The Cid Mutation Encodes a Chimeric Protein Whose Activity Is Regulated

Developmental Biology 208, 147–156 (1999) Article ID dbio.1998.9185, available online at http://www.idealibrary.com on

View metadata, citation and similar papers at core.ac.uk brought to you by CORE D The ci Mutation Encodes a Chimeric Protein provided by Elsevier - Publisher Connector Whose Activity Is Regulated by Wingless Signaling

Tonia Von Ohlen,*,1 and Joan E. Hooper*,†,2 *Program in Cell and Developmental Biology and †Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

The Drosophila cubitus interruptus (ci) gene encodes a sequence-speciﬁc DNA-binding protein that regulates transcription of Hedgehog (Hh) target genes. Activity of the Ci protein is posttranslationally regulated by Hh signaling. In animals homozygous for the ciD mutation, however, transcription of Hh target genes is regulated by Wingless (Wg) signaling rather than by Hh signaling. We show that ciD encodes a chimeric protein composed of the regulatory domain of dTCF/Pangolin (Pan) and the DNA binding domain of Ci. Pan is a Wg-regulated transcription factor that is activated by binding of Armadillo (Arm) to its regulatory domain. Arm is thought to activate Pan by contributing a transactivation domain. We ﬁnd that a constitutively active form of Arm potentiates activity of a CiD transgene and coimmunoprecipitates with CiD protein. The Wg-responsive activity of CiD could be explained by recruitment of the Arm transactivation function to the promoters of Hh-target genes. We suggest that wild-type Ci also recruits a protein with a transactivation domain as part of its normal mechanism of activation. © 1999 Academic Press Key Words: cubitus interruptus; Hedgehog; Wingless; Drosophila.

INTRODUCTION adjacent anterior compartment cells to activate transcription of its target genes. wg and gooseberry (gsb) are acti- Developmental patterning involves a variety of key sig- vated anterior to Hh at the parasegment border (Baker, naling molecules such as the Drosophila segmentation 1987; Hidalgo and Ingham, 1990), while patched (ptc)is genes wingless (wg)3 and hedgehog (hh). Both hh and wg activated, at both the parasegment and segment borders encode secreted proteins that provide positional informa- (Hooper and Scott, 1989; Nakano et al., 1989). Ci is a tion along the anterior/posterior (A/P) axis within each transcriptional mediator of Hh signaling (Alexandre et al., segment of the Drosophila embryo. Each protein activates a 1996; Von Ohlen et al., 1997; Von Ohlen and Hooper, 1997; signal transduction pathway that affects transcription of reviewed in Ruiz i Altaba, 1997). Ci is a member of the Gli target genes in cells receiving the signal. During early germ family of sequence specific DNA binding proteins (Ruppert band extension, wg and hh are expressed in adjacent cells et al., 1988). It is expressed throughout the anterior com- flanking the parasegment border and act in a positive partment in a pattern complementary to that of hh (Eaton feedback loop to maintain each other’s expression. The net and Kornberg, 1990; Slusarski et al., 1995). In the absence of effect is to stabilize discrete sources of these organizing Hh signaling, Ci is proteolytically processed to a Ci75 form signals which then pattern the rest of the segment during which blocks transcription of Ci-regulated genes including later development. hh, decapentaplegic (dpp), and ptc (Aza-Blanc et al., 1997). Hh is made in the posterior compartment and acts on In the presence of Hh signaling, proteolysis is blocked (Aza-Blanc et al., 1997) and transcription is activated by an 1 Present address: Institute of Neurosciences, University of Or- unprocessed form of Ci which remains to be identified egon, Eugene, OR 97403. 2 (Alexandre et al., 1996, Domıńguez et al., 1996). Thus, the To whom correspondence and reprint requests should be ad- choice between Ci-mediated transcriptional activation and dressed at Department of Cellular and Structural Biology, UCHSC Box B111, 4200 E. 9th Ave., Denver, CO 80262. Fax: (303) 315-4729. repression is regulated by Hh signaling. D E-mail: [email protected]. The ci mutation produces an abnormally large Ci pro- 3 Italics refer to the gene or its transcript. The protein product is tein that is expressed at low levels relative to wild type capitalized and not italicized. (Orenic et al., 1990; Slusarski et al., 1995). Unlike wild-type

0012-1606/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 147 148 Von Ohlen and Hooper ci whose expression is restricted to the anterior compart- These values closely approximate those for the predicted 3:3:1:1 ment (Eaton and Kornberg, 1990; Orenic et al., 1990), ciD ratio. Hs-wg; ciD/ϩ larvae were generated by mating Hs-wg/TM3, D mRNA is expressed in both anterior and posterior compart- Sb to ci /M62f. Third instar larvae from this cross were heat ments (Slusarski et al., 1995). The activity of the ciD shocked for 30 min at 37°C, allowed to recover at 25°C for 1 h, then mutation is very different from ci loss of function muta- dissected and processed for in situ hybridizations according to Ϫ previously described methods (Tautz and Pfeiffle, 1989; Jiang et al., tions (Orenic et al., 1987). While ci embryos lose expres- 1991). UASciD⌬ transgenic flies were made according to standard sion of Hh target genes, ciD homozygous embryos express procedures (Spradling and Rubin, 1982). Two independent lines wg and gsb in broad irregular stripes that extend anterior to were tested and produced identical phenotypes. their normal domains (Hidalgo and Ingham, 1990). ptc Molecular biology. Total RNA was isolated from 0–6 h em- expression also expands, filling most of the anterior com- bryos collected from ciD/M62f adults in TRIZOL according to the D partment (Hidalgo and Ingham, 1990). In hh; ci double manufacturer’s specifications (Gibco BRL). First strand synthesis mutant embryos the ectopic expression of hh target genes was performed with Superscript II Reverse Transcriptase (Gibco persists, just as in ciD embryos (Von Ohlen et al., 1997). BRL) with a reverse oriented primer corresponding to bp 556–570 of Therefore, ciD does not require hh signaling to activate ci cDNA, RT-1 (5Ј-GAGCTTGAAACGTCA-3Ј). Second strand expression of target genes. synthesis was performed as in Gubler and Hoffman (1983). The In addition to the aberrant function of ci, the ciD mutants double-stranded product was blunt ended, phosphorylated, and are null for pangolin (pan) (Brunner et al., 1997; van de ligated to double-stranded linker adapter as in Orlicky and Nordeen (1996). This product was then PCR amplified with a linker-specific Wetering et al., 1997). pan, also known as dTCF is a primer and a nested ci-specific primer (RT-2; bp 530–548; 5Ј- transcriptional effector of the Wg signaling pathway and ATGTTGGTTTATGCATGG-3Ј). PCR conditions were: 3 mM lies adjacent to ci on the fourth chromosome. Zygotic loss MgCl2 25 cycles at 94°C for 30 s, 48°C for 30 s, 72°C for 2:30 min. of pan function contributes little to the embryonic pheno- PCR products were ligated into pCRtmII (Invitrogen). Six hundred D type of ci because it is masked by maternally contributed and fifty colonies were screened with a third Ci-specific primer, pan message. Current models suggest that Pan is activated RT-3 (5Ј-GATCCTGGTAAAACAGTA-3Ј). DNA from 150 posi- by binding of Armadillo (Arm) to its amino terminal do- tive colonies was digested with EcoRI. Twelve of these with main. The resulting protein complex is transcriptionally different restriction patterns from each other were sequenced using active with Arm providing a transactivation domain and Sequenase kit (U.S. Biochemical). One of these contained an open Pan providing a DNA binding domain (reviewed in Nusse, reading frame fused in frame to Ci. A probe specific for this novel 1997). sequence identified seven clones from 450 colonies. The new sequence was derived from near the 5Ј end of pangolin/dTCF (pan) In an effort to understand the hh-independence of ciD D (Brunner et al., 1997; van de Wetering et al., 1997). To obtain the activity, we have extended our studies of ci . Unexpectedly, D D entire coding sequence of ci , we performed an additional RT-PCR we find that ci is not constitutively active but instead is reaction using a pan-specific primer corresponding to bp 204–221 regulated by Wg signaling. We have cloned the cDNA of pan cDNA (5Ј-AGGTAAATACGACCTAAC-3Ј) and RT-3. This D associated with the ci mutation and found that the result- product was then ligated into pCRtmII (InVitrogen) and sequenced. ing protein product constitutes a Pan/Ci fusion. The CiD A full-length ciD cDNA was reconstructed by cloning the fusion protein includes the Arm binding domain of Pan, and C-terminal of ci cDNA1 (Dr. R. Holmgren, Northwestern Univer- coimmunoprecipitates with activated Arm protein. Thus, sity), which encodes a protein truncated 4 amino acids after N703 Wg signaling appears to activate CiD by contribution of a (Aza-Blanc et al., 1997) into the PstI and NotI sites of Bluescript ϩ transactivation domain. We speculate that recruitment of a KS (Stratagene). The N-terminal of CiD was then added as a PstI D transactivation domain may also be key to activation of fragment from pCRtmII. ci was then subcloned as Bsp120I/NotI fragment into the NotI site of pUAST (Brand and Perrimon, 1993) wild-type Ci by Hh. and the BamHI site of pRm-1 (Bunch et al., 1988). ArmS10 cDNA (Dr. M. Peifer, UNC) was recloned into the BamHI site of pRm-1 (Bunch et al., 1988). MATERIALS AND METHODS Immunoprecipitations. S2 cells were stably cotransformed with Ci75 and ArmS10 or with CiD⌬ and ArmS10. Each of the cDNAs Drosophila stocks, crosses, and analysis. Hs-wg flies were was under control of the metallothionein promoter of pRm-1 obtained from Dr. R. Nusse (Stanford University). wgts flies were (Bunch et al., 1988). cDNA expression was induced for 24 h with 50 ϳ 8 obtained from Dr. A. Bejsovic (Northwestern University). UAS- mM CuSO4 and then 10 cells were harvested, lysed on ice for 10 armS10 flies were obtained from Dr. M. Peifer (UNC, Chapel Hill). min in IP buffer (150 mM NaCl, 10 mM Tris, pH 7.5, 1% Triton Hs-wg/ϩ; ciD/ciD embryos were generated by mating Hs-wg/ϩ; X-100), and the insoluble fraction removed by centrifugation 5 min ciD/ϩ to ciD/M62f. Embryos from this cross were heat shock treated 15,000g. Following preclearing with Protein A–agarose (Gibco as in (Noordermeer et al., 1992), then processed for expression of BRL), lysates were incubated with either Myc antibody (25 ␮l 9E10 wg 5ЈUTR (Noordermeer et al., 1992) or gsb by in situ hybridization hybridoma supernatant; Evan et al., 1985) or Ci N-terminal anti- (Tautz and Pfeiffle, 1989; Jiang et al., 1991). The Hs-wg; ciD/ciD body (0.1 ␮l, gift of Dr. T. Kornberg, UCSF, Aza-Blanc et al., 1997). phenotype was identified as that expression pattern present in 1/8 Immune complexes were recovered with Protein A–agarose, sub- of the progeny that was neither wild type, ciD/ciD, nor Hs-wg. For jected to SDS–PAGE, and transferred to PVDF membranes. Com- gsb mRNA expression, 138 embryos were scored. A ␹2 value of 4.09 ponents of the immune complex were then detected with Ci and a P value of 0.25 were obtained. For wg in situs, 333 embryos N-terminal antibody (1:50,000), HRP–protein A (1:10,000, Zymed), were scored. A ␹2 value of 2.75 and a P value of 0.4 were obtained. and chemiluminescent reaction (New England Nuclear). Blots were

then reprobed with Myc antibody (1:10) and HRP-anti Mouse (1:2,000, Amersham).

RESULTS

Regulation of ciD activity. ciD is a neomorphic allele (Orenic et al., 1987) that results in abnormally broad expression of wg and gsb (Hidalgo and Ingham, 1990). Previous analysis of hh; ciD double mutants demonstrated that ciD activates wg and gsb expression at the parasegment border in the complete absence of hh activity (Von Ohlen et al., 1997). However, careful examination of ptc expression in ciD mutants suggests that ciD is not simply a constitutively active form of ci. ciD mutants express ptc broadly at the parasegment border but lose ptc at the segment border (Hidalgo and Ingham, 1990; Von Ohlen et al., 1997). ptc in the embryo is not an ideal reporter for Hh and Ci activity, since there is significant ptc expression in the ectoderm hh or ci null embryos (e.g., Hidalgo and Ingham, 1990; Von Ohlen et al., 1997). We have used roadkill (rdx), a newly identified Hh target gene (J.E.H. and Dr. Kent, in prepara- tion) to confirm this curious lack of ciD activity at the segment border. rdx is expressed in two stripes per segment in the embryonic ectoderm (Fig. 1A). These stripes flank the domain of hh expression (data not shown) and are therefore coincident with the ptc stripes at the segment and parasegment borders. rdx expression in the ectoderm requires hh activity (Fig. 1B). Like ptc, rdx is maintained in ciD mutants at the parasegment border but is lost at the segment border D (Fig. 1C). Therefore, ciD has opposite effects on the expres- FIG. 1. ci activity is independent of Hh signaling. All panels are rdx sion of Hh target genes at the segment and parasegment mRNA expression at the ventral surface of stage 11 embryos. Anterior is to the left. (A) In wild-type embryos rdx is expressed in borders. At the parasegment border, ciD is active in the D the ectoderm in two stripes per segment, flanking the hh expres- absence of Hh while at the segment border ci is inactive, sion domain. In the underlying mesoderm, out of focus expression even in the presence of Hh. spans the hh expression domain. (B) rdx expression is lost in the The expression of the Hh target gene ptc in wing imaginal ectoderm and mesoderm in hhGS1 embryos. It can still be detected discs suggests that ciD activity is regulated by Wg signaling in the large neuroblasts underlying the ectoderm. (C) In ciD mutant (Fig. 2). In wild-type discs, ptc is expressed along the A/P embryos rdx expression is retained in only one stripe per segment. compartment border and is regulated by Hh signaling in a The retained stripe is positioned in the middle of the segment in manner similar to that observed in the embryo (Basler and the vicinity of the parasegment border, not near the tracheal pits Struhl, 1994; Tabata and Kornberg, 1994; Fig. 2A). In ciD/ϩ (small arrows) or the deep segment grooves of the gnathal segments wing discs, Ci antigen is misexpressed in the posterior (large arrows) that mark the segment border. Thus the stripe posterior to the hh expression domain is lost while the anterior compartment (Slusarski et al., 1995). Wild-type ci is not stripe is retained. expressed in the posterior compartment, so any effects there must reflect pure ciD activity. ptc is ectopically expressed in the posterior compartment of ciD/ϩ wing imaginal discs in two pennant shaped regions flanking the to the D/V boundary suggests that Wg might regulate ciD dorsal/ventral (D/V) boundary (Fig. 2B). The expression of activity. If Wg signaling activates ciD then in the absence of dpp and hh, two other ci-regulated targets (Domın´guez et wg, ectopic ptc expression should disappear in the posterior al., 1996; Tabata and Kornberg, 1994) is not changed (data compartment. We used a wgts allele to remove wg activity not shown). In late third instar wing imaginal discs the D/V in the discs during the third larval instar. In wgts; ciD/ϩ boundary, the prospective wing margin, is a source of Wg discs (Fig. 2C) ptc expression is retained in the anterior signaling (Couso et al., 1993; Diaz-Benjumea and Cohen, compartment along the A/P boundary only near the center 1994; Fig. 2E). Moreover, cells at the D/V boundary express of the disc. Since anterior compartment ptc expression very low levels of the putative Wg receptor frizzled2, and so reflects the combined activities of wild-type ci and of ciD, should be relatively insensitive to Wg (Cadigan et al., 1998). this suggests that ciD can interfere with the activity of The restriction of ectopic ptc expression to regions adjacent wild-type ci. In the posterior compartment where expres-

FIG. 2. wg signaling regulates ciD activity. All panels are ptc mRNA expression in wing imaginal discs from late third larval instar. Anterior is to the left. (A) In wild type, ptc is expressed strongly along the A/P boundary and weakly throughout the anterior compartment. Similar ptc expression is seen in HS-wg discs (not shown). (B) In ciD/ϩ, ptc is ectopically expressed in the posterior compartment in the region bordering the D/V boundary. Expression in the anterior compartment and at the A/P boundary is normal. (C) In wgts/wgts; ciD/ϩ wing discs grown at the permissive temperature (17°C) and then shifted to the restrictive temperature (25°C) for 24 h lose virtually all ectopic ptc expression in the posterior compartment. In addition, ptc expression is repressed along the A/P boundary away from the D/V boundary. (D) In Hs-wg; ciD/ϩ wing discs, ubiquitous Wg overexpression leads to ptc expression throughout the posterior compartment. (E) Diagram of wing disc showing normal expression domains of wg along the D/V boundary (black), ptc along the A/P boundary (dark gray), and hh in the posterior compartment (light gray).

sion of ptc reflects pure ciD activity, expression flanking the encodes a chimeric protein where the first 246 amino acids D/V boundary is lost. We conclude that ciD requires wg of Pan are substituted for the first 12 amino acids of Ci. The signaling to activate gene expression. junction between Pan and Ci sequences corresponds to the If wg activates ciD, then ubiquitous Wg should drive ninth intron of Pan (Dooijes et al., 1998) and the first intron ubiquitous ptc expression in ciD/ϩ wing discs. Using a of Ci (data not shown). The additional 234 amino acids of heat-shock inducible wg transgene (Hs-wg) to drive ubiqui- Pan protein could account for the increased size of the CiD tous Wg expression in ciD/ϩ discs, we found enhanced protein relative to wild-type Ci seen by Western analysis expression of ptc in the posterior compartment (Fig. 2D). (Slusarski et al., 1995). The ciD chromosome is null for pan While the two pennant-shaped domains remain, we also (Brunner et al., 1997; van de Wetering et al., 1997), so it is detect ptc expression throughout the posterior compart- no surprise that pan sequences are involved in the ciD ment. Hs-wg had no effect on ptc expression in the absence rearrangement. of ciD (data not shown). We conclude that in posterior If the novel transcript were the product of the ciD muta- compartment of ciD/ϩ wing discs, Ci target gene expression tion, then it should segregate from wild-type ci transcripts is limited to cells receiving the wg signal. Since ciD is consistent with classical Mendelian genetics. To test this ubiquitously expressed (Slusarski et al., 1995) this suggests we performed RT-PCR on total RNA isolated from indi- that the unusual activity of ciD is due to posttranscriptional vidual progeny of ciD/ϩ heterozygous parents (Fig. 3). Using activation in response to wg signaling. PCR primers specific for either the wild-type or novel ciD encodes a fusion protein. ciD heterozygotes produce transcript we observed that 4 of 12 embryos expressed only a Ci protein about 20 kDa larger than authentic Ci, as the wild-type transcript, 3 of 12 expressed only the putative detected on Western blots (Slusarski et al., 1995). The ciD ciD transcript, and 5 expressed both. Given the small mutant chromosome includes a genomic rearrangement in sample size, this distribution is not significantly different the first intron of ci (Schwartz et al., 1995; Slusarski et al., from the expected 1:1:2 ratio for the genotypes, ϩ/ϩ: 1995; Locke and Tartof, 1994). These observations sug- ciD/ciD: ciD/ϩ. Thus the novel transcript appears to segre- gested that the protein product of the ciD locus (CiD) might gate from wild-type ci transcripts in a manner consistent be a fusion of Ci and some other protein (Slusarski et al., with it being the product of the ciD mutation. 1995). To clone the ciD cDNA we isolated total embryonic Expression of the ciD transcript phenocopies the ciD RNA from a heterozygous population and utilized a modi- mutation. If the novel transcript we have identified were fied 5Ј RACE protocol. Of 1100 clones, we found eight responsible for the ciD phenotype then its expression in independent PCR products with the same novel open read- transgenic embryos should mimic the phenotype observed ing frame (ORF) fused to ci coding sequences at the pre- in ciD mutant embryos. Overexpression of wild-type Ci will dicted splice junction (Fig. 3). Sequence analysis revealed activate target gene expression even the absence of Hh that the novel ORF is derived from the segment polarity stimulation (Hepker et al., 1997; Alexandre et al., 1996). We gene pangolin (pan) (Fig. 3). This candidate ciD cDNA generated a transgene (CiD⌬) lacking the C-terminal do-

main required for Ci to activate transcription (Alexandre et al., 1996; Hepker et al., 1997; Aza-Blanc et al., 1997) to eliminate effects simply due to overexpression. We used ptcGal4 and the Gal4/UAS conditional expression system (Brand and Perrimon, 1993) to drive expression of CiD⌬ in anterior compartment cells. Western blotting showed that the CiD⌬ transgene was expressed at levels equivalent to that of endogenous Ci (data not shown). CiD⌬ expression caused a phenotype similar to that seen in ciD mutant embryos. Expression of wg, gsb, and ptc was expanded anteriorly from the parasegment border (compare Figs. 4A–4C to 4D–4F), consistent with the range of wg signaling as defined posteriorly by activation of en expression (e.g., Bejsovec and Martinez Arias, 1991). In wg mutant embryos CiD⌬ failed to activate transcription of wg and gsb in the ectoderm (Figs. 4G and 4H). Thus CiD⌬ like the ciD mutant requires wg signaling to activate transcription. We conclude that the transcript we have identified is produced by the ciD mutation and is responsible for generating the ciD phenotype. In the posterior compartment of the wing the ciD mutant has a dominant phenotype. This includes interruption of the fourth longitudinal wing vein (Hochman, 1976; Slusar- ski et al., 1995) and ectopic activation of ptc (Fig. 2B). To ask whether our transgene recapitulates this aspect of the ciD phenotype, we used MS1096Ga14 (Domın´guez et al., 1996) to express CiD⌬ throughout the wing pouch of imaginal discs. Expression of CiD⌬ caused a complete loss of the fourth wing vein, extreme deformities of the third and second wing veins, and overgrowth of the anterior wing (Fig. 5D). ptc was ectopically expressed in both the anterior and posterior compartments in two stripes parallel to the D/V boundary (Fig. 5E). In addition, dpp was ectopically expressed in two broad stripes parallel to the D/V boundary, with stronger expression in the anterior compartment than in the posterior (Fig. 5F). These phenotypes can be inter- preted as an exaggeration of the ciD/ϩ mutant phenotypes. The ciD transcript is expressed at much higher levels in transgenic animals than in heterozygous mutants, so its phenotype should be stronger in the transgenic animals. Alternatively, the phenotypic differences might reflect regulatory differences due to the Hh-responsive carboxy terminal domain that is deleted in the CiD⌬ transgene.

DNA binding domain of Ci (zinc ﬁngers) along with its cytoplasmic anchor and CBP binding domains. Middle panel: Sequence of the N-terminus of the CiD cDNA and protein. The arrowhead indicates the junction between Pan and Ci sequences. Circles indicate 5Ј ends of individual RT-PCR clones. Black circles indicate one FIG. 3. Cloning of ciD revealed a Pan/Ci fusion protein. Top panel: clone and gray circles indicate multiple clones. Bottom panel: Protein domain structure for Pan, Ci, and CiD protein products. Mendelian segregation of ciD (top) and wild-type ci (bottom) tran- The arrowhead and number above each protein indicates the scripts in single embryos from ciD heterozygous parents. Lanes location and amino acid of the chimeric protein fusion. The CiD 1–12, RT-PCR from 12 individual embryos. Lane 13, no RT, RNA fusion protein includes the Arm binding domain of Pan but template. Lane 14, PCR, Ci cDNA as template. Lane 15, RT-PCR, excludes Pan’s DNA binding domain (HMG box). It includes the no template RNA or DNA.

FIG. 4. UASciD⌬ recapitulates the ciD mutant phenotype. (A, D, G) wg mRNA, ventral view, stage 11. (B, E, H) gsb mRNA, ventral view, stage 11. (C, F) ptc mRNA, lateral view, stage 11. (A–C) In ciD homozygous mutant embryos, Hh target gene expression expands two to three cells anterior of the parasegment border. (D–F) In ptcGal4/UASciD⌬ embryos, transgenic CiD expression in the anterior compartment drives strong expression of Hh target genes three to four cells anterior of the parasegment border. (G, H) When wg activity is eliminated in wg; ptcGal4/UASciD⌬ embryos, the ciD transgene no longer activates expression of Hh target genes in the ectoderm. Residual Hh-independent expression is seen in the underlying neuroblasts. The lack of target gene expression in the normal domain indicates that the ciD transgene is interfering with transcriptional activation by wild-type Ci.

In addition to transcriptional activation near the D/V Pan. A current model suggests that activation of Wg signal- boundary, CiD⌬ caused transcriptional repression. hh ing leads to accumulation of Arm, which then binds to Pan was repressed in the posterior compartment in a pattern and reconstitutes an active transcription factor (reviewed in complementary to that of the ectopic ptc (not shown). Nusse, 1997). If Wg regulates CiD through Arm, then Arm Overexpression of wild-type Ci will likewise repress hh should substitute for Wg in activating CiD. To test this, we expression, though the mechanism is completely obscure used a UAS-driven transgene expressing ArmS10, a constitu- (Domın´guez et al., 1996). ptc and dpp expression was tively active form of Arm (Pai et al., 1997). When expressed suppressed in the normal domain at the compartment in the wing pouch, ArmS10 led to overgrowth of the wing and border, both in the center of the disc and away from the mispatterning of the wing margin (data not shown). The ⌬ D/V boundary (Figs. 5E and 5F). Apparently CiD that is expression of ptc was unaffected (Fig. 5G). When ArmS10 and not stimulated by Wg has a dominant negative activity CiD⌬ were coexpressed in the wing pouch, the resulting and can block transcription by wild-type Ci. This domi- pupae failed to eclose. The wing imaginal discs were over- nant negative activity is similar to that of Ci75 (Aza- grown and distorted, and ptc was expressed at high levels Blanc et al., 1997), which like CiD⌬ is deleted for throughout the wing pouch (Fig. 5H). Thus, Arm is a potent C-terminal regulatory sequences. It is also similar to the activator of CiD. repressor activity of Pan/TCF in the absence of Wg Since CiD has acquired an Arm binding domain from stimulation (Cavallo et al., 1998; Waltzer and Bienz, 1998). The Pan sequences acquired by CiD overlap par- Pan, it is likely that Arm activates CiD through direct tially with the binding site of corepressor Groucho and binding to the chimeric CiD protein. To test this, we S10 ⌬ include a Lysine acetylated by the corepressor, CBP coexpressed Arm with either Ci75 or CiD in S2 cells. (Cavallo et al., 1998; Roose et al., 1998; Waltzer and By coimmunoprecipitation and Western blots, we found ⌬ Bienz, 1998). Thus the repressor activity of CiD⌬ could that CiD was associated with Arm while Ci75 was not be a default due to lack of activation by Wg or could (Fig. 6). Thus, Arm protein physically interacts with CiD involve active corepression by Groucho and/or CBP. but not with wild-type Ci. Since Arm binding to Pan is Genetic and physical interaction between ciD and arm. thought to contribute the transactivation function nec- The unusual activity of the ciD allele appears to be due to its essary to activate transcription (reviewed in Nusse, regulation by Wg rather than by Hh (Figs. 1 and 2). CiD 1997), Arm binding to CiD could likewise provide the protein, the product of the ciD allele, has acquired the Arm transactivation function that activates Ci target genes in binding domain of the Wg regulated transcription factor, response to Wg signaling.

FIG. 5. The UASciD⌬ phenotype is enhanced by constitutively activated Armadillo. (A) Wild-type wing. (B) ptc mRNA expression in wild-type imaginal disc. (C) dpp mRNA expression in wild-type imaginal disc. (D–F) UASciD⌬ transgene expressed throughout the wing pouch using MS1096Gal4 as a driver. (D) Gross veination defects include distal loss of the fourth and second wing veins and realignment of vein 3 parallel to the wing margin. The third wing vein is identified by campaniform sensilla, which normally form along its length (reviewed in Campuzano and Modolell, 1992). (E) ptc mRNA is ectopically expressed in two stripes flanking the presumptive wing margin. In addition, it is repressed in its normal domain at the compartment border. (F) In the anterior compartment, dpp mRNA is activated flanking the presumptive wing margin and is repressed at the compartment border. (G) UASarmS10, a constitutively active form of Arm, driven by MS1096Gal4 has no effect on ptc mRNA expression. (H) Coexpression of UASarmS10 enhances the CiD⌬ phenotype, including overgrowth of the wing pouch and overexpression of ptc mRNA.

DISCUSSION gests that Wg signaling activates CiD through direct association with Arm. In the current model for Wg signal- We have shown that the protein product of the ciD ing cytoplasmic Arm protein accumulates, binds to Pan, mutation encodes a chimeric protein where the Arm- and forms a functional transcription factor (reviewed in binding regulatory domain of Pan is fused to virtually the Nusse, 1997). By analogy with Pan, the CiD–Arm com- entire Ci protein. The result is a CiD protein that should plex might be transcriptionally active because CiD pro- recognize Ci target elements in promoters and should be vides the DNA binding domain and Arm provides the regulated by Wg signaling through physical interaction transactivation domain. with Arm. We have shown that increased wg expression Regulation of Ci activity. In wild-type imaginal discs, in vivo enhances the ciD phenotype while the ciD pheno- Ci protein is detectable in two forms, a full-length form, type is attenuated in the absence of wg activity. In other Ci155, and a truncated form, Ci75 (Aza-Blanc et al., 1997). words, Wg signaling regulates activity of CiD. Arm over- Ci75 functions as a transcriptional repressor. In cells at a expression enhances activity of a ciD transgene and Arm distance from the compartment border and thus away from protein directly associates with CiD protein. This sug- Hh signaling, Ci protein is proteolytically processed to

FIG. 6. Physical interaction of CiD and Armadillo proteins. S2 cells expressing either Ci75 ϩ ArmS10 or CiD⌬ϩArmS10 cDNAs were immunoprecipitated either with antibody recognizing Ci N-terminal domain (CiN) or antibody recognizing the Myc epitope tag of ArmS10. Western blots of the immunoprecipitates were probed for CiN and then reprobed for Myc. CiD protein but not Ci protein is detected in the Myc immunoprecipitates. Myc epitope is detected in the CiN immunoprecipitate from CiD expressing cells, but not from Ci-expressing cells.

make Ci75. This suggests that one function of Hh signaling hh-dependent step, which is independent of inhibition of is to inhibit processing of Ci to the repressor form. processing. But how does Hh signaling activate transcription of target We propose that the unusual activity of ciD is due to genes? One possibility is that Hh simply acts to inhibit Wg-regulated association with Arm protein. We further processing of Ci to the repressor form. CiD activity is suggest that activation of wild-type Ci by Hh also involves apparently not regulated by processing. In the posterior association with a protein partner that bestows transacti- compartment of ciD/ϩ discs, CiD does not appear to be vation function. Wild-type Ci is found in a cytoplasmic extensively processed, yet it is not constitutively active. complex with Cos and Fu, two proteins known to play a Instead it activates transcription in response to Wg signal- role in transducing the Hh signal (Aza-Blanc et al., 1997; ing. Processing of Ci to Ci75 on a cell-by-cell basis is Sisson et al., 1997; Robbins et al., 1997). Association with detected by immunofluorescence as loss of C-terminal this complex may control processing of Ci and/or provide epitope (Aza-Blanc et al., 1997). Since C-terminal epitope of Ci with what it needs to activate transcription. Creb Ci is detectable in all posterior compartment cells of ciD/ϩ binding protein (CBP) is required for transcriptional activa- wing imaginal discs (Slusarski et al., 1995), CiD protein is tion by Ci, is able to bind to the carboxy terminal region of not significantly processed. Yet ptc expression is activated Ci (Akimaru et al., 1997), and has transactivation function in only the posterior compartment cells responding to wg (Ogryzko et al., 1996; Bhattacharya et al., 1996; Dai et al., signaling (Fig. 2). These results suggest that activation of 1996; Kwok et al., 1994). Hh-regulated association with CiD is not mediated by inhibition of processing. Instead, CBP could provide a mechanism for activation of Ci just as Wg-regulated association with Arm provides a likely Wg-regulated association with Arm provides a mechanism mechanism for activation of CiD. for activation of CiD. Manipulation of PKA activity in embryos further sup- The ciD mutation provides an example of how mutation ports the suggestion that inhibition of processing is not can lead to profound changes in development and morphol- sufficient to activate Ci (Ohlmeyer and Kalderon, 1997). ogy. A single chromosome rearrangement causes a whole Increasing PKA activity in embryos by expression of a cadre of developmentally important genes (wg, gsb, ptc, constitutively active PKA catalytic subunit facilitated pro- dpp) to switch allegiance from Hh regulation to Wg regula- cessing of Ci while reducing PKA activity by expression of tion. The result is profound effects on morphology (e.g., a regulatory subunit blocked processing to the repressor realignment of the A/P axis in the distal wing) if the ciD form. In these experiments blocking processing was not product is expressed at moderate levels (e.g., in our trans- sufficient for Ci mediated transcriptional activation. The genic flies). Low levels of expression allow the mutation to activity of smoothened (smo) and hh, two regulators of Ci be carried in a heterozygous population without serious activity, was still required for activation of wg expression. deleterious effects. In anterior compartment, CiD does not This implies that transcriptional activation by Ci requires a influence target gene expression because its effects are

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Characterization of the ciD Mutation 155 overwhelmed by wild-type Ci. In posterior compartment Campuzano, S., and Modolell, J. (1992). Patterning of the Drosoph- where wild-type ci is not expressed, low expression of ciD ila Nervous System: the achaete-scute gene complex. Trends has only minor effects. Thus a population carrying this Genet. 8, 202–208. mutation could be a reservoir from which additional muta- Cavallo, R. A., Cox, R. T., Moline, M. M., Roose, J., Polevoy, G. A., tion changing time, place, or level of expression would Clevers, H., Peifer, M., and Bejsovec, A. (1998). Drosophila Tcf generate radical new morphologies. and Groucho interact to repress Wingless signalling. Nature 395, 604–608. Couso, J. P., Bate, M., and Martinez-Arias, A. (1993). A wingless- Dependent Polar Coordinate System in Drosophila Imaginal ACKNOWLEDGMENTS Discs. Science 259, 484–489. 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