Suppressor of Fused Opposes Hedgehog Signal Transduction by Impeding Nuclear Accumulation of the Activator Form of Cubitus Interruptus

Development 127, 4001-4010 (2000) 4001 Printed in Great Britain © The Company of Biologists Limited 2000 DEV2584

Suppressor of Fused opposes Hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus

Nathalie Méthot and Konrad Basler* Institut für Molekularbiologie, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland *Author for correspondence (e-mail: [email protected])

Accepted 30 June; published on WWW 22 August 2000

SUMMARY

Hedgehog controls the expression of key developmental constitutively active form of Ci in a Hh-dependent manner, genes through the conversion of the transcription factor suggesting that Fu enhances the activity rather than the Cubitus interruptus (Ci) into either an activator (Ci[act]) formation of Ci[act]. Su(fu) reduces the nuclear or a repressor (Ci[rep]) form. Proteolytic cleavage of full- accumulation of the constitutively active form of Ci, length Ci is important for the generation of Ci[rep], but arguing that Su(fu) can function subsequent to Ci[act] little is known about how Ci[act] arises in response to Hh. formation. We propose that Hh induces target gene Here we examine Hh signal transduction components for expression by a two-step mechanism in which Ci[act] is ﬁrst their role in the conversion of full-length Ci into either formed and then accumulates in the nucleus via Fu-induced Ci[act] or Ci[rep]. We report that Cos2, PKA and Fused neutralization of Su(fu) activity. are necessary for the generation of Ci[rep], whereas the inhibition of either Cos2 or PKA activity is a prerequisite Key words: Hedgehog signaling, Nuclear entry, Transcriptional for Ci[act] formation. Fused (Fu) kinase stimulates a regulation, Cubitus interruptus, Drosophila

INTRODUCTION (Ci; Orenic et al., 1990) and the transcriptional activation of target genes. Several proteins, such as Cos2, PKA, Su(fu) and Members of the Hedgehog (Hh) family of secreted signaling Slimb, prevent or downregulate signaling, but their mode of proteins function as potent organizers in animal development. action is not well understood. Some evidence suggests that Drosophila Hh controls pattern formation during segmentation Cos2 tethers Ci to the cytoplasm (Robbins et al., 1997, Sisson and limb development. Vertebrate Hh homologues play key et al., 1997), whereas Slimb and PKA are required for the roles in the morphogenesis of the neural tube, somites, axial proteolytic processing of Ci (Jiang and Struhl, 1998; Chen et skeleton, limbs, lung and skin (reviewed in Neumann and al., 1998). Cleavage of Ci occurs in the absence of Hh (Aza- Cohen, 1997; Ingham, 1998; Ruiz i Altaba, 1999; McMahon, Blanc et al., 1997) and results in the release of a C-terminally 2000). Most if not all Hh signaling is mediated through the truncated Ci (here referred to as Ci[rep]), that translocates to transcriptional effectors of the Gli/Ci family (reviewed by Aza- the nucleus and inhibits transcription of target genes such as Blanc and Kornberg, 1999). Major interest is devoted therefore decapentaplegic (dpp) and hh itself (Dominguez et al., 1996; to the mechanisms by which Hh signaling regulates the activity Aza-Blanc et al., 1997; Méthot and Basler, 1999). Reception of Gli/Ci proteins. of Hh prevents Ci[rep] formation, and activates the The function of Hh is best understood in the developing transcription of several Hh target genes such as dpp and ptc Drosophila wing imaginal disc, a tissue that is formed by (Aza-Blanc et al., 1997; Méthot and Basler, 1999). Recently, two cell populations, the anterior (A) and posterior (P) it has been shown that inhibition of Ci processing is not compartments. The selector gene engrailed (en) is expressed sufficient to activate Hh-target gene transcription. An in P cells where it permits the synthesis of Hh while at uncleavable form of Ci (CiU) still requires Hh input to induce the same time preventing a response to Hh (reviewed by ptc expression in wing imaginal discs (Méthot and Basler, Lawrence and Struhl, 1996). Hh diffuses into the A 1999), and the proteosome inhibitor lactacystin does not compartment and initiates a signaling cascade by binding to potentiate the response to Hh in tissue culture cells (Chen C.- its receptor, the multi-transmembrane domain protein Patched H. et al., 1999). These results suggest that a Hh-dependent (Ptc). Hh neutralizes the inhibitory hold of Ptc on the co- event other than prevention of cleavage permits full-length Ci receptor protein Smoothened (Smo), which then signals to to function as a potent transcriptional activator (Ci[act]). downstream effectors (reviewed in Alcedo and Noll, 1997; Several studies identiﬁed phosphorylation of Ci by protein Ingham, 1998; Strigini and Cohen, 1999). Signaling results in kinase A (PKA) as a key event in mediating cleavage (Chen the stabilization of the zinc ﬁnger protein Cubitus interruptus et al., 1998; Chen Y. et al., 1999; Price and Kalderon, 1999; 4002 N. Méthot and K. Basler

Wang and Holmgren, 1999; Wang et al., 1999). Elegant grown to an optical density of 0.5 to 0.8 before induction with 1 mM genetic experiments have indicated that the serine-threonine isopropyl-1-thio-β-D-galactopiranosid (IPTG). Cells were grown for kinase Fu plays a role in the conversion of Ci into a 3 hours at 30°C before harvesting by centrifugation. The pellet was transcriptional activator (Ohlmeyer and Kalderon, 1998). resuspended in lysis buffer (phosphate-buffered saline, 1 mM EDTA, Our previous studies focussed on the demonstration that 1 mM DTT and 1 mM phenylmethylsulfonyl fluoride). Cells were the formation of both Ci[act] and Ci[rep] is tightly controlled lysed on ice by five sonication cycles. Cellular debris was removed by centrifugation. GST-fusion proteins were purified on glutathione- by Hh signaling (Méthot and Basler, 1999). Here we agarose (Pharmacia). Ci deletion mutants (as specified in the figure examined Hh signal transduction components for their role in legend) were labeled with [35S]methionine using the T7 TNT in vitro the conversion of full-length Ci into either Ci[act] or Ci[rep]. translation system (Promega). Translation reactions (20 µl) were We found that the formation of Ci[rep] requires PKA, Cos2 diluted to 300 µl in binding buffer (20 mM Hepes pH 7.4, 200 mM and Fu, while the generation of Ci[act] proceeds through the KCl, 1 mM DTT, 1 mM MgCl2, 1% NP40, 10% glycerol) and neutralization of PKA and Cos2 activity. Although Ci can incubated for 1 hour at room temperature with 1 µg of GST-fusion bypass PKA and gain constitutive Ci[act] activity by protein. Glutathione-agarose beads were added, and the mixture was mutations in its PKA phosphorylation sites, we have observed incubated for an additional 15 minutes on a rotator. Beads were that this activity can be further stimulated by Hh signaling. washed five times in binding buffer, before loading on an SDS- Both Su(fu) and Fu alter the transcriptional output of this polyacrylamide gel. Gels were fixed, treated for fluorography and exposed overnight on Kodak XAR-5 film. mutant form of Ci, by regulating its nuclear-cytoplasmic localization. The accumulation of full-length Ci in the Marked clones of mutant cells and immunochemistry nucleus is Hh-dependent and is blocked by excess Su(fu). We Alleles used in this study were: smo1, smo3 (Nüsslein-Volhard et al., propose that Fu kinase stimulates the Hh pathway, not by 1984); DCOE95 (Lane and Kalderon, 1993); slimb1 (Jiang and promoting the formation of Ci[act], but rather by facilitating Struhl, 1998); cos25 (Forbes et al., 1993); fu1, fuA, fumH63 and its entry into the nucleus. Su(fu)LP (Préat et al., 1993). Clones of mutant cells were generated by Flp-mediated mitotic recombination (Xu and Rubin, 1993). Larvae were heat shocked in late first instar for 30 minutes at 35°C and heat shocked again in late 3rd instar phase to induce marker MATERIALS AND METHODS gene expression (hsp70-GFP or hsp70-CD2). Following a 1 hour recovery period, larvae were dissected, discs were fixed and stained Transgenes with the appropriate antibodies. The following antibodies were used: Ectopic expression of Ci, Ci mutants, mycSu(fu), mycFu and rat monoclonal anti-Ci (gift from R. Holmgren); mouse monoclonal mycCos2 in wing imaginal discs was achieved with the UAS-Gal4 anti-Ptc (gift from I. Guerrero); mouse monoclonal and rabbit system (Brand and Perrimon, 1993). UAS-Ci and UAS-CiPKA (in this polyclonal anti-GFP (Clonetech); mouse monoclonal anti-CD2 paper referred to as CiPKA1), where Ci codons for amino acids 837, OX34 (Serotech); mouse monoclonal (Promega) and rabbit 838 and 839 were mutated to encode alanine residues, have been polyclonal (Cappel) anti-βGal; mouse monoclonal anti-myc 9E10; described in Méthot and Basler (1999). The cDNA for CiPKA4 is a gift Alexa 488 and 594 secondary antibodies (Molecular Probes). from C. Dahmann and encodes a mutant protein in which the Presence of Ci[rep] was assayed genetically in vivo by its effect following serine residues are replaced by alanines: S838, S855, S856, on hh-lacZ transcription. The final genotype of larvae were the S891, S892 and S962. The cDNA for CiGFP was made by fusing an following: EGFP (Clontech) cDNA in frame to the 3′ end of Ci cDNA. GFPCi75 Fig. 1A: y w hsp70-flp; smo3 FRT39/hsp70-CD2 FRT39; UAS-Ci/ DNA was obtained by fusing EGFP DNA to the 5′ end of Ci cDNA C765 hh-lacZ that had been truncated at the unique EcoRV site. All constructs that Fig. 1B: y w hsp70-flp; smo3 DCOE95 FRT39/hsp70-CD2 FRT39; were obtained following a PCR step or blunt ending of restriction sites UAS-Ci/ C765 hh-lacZ were sequenced to verify integrity. DNA templates used for in vitro Fig. 1C: y w hsp70-flp; smo1 FRT42 cos25/smo3 FRT42 P[smo+, translations were obtained from Ci cDNA coding for a triple HA tag hsp70-GFP]; UAS-Ci/ C765 hh-lacZ. at its 5′ end (Méthot and Basler, 1999), and truncated at its unique Fig. 1D: y w f fuA; smo3 FRT39/hsp70-CD2 FRT39; UAS-Ci hh- EcoRV site, to generate pBS-Ci440. The in vitro translated product lacZ/ C765 hsp70-flp derived from this construct consists of a triple HA tag followed in Fig. 3D: y w hsp70-flp; smo3 FRT39/hsp70-CD2 FRT39; UAS- frame by Ci amino acids 5 to 440. Deletions (∆212-268 and ∆268- CiPKA4/ C765 hh-lacZ. 346) were generated by PCR mutagenesis and introduced into pBS- To determine the Hh-dependency of CiPKA4, larvae with the Ci440. Fused and Su(fu) cDNAs were amplified from Drosophila following genotype were analyzed: embryonic cDNA and cloned in frame 5′ to a myc tag. The resulting Fig. 3C: y w hsp70-flp; smo3 FRT39 ptc-lacZ/hsp70-CD2 FRT39; vectors [pBS-mycFu and pBS-mycSu(fu)] were transferred as UAS-CiPKA4/ C765. Asp718-XbaI fragments into pUAST. A construct coding for GST- Other marked clones: mycSu(fu) was derived from pBS-mycSu(fu), where an EcoRI- Fig. 2A,B: cos25: y w hsp70-flp; dpp-lacZ FRT42 hsp70-CD2/ blunted Asp718 fragment was transferred into pGEX3X (Pharmacia) FRT42 cos25 digested with BamHI, blunted and redigested with EcoRI. Cos2 cDNA Fig. 2C: PKAE95: y w hsp70-flp; FRT40 DCOE95 / 2x hsp70-πmyc (gift of K. Ho and M. Scott) was cloned in frame 3′ to a myc-tag, and FRT40 ptc-lacZ transferred to pUAST. Fig. 2D fuA: y w f fuA FRT19/ y w hsp70-GFP FRT19; ptc-lacZ / Reporter genes used in this study were dppP10638 (Zecca et al., +; hsp70-flp. 1995), hhP30 (Lee et al., 1992), ptc(10.8L)A (Chen and Struhl, Clones expressing mycFu, mycSu(fu) and mycFu∆N were 1996). Gal4 drivers used were C765 (Nellen et al., 1996), produced by mild heat shock (30 minutes at 34°C) of larvae act5c>CD2>Gal4 (Pignoni and Zipursky, 1997) and apterous-Gal4 containing the respective UAS construct and the act5c>CD2>Gal4 (Calleja et al., 1996). transgene (Pignoni and Zipursky, 1997). To inhibit nuclear export, discs from 3rd instar larvae were dissected and cultured for 8 hours in Protein purification, GST pull-down assays C8 media (Bhanot et al., 1996) in the presence or absence of 30 nM pGEX-mycSu(fu) was transformed into E. coli BL21. Bacteria were leptomycin B. Fu stimulates the activator form of Ci 4003

RESULTS Hence, the activities of PKA and Cos2 are required for Ci[rep] formation. Formation of Ci[rep] requires Cos2, PKA and Fu The behavior of certain fu alleles suggests that Fu, in Ci functions as a potent repressor of hh expression in the addition to its stimulatory function, also plays an inhibitory absence of Hh signal transduction (Aza-Blanc et al., 1997; role in the Hh pathway (Préat et al., 1993). Two types of fu Méthot and Basler, 1999). Posterior cells that ectopically alleles exist. Molecular analysis of these two classes has shown express Ci transduce the Hh signal and do not form Ci[rep] due that fu type I alleles bear mutations in the kinase domain, which to their exposure to the Hh signal. Removal of Smo function, are thought to reduce or abolish kinase activity, while fu type however, prevents such P cells from transducing Hh and results II alleles lack the C terminus of the protein (Thérond et al., − in the formation of Ci[rep] and inhibition of hh transcription 1996). We assayed Ci[rep] formation in posterior smo cells (Fig. 1A). To determine which components of the Hh signal that expressed Ci and were either mutant for a fu type I (fu1) transduction cascade are required for the generation of Ci[rep], or a type II (fuA) allele. hh transcription was eliminated in smo3 we expressed Ci in P cells and removed the function of Smo clones of fu type I discs (data not shown), but not of fu type II together with that of either Cos2 or PKA. In contrast to smo− discs (Fig. 1D). Thus, the catalytic function of Fu is dispensible single mutant clones, hh transcription was not reduced in smo− for Ci[rep] formation, whereas the function of the C terminus PKA− (Fig. 1B) or smo− cos2− (Fig. 1C) double mutant clones. is required for Ci[rep]. Formation of Ci[act] is prevented by PKA and Cos2 We found that absence of Cos2 or PKA activity prevents formation of Ci[rep]. It has been shown earlier that mere prevention of Ci cleavage does not suffice for Ci[act] formation (Méthot and Basler, 1999). To test whether, in these mutant contexts, prevention of Ci[rep] formation is linked to the generation of Ci[act], we removed PKA or Cos2 and assessed Ci[act] activity. While the repression of hh transcription is indicative for the presence of Ci[rep], the upregulation of ptc transcription serves as an indicator for the presence of Ci[act] (Méthot and Basler, 1999). We examined ptc expression in A cells mutant for cos2 or PKA and found, in both cases, ptc to be upregulated (Fig. 2B,C). These results indicate that A cells mutant for cos2 or PKA generate Ci[act], and suggest that neutralization of the activities or effects of either Cos2 or PKA is an important step for the formation of Ci[act]. Interestingly, although the C terminus of Fu is required for Ci[rep] formation, its absence did not lead to ectopic ptc-lacZ expression and Ci[act] formation (Fig. 2D). Ci protein resistant to phosphorylation by PKA is still subject to Hh control Ci[act] is generated in cells that lack PKA activity (Li et al., 1995; Jiang and Struhl, 1995; this work). An equivalent situation can be created by mutating the PKA phosphorylation sites of Ci. We mutated one (CiPKA1) or four (CiPKA4) PKA phosphorylation sites and compared the ability of these mutants to activate ptc-lacZ in wing imaginal discs. Ubiquitous weak expression of wild-type Ci led to ptc-lacZ activation only in Hh-exposed cells (Fig. 3A). This indicates that under physiological conditions, transcriptional activity of Ci is under the control of Hh. In contrast, CiPKA1 activated ptc-lacZ in all Fig. 1. Cos2, PKA and Fu are required to make Ci[rep]. Wild-type Ci cells, regardless of their exposure to Hh (Fig. 3B). Thus, is expressed ubiquitously in the wing imaginal disc with the C765 CiPKA1 is constitutively active in vivo. Identical results were Gal4 driver. Repression of hhZ (red) indicates the presence of recently described by several groups, and are taken as Ci[rep]. smo3 (A,D) smo3 PKA (B) and smo3 cos25 (C) clones were indication for a role of PKA in Ci activation (Chen Y. et al., generated by mitotic recombination and are marked by the absence 1999; Price and Kalderon, 1999, Wang et al., 1999). of green staining (CD2: A,B,D; GFP: C). In this figure and all Interestingly, closer examination of discs ubiquitously subsequent figures, anterior is left and dorsal is up. (A) Removal of expressing CiPKA1 reveals that P cells express ptc-lacZ at smo activity in posterior cells that express Ci leads to downregulation of hhZ, which reflects Ci[rep] formation. (B,C) Posterior cells doubly higher levels than A cells (Figs 3B, 4I). This suggests that the PKA1 mutant for smo and PKA (B) or smo and cos2 (C), and expressing transcriptional activity of Ci can be further enhanced by wild-type Ci, continue to express hhZ. (D) The C terminus of Fu is Hh signaling. We have obtained similar results with CiPKA4 required to make Ci[rep]. smo3 clones generated in fuA discs that (Figs 3C, 4C). To test whether it is the reception of the Hh ubiquitously express Ci. Transcription of hhZ is unaffected. signal that stimulates the basal activity of CiPKA, we removed 4004 N. Méthot and K. Basler

Fig. 2. PKA and Cos2 inhibit Ci[act] formation. cos25 and fuA clones are marked by the absence of green staining (A: anti-CD2; D: anti- GFP). Clones of cos25 ectopically express both dppZ (A′) and Ptc protein (B) to near maximal levels. Clones lacking PKA activity express ectopic Ptc (C, red). Hence, lack of Cos2 and PKA promote formation of Ci[act]. fuA cells (D) do not transcribe ptcZ (D′).

Fig. 3. Ci mutated at its PKA phosphorylation sites exhibits both Hh- the function of Smo from a subset of P cells expressing CiPKA4. independent and Hh-dependent activities. Clones mutant for smo Such cells transcribed ptc-lacZ at lower levels than their smo+ (C,D) are marked by the absence of green staining. β-galactosidase neighbors, levels that were similar to those found in anterior for reporter genes (dppZ, ptcZ or hhZ) is visualized in red. (A) Wing smo+ CiPKA cells (Fig. 3C). Thus Ci protein that is imaginal disc ubiquitously expressing low levels of wild-type Ci protein (green) under control of the C765 Gal4 driver. Higher green constitutively active due to mutated PKA phosphorylation sites staining in the anterior (A) compartment results from the sum of is further stimulated in its transcriptional activity by the Hh endogenous and transgenic Ci protein. ptcZ (red) is transcribed only signal. in the posterior half of the disc where cells receive Hh. Some exogenous dppZ activity is seen in A and P cells. (B) Weak PKA Fu kinase activity stimulates Ci expression of CiPKA1 (green) results in ectopic ptcZ expression (red) It is possible that the reduction of ptc-lacZ expression in in the entire wing disc. ptcZ expression is higher in P cells than in A posterior smo− CiPKA4 cells is due to formation of some cells. dppZ expression is high in A cells but absent in P cells due to Ci[rep], which could compete with the Ci[act] activity of repression by engrailed (Sanicola et al., 1995; Méthot and Basler, PKA 1999). (C) High ptcZ levels in CiPKA-expressing P cells are due to Ci . We used the repressor assay described above to test 3 PKA4 whether CiPKA4 can be converted to a transcriptional repressor, Hh signaling. smo clones in P cells that express Ci transcribe − ptcZ, but at reduced levels. The loss of Smo function has no effect on and found that hh transcription in smo posterior cells was ptcZ expression in A cells. (D) CiPKA4 cannot make the repressor PKA4 essentially unaffected (Fig. 3D). This indicates that Ci form of Ci, as hhZ expression is not reduced in CiPKA-expressing P cannot be converted to Ci[rep] and that the reduction of ptc cells upon loss of smo function. expression in smo− CiPKA4 cells is not due to the presence of Ci[rep]. Next, we searched for components of the Hh pathway that could still be seen near the AP compartment boundary in fu1 could stimulate the basal activity of CiPKA. Fu is one of the few discs, and may be the result of cumulative activities of proteins that have a positive input on Hh signaling (Préat et al., endogenous Ci[act] and CiPKA. We conclude that Fu kinase 1990, Ohlmeyer and Kalderon, 1998; Thérond et al., 1999). enhances the basal activity of CiPKA. We examined ptc-lacZ levels in wing imaginal discs that ubiquitously expressed CiPKA1, in either a wild-type or fu1 Fu stimulates CiPKA by inhibiting Su(fu) activity background. ptc-lacZ expression was clearly reduced in P cells fu is tightly linked to Su(fu), both genetically and of fu1 discs (compare Fig. 4A and B). Similar results were also biochemically (Préat, 1992; Préat et al., 1993; Monnier et al., obtained with CiPKA4 (Fig. 4I,J). Slightly elevated ptc-lacZ 1998). To test whether the modulation of CiPKA activity Fu stimulates the activator form of Ci 4005

Fig. 4. Fu is needed to fully activate CiPKA. The Hh-dependent, high ptcZ levels (red) induced by CiPKA1 in P cells (A) are suppressed in discs mutant for fu (B). Stainings in A and B were done in parallel. The higher amounts of Ci protein (green) seen in B may be due to stabilization of Ci by Su(fu) (Ohlmeyer and Kalderon, 1998). (C,D) Co-expression of Su(fu) (D), but not GFP (C), reduced the ptcZ transcription induced by CiPKA4. (E) ptcZ expression in Su(fu) mutant discs expressing CiPKA1. Anterior and posterior ptcZ levels are similar. However, in most discs examined, P cells still expressed slightly more ptcZ. (F) GST pull-down assay with N-terminal deletion mutants of Ci and GST-Su(fu). Deletion of Ci amino acids 212-268 impaired the association between Ci and GST-Su(fu). In vitro translated Ci N-terminal fragments (wild-type aa 5- 440 (lanes 1, 4, 5), or deleted between aa 212- 268 (lanes 2, 6, 7) or 268-346 (lanes 3, 8, 9)), were pulled-down with either GST (lanes 4, 6, 8) or GST-Su(fu) (lanes 5, 7, 9). (G,H) Subcellular localization of CiPKA4 (green) in wild-type salivary glands (G) or Su(fu)LP mutant glands (H). Induction of ptcZ (red) by CiPKA4 marks nuclei. A marked increase in nuclear CiPKA4 in observed in salivary glands that lack Su(fu). (I,J) The Hh-stimulated activity of CiPKA4 (I) is strongly reduced in fumH63 mutant discs (J). (K,L) Ci∆NPKA4 combines mutations at the PKA phosphorylation sites and the 212-268 deletion. This mutant protein is constitutively active (K). The lower transcriptional activity of Ci∆NPKA4 relative to CiPKA4 is probably due to lower expression levels of the protein. The Hh-stimulated activity of Ci∆NPKA4 is reduced in fumH63 discs (L), but much less so than that of CiPKA4 (compare J and L). involves Su(fu), we ubiquitously expressed CiPKA4 together To determine whether Su(fu) negatively acts on CiPKA4 by with myc-tagged Su(fu) or GFP as negative control. CiPKA4 (in direct protein-protein interaction, we created a mutant form of the presence of GFP) induced ptc-lacZ expression everywhere Ci with impaired Su(fu) binding. Su(fu) interacts with Ci in the wing imaginal disc but at higher levels in the P within a region that encompasses amino acids 244-346 compartment (Fig. 4C). Co-expression with mycSu(fu) (Monnier et al., 1998). Indeed, an N-terminal fragment of Ci abolished ptc-lacZ expression in the A compartment and (amino acids 5-440) interacts with GST-Su(fu) (Fig. 4F, lane reduced ptc-lacZ levels in P cells (Fig. 4D). Thus Su(fu) 5). A deletion removing amino acids 212-268 of Ci almost inhibits the activity of CiPKA4. This results is strengthened by abrogated Su(fu) binding to an N-terminal in vitro translated the converse experiment, where the absence of Su(fu) (in product of Ci (Fig. 4F, lanes 6 and 7). Removal of amino acids Su(fu)LP homozygous animals) reduced the difference in ptc- 268 to 346 also reduced Su(fu) binding, but to a lesser extent lacZ levels between A and P CiPKA1-expressing cells (Fig. 4E). (lanes 8 and 9). The ∆212-268 deletion was introduced into 4006 N. Méthot and K. Basler

CiPKA4, to create Ci∆NPKA4. This mutant was constitutively nuclei of posterior cells (Fig. 5A, green). Treatment of wing active, with P cells expressing higher ptc-lacZ levels than A discs with LMB resulted in a dramatic accumulation of GFP cells (Fig. 4K). The activity of Ci∆NPKA4 was slightly reduced fluorescence in the nuclei of P cells or A cells close to the Hh when introduced into a strong fu background (fumH63; compare source. A weaker accumulation can also be observed in A Fig. 4K and L), but the reduction was much less pronounced cells far from the Hh source (Fig. 5B). Several conclusions compared to that observed for CiPKA4 (compare Fig. 4I and J). can be drawn from these results. In the absence of Hh, some We conclude that inhibition of Su(fu) activity by Fu kinase is Ci must enter the nucleus, as accumulation in A cells is seen an important step towards stimulating the basal activity of in the presence of LMB. It is probably exported rapidly, since CiPKA4 (and by analogy Ci[act]). Ci, Fu and Cos2 shuttle in and out of the nucleus Regulation of Ci nuclear accumulation may be an important control point in the Hh signaling cascade. It has been shown recently that human GLI1 and Ci accumulate in the nuclei of tissue culture cells treated with the nuclear export inhibitor drug leptomycin B (LMB; Kogerman et al., 1999; Chen, C.-H. et al., 1999). To test if LMB has similar effects in vivo, Ci tagged at its C terminus with GFP (CiGFP) was expressed in the dorsal compartment of wing imaginal discs using an apterous-Gal4 driver (Calleja et al., 1996). Nuclear β-galactosidase derived from the ptc- lacZ reporter was used as a marker for nuclei and CiGFP activity. Under these conditions, CiGFP induced ptc-lacZ in P compartment cells only, indicating that the protein is active and under the control of Hh signaling (Fig. 5A, inset). A higher magnification shows that GFP fluorescence was largely excluded from the nuclei of untreated anterior cells, whereas some GFP fluorescence was observed in some

Fig. 5. Inhibition of nuclear export causes an accumulation of Ci and Fu in the nucleus. (A,B) GFP ﬂuorescence (green); (C-H) anti-myc staining (green). Nuclear β-galactosidase (red) from dppZ, ptcZ or hhZ reporters was used for orientation in the disc and to mark nuclei. (A) Expression of CiGFP in dorsal wing disc cells using the apterous-Gal4 driver (aptGal4; Calleja et al., 1996). ptcZ is made in the dorsal posterior compartment, where Hh and CiGFP are present concurrently. Inset shows the entire wing disc. The stripe of endogenous ptcZ expression (red) in the ventral compartment marks the approximate position of the AP boundary. Some CiGFP ﬂuorescence (green) is seen in the nuclei of a few posterior cells. (B) Leptomycin B (LMB) treatment causes a marked accumulation of CiGFP in nuclei of P cells, and a slight accumulation in those of A cells. (C,D) Subcellular localization of mycSu(fu) in untreated (C) or LMB-treated (D) wing discs. No substantial nuclear accumulation of mycSu(fu) is observed. (E,F) Subcellular localization of mycFu in untreated (E) or LMB- treated (F) discs. MycFu accumulates in the nuclei of LMB-treated disc cells. (G,H) Subcellular localization of mycCos2 in untreated (G) or LMB- treated (H) discs. MycCos2 accumulates in the nuclei of LMB-treated discs. Fu stimulates the activator form of Ci 4007

Fig. 6. Su(fu) prevents nuclear entry of Ci75 and full-length Ci (both in green by GFP fluorescence). Anti-Ci stain (red) marks the cytoplasm (A,B). mycSu(fu) is used as cytoplasmic marker in B (red) and D (blue). (A) Clones of cells expressing Ci75 tagged at its N terminus by GFP. GFPCi75 is located mostly in the nucleus. (B) Co-expression of Su(fu) with GFPCi75 reduces the amount of nuclear GFPCi75. (C) Nuclear accumulation of CiGFP in the nuclei of LMB-treated wing disc cells. (D) Co-expression of CiGFP (green) and Su(fu) (blue) reduces the amount of nuclear CiGFP in LMB treated discs, and hinders ptcZ expression (not shown). no nuclear Ci is detected without LMB treatment. In the with a GFP-tagged form of Ci75, which has been shown to be presence of Hh, more Ci enters the nucleus, but it is also mainly nuclear (Aza-Blanc et al., 1997; Hepker et al., 1997, rapidly exported, explaining why little nuclear accumulation Fig. 6A). Expression of mycSu(fu) reduced the amount of is seen in untreated P cells. GFPCi75 that accumulates in the nucleus (Fig. 6B). We also Next, we studied the effect of LMB on the subcellular performed these experiments with full-length Ci (CiGFP). As localization of Fu and Su(fu). dpp-lacZ was used as a marker shown before, CiGFP accumulates in LMB-treated nuclei, for nuclei and the AP compartment boundary, and mycSu(fu) particularly in those exposed to Hh (Fig. 6C). Co-expression and mycFu were expressed in cell clones using an of Su(fu) in LMB-treated discs reduced the amount of nuclear act5c>CD2>Gal4 transgene (Pignoni and Zipursky, 1997). CiGFP, both in A and P cells (Fig. 6D). We conclude that Su(fu) was largely confined to the cytoplasm of untreated or Su(fu) downregulates the Hh pathway by preventing nuclear LMB-treated wing imaginal discs (Fig. 5C,D). Fu was also accumulation of Ci[act]. cytoplasmic in untreated cells (Fig. 5E), but showed some accumulation in the nuclei of LMB treated cells (Fig. 5F). In contrast to Ci, the quantity of nuclear Fu did not depend on Hh DISCUSSION exposure (data not shown). The subcellular localization of myc-tagged Cos2 was also examined. mycCos2 was mainly Ci[act] and Ci[rep] are two distinct forms of Ci whose cytoplasmic in untreated imaginal disc cells (Fig. 5G), but generation is strictly under the control of Hh. The mechanism partly nuclear in LMB-treated cells (Fig. 5H). As with Fu, no by which full-length Ci is converted into Ci[act] is unknown, difference in nuclear accumulation between A and P cells as are the precise events leading to the formation of Ci[rep]. could be detected. Thus, Fu and Cos2 can enter the nucleus, Using the Ci target genes ptc, dpp and hh as diagnostic tools but the rate of entry does not change with Hh signaling. for the presence of Ci[act] or Ci[rep], we demonstrate that PKA and Cos2 prevent Ci[act] formation and promote the generation Su(fu) prevents nuclear accumulation of Ci of Ci[rep]. The structural integrity of Fu also contributes to A possible mechanism by which Fu stimulates and Su(fu) Ci[rep] formation. Loss of PKA activity leads to the counteracts Ci[act] could be the promotion or impediment of appearance of Ci[act], an effect that can be mimicked by nuclear Ci[act] accumulation, respectively. We compared the mutating PKA phosphorylation sites of Ci. The key finding of subcellular distribution of CiPKA in cells expressing or lacking our present study is the discovery that, although such mutant Su(fu). Strikingly, we found that, while wild-type cells show a forms of Ci are constitutively active, their activity can be cytoplasmic distribution of CiPKA4, this protein is mostly further stimulated by Hh signaling. Our results indicate that nuclear in Su(fu) mutant salivary gland cells (Fig. 4G,H). this effect is mediated by Fu and counteracted by Su(fu). We Identical results were obtained with wild-type Ci fused N suggest that Fu, rather than being involved in Ci[act] formation terminally to GFP (data not shown). This suggests that Su(fu) per se, stimulates the Hh pathway by permitting nuclear influences the nuclear localization of Ci. We further tested the accumulation of Ci[act]. Below we describe arguments that effect of Su(fu) on Ci localization by overexpressing Su(fu) support this proposal. 4008 N. Méthot and K. Basler

The roles of Fu and Su(fu) the activity of the Fu kinase is only required to maximize the Su(fu) is not involved in the formation of Ci[rep] or Ci[act], as output of an already activated form of Ci, for example by no ectopic dpp or ptc expression was detected in Su(fu)LP clones opposing cytoplasmic tethering of Ci[act] by Su(fu). The (data not shown). Indeed, Ohlmeyer and Kalderon (1998) report precise mechanism of how these components act is not no change in the amount of Ci75 in Su(fu)LP discs. Rather, Su(fu) understood. No substrate for the Fu kinase has been identified appears to restrict the activities of Ci[act]. This is evident from and the significance of nuclear Fu protein is unclear. the observation that Su(fu) overexpression substantially curbs the transcriptional activity of constitutively active CiPKA, and is Ci[rep] formation suggestive of Su(fu) acting after Ci[act] formation. There are Slimb, Cos2, PKA and Fu are required for the conversion of several ways by which Su(fu) could fulfill such a role. One full-length Ci into the repressor form. Basal levels of dpp are possibility is that it impedes entry of Ci[act] into the nucleus. transcribed in anterior slimb1 clones, indicating absence of Alternatively, Su(fu) might promote nuclear export of Ci[act]. It Ci[rep] in these cells. No Ci[rep] could be detected by our is difficult to distinguish between these two possibilities. The repressor assay in cells mutant for cos2 or PKA. Moreover, we observation that little Su(fu) accumulated in the nuclei of LMB- found that an intact C terminus of Fu is required to generate treated cells suggests that Su(fu) functions primarily in the Ci[rep], whereas Fu kinase activity is dispensable. cytoplasm and hence might exert a negative effect on Ci[act] by preventing its nuclear entry. It cannot be excluded, however, that Ci[act] formation a minor fraction of Su(fu) negatively affects the activity, stability slimb1 mutant cells do not ectopically express ptc (N.M. and or localisation of Ci[act] in the nucleus. K.B., unpublished). Similar results were recently reported by Fu, as the main regulator of Su(fu) activity, is also controlled Wang et al. (1999), who have also used a slimb null allele. Thus by Hh. In fu1 discs, CiPKA expression leads to similar levels of Slimb seems to play no role in the generation of Ci[act]. PKA ptc transcription in A and P cells but, in fu+ discs, CiPKA and Cos2, in contrast, appear to be components dedicated to expression causes higher ptc levels in P cells. In other words, prevent Ci[act] formation, as cells lacking these proteins Fu enhances CiPKA activity only in Hh-exposed cells. From ectopically express ptc. This suggests that some of the events this, it can be concluded that Fu activity is subject to Hh leading to Ci[act] include the neutralization of PKA and Cos2 control. activities. PKA directly phosphorylates Ci in vitro and One puzzling aspect regarding Su(fu) is that it is dispensable mutations at the PKA phosphorylation sites of Ci have a for viability. Animals that lack Su(fu) protein do not exhibit profound effect on Ci activity (Chen et al., 1998; Chen Y. et Hh-independent Ci[act] activity. This paradox can be partly al., 1999; Wang et al., 1999; Price and Kalderon, 1999; this explained by viewing Su(fu) only as a partial inhibitor of study). Since no change in catalytic activity has been reported Ci[act] activity, which exerts its function subsequent to Ci[act] for PKA upon Hh signaling (Ohlmeyer and Kalderon, 1997), formation. Other elements, discussed below, ensure tight it is thought that other events, such as protection of Ci from control over the generation of Ci[act]. phosphorylation by PKA, or dephosphorylation of Ci, may be The problem of how full-length Ci protein is converted into involved in activation. Indeed, Chen C.-H. et al. (1999) provide Ci[act] is more challenging. Fu has been implicated in this pharmacological evidence for specific phosphatases that process (Ohlmeyer and Kalderon, 1998; this work), but as in modulate the phosphorylation state of Ci. the case of Su(fu), Fu kinase activity is partially dispensable in wild-type discs and entirely dispensable in animals lacking Su(fu) (Préat, 1992; Préat et al., 1993). This suggests that the Hh Fu kinase functions only to prevent Su(fu) from negatively acting on the Hh pathway. If we accept that Su(fu) acts subsequent to Ci[act] Fu (kinase) formation, we must conclude that the same is Cos2 true for the Fu kinase. In short, we propose that PKA Fu (C-term) Su(fu) Fig. 7. Model in which maximal activation of Hh target genes occurs in a two-step process. Complex formation with Fu, Cos2 and microtubules serves to tether Ci to the cytoplasm and to locate Ci to the site Ci Ci[act] Ci[act] induction of of Slimb-dependent proteolytic processing. Hh complexed wg, dpp, ptc stimulates the release of this complex from microtubules, leading to the formation of Ci[act]. Slimb The latter can still be partially inhibited through the action of Su(fu), either by suppressing nuclear repression of import (as indicated in the diagram) or by enhancing Ci[rep] Ci[rep] hh, dpp nuclear export (not indicated). Hh stimulation of Fu kinase counteracts this effect and allows Ci[act] to accumulate in the nucleus. Nuclear Ci is rapidly exported, both in Hh receiving cells as well as in non-Hh receiving cells (see also Chen C.-H. et al., 1999). Fu stimulates the activator form of Ci 4009

The role of complex formation Bhanot, P., Brink, M., Harryman Samos, C., Hsieh, J.-C., Wang, Y., We have shown that PKA and Cos2 prevent Ci[act] formation Macke, J. P., Andrew, D., Nathans, J. and Nusse, R. (1996). A new member of the frizzled family from Drosophila functions as a Wingless and that the same components are required for Ci[rep] receptor. Nature 382, 225-230. formation. This observation closely links the two events. Cos2, Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means Fu and Ci are found in a large cytoplasmic complex that is of altering cell fates and generating dominant phenotypes. Development 118, associated with microtubules (Sisson et al., 1997; Robbins et 401-415. Calleja, M., Moreno, E., Pelaz, S. and Morata, G. (1996). Visualization of al., 1997). Fu derived from type II alleles, lacking the C- gene expression in living adult Drosophila. Science 274, 252-255. terminal portion, fails to locate to this complex (Robbins et al., Chen, C.-H., von Kessler, D., Park, W., Wang, B., Ma, Y. and Beachy, P. 1997). Indeed, Ci[rep] is not generated in cells expressing only A. (1999). Nuclear trafficking of Cubitus interruptus in the transcriptional type II-mutant Fu protein. In addition, exposure to Hh releases regulation of Hedgehog target gene expression. Cell 98, 305-316. Cos2 from microtubules (Robbins et al., 1997). This links Chen, Y., Gallaher, N., Goodman, R. H. and Smolik, S. M. (1998). Protein kinase A directly regulates the activity and proteolysis of Cubitus Ci[rep] formation to complex formation. Together, these interruptus. Proc. Natl. Acad. Sci. USA 95, 2349-2354. ﬁndings lead us to surmise that complex formation fulﬁlls two Chen, Y. and Struhl G. (1996). Dual roles for Patched in sequestering and roles: one is to tether Ci to microtubules, thereby preventing transducing Hedgehog. Cell 87, 553-563. nuclear entry. The other is to localize Ci to the site of Chen, Y., Cardinaux, J.-R., Goodman, R. H. and Smolik, S. M. (1999). proteolytic processing for the formation of Ci[rep]. Hh Mutants of cubitus interruptus that are independent of PKA regulation are independent of hedgehog signaling. Development 126, 3607-3616. signaling would promote the formation of Ci[act] by releasing Dominguez, M., Brunner, M., Hafen, E. and Basler, K. (1996). Sending and this complex (or Ci) from microtubules, and as a consequence receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus would prevent the cleavage of Ci. Upon release, Ci[act] would interruptus. Science 272, 1621-1625. be subjected to Su(fu) action, possibly by cytoplasmic Forbes A. J., Nakano Y., Taylor A. M. and Ingham P. W. (1993). Genetic analysis of hedgehog signaling in the Drosophila embryo. Development1993 tethering. Stimulation of Fu kinase activity by Hh inhibits Supplement, 115-124. 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