interacting with vri affect different types of actin, muscle and non-muscle actin. It will be interesting to search for the direct targets of Vri to understand its implication in locomotion and cytoskeletal integrity.

	ACKNOWLEDGMENTS

 
We thank A. Kropfinger for helpful comments on the manuscript, D. Montero for performing scanning electron microscopy, C. Dubucs for performing most of the sequences, and M. Bienz, C. Lehner, P. Leopold, M. B. O'Connor, J. Silber and the Bloomington Stock Center for providing Drosophila stocks. This work was supported by the Centre National de la Recherche Scientifique, by a grant from the Association pour la Recherche sur le Cancer (ARC) N°9894 to R.T., and by grants from the Ministère de l'Enseignement, de la Recherche et de la Technologie (MERT), the ARC and the Fondation pour la Recherche Médicale to S.S.

	Footnotes

 
^* Present address: European Molecular Biology Laboratory, Meyerhofstr.1, 69117, Heidelberg, Germany

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