826 Research Article

IntroductionHedgehog (Hh) is a secreted protein that patterns and specifiescell fate in several different tissues. In Drosophila, response toHh is mediated by Cubitus interruptus (Ci), a transcriptionfactor with either activator or repressor functions (Dominguezet al., 1996; Aza-Blanc et al., 1997; Methot and Basler, 1999).The processing of Ci into activator or repressor is achievedthrough the Hh cytoplasmic complex that includes the kinesin-like protein, Costal-2 (Cos2), the serine-threonine kinase Fused(Fu), and Suppressor of Fused [Su(fu)] (Robbins et al., 1997;Sisson et al., 1997; Stegman et al., 2000). Secreted Hh bindsto its receptor Patched (Ptc). As a result, Fu and Cos2 arehyperphosphorylated (Therond et al., 1996; Robbins et al.,1997; Nybakken et al., 2002), the cytoplasmic complexdissociates from microtubules, and full-length Ci, Ci155,translocates into the nucleus as a transcriptional activator(Ohlmeyer and Kalderon, 1998; Chen et al., 1999; Wang andHolmgren, 1999). In the absence of Hh, Ci is proteolyzed toits repressor form, Ci75. This proteolysis involvesphosphorylation of Ci by PKA, GSK3 and CKI and the activityof the F-box protein, Slimb (Aza-Blanc et al., 1997; Robbinset al., 1997; Jiang and Struhl, 1998; Chen et al., 1999; Methotand Basler, 2000).

The fate of Ci is controlled by Ptc, a twelve-passtransmembrane protein, and Smoothened (Smo), a seven-passtransmembrane protein. In the absence of Hh, Ptc suppressesSmo and this triggers the proteolysis of Ci. When present, Hh

relieves the Ptc-mediated suppression of Smo, leading tophosphorylation and stabilization of Smo, activation of Ci, anddegradation of Ptc and the Hh ligand (Aza-Blanc et al., 1997;Denef et al., 2000; Alcedo et al., 2000; Zhang et al., 2004).The Smo C-terminal tail has been shown to directly bind toCos2 (Ogden et al., 2003), and upon Hh inactivation of Ptc,activates signaling through Cos2 and the associated Hhcytoplasmic components (Hooper, 2003; Lum et al., 2003; Jiaet al., 2003; Ogden et al., 2003).

Sex lethal (Sxl) functions as the master switch in sexdetermination, controlling somatic sexual development anddifferentiation in Drosophila. It is activated in females but isinactive in males. These two modes of expression aremaintained throughout the life cycle (Sanchez and Nöthiger,1983; Cline, 1984). Sxl promotes female differentiation byregulating transformer (Boggs et al., 1987; McKeown et al.,1987) and the dosage compensation process (reviewed byLucchesi et al., 2005).

Previously, we showed that Sxl enhances the Hh signal andproposed that this was the mechanism by which Sxl generatesthe larger female body size (Horabin, 2005). In the wing discanterior compartment, Sxl responds to the presence of Hh in aPtc-dependent manner but Smo activity is not required (Horabinet al., 2003). Here we show that Sxl is part of the cytoplasmicHh complex that is tethered to the Smo carboxyl tail. Weexamined whether Ptc is also a member of the Hh signalingcomplex, and found that Ptc and Smo can be co-

Hedgehog acts as an organizer during development. Itssignaling involves the receptor Patched, signal transducerSmoothened and a cytoplasmic complex containing thetranscription factor Cubitus interruptus tethered to theSmoothened carboxyl tail. Without Hedgehog, Patchedrepresses Smoothened resulting in proteolysis of Cubitusinterruptus to its repressor form. With Hedgehog, Patchedrepression of Smoothened is relieved and Cubitusinterruptus is activated. Sex-lethal, the master switch forsex determination in Drosophila, has been shown toassociate with Cubitus interruptus and the cytoplasmiccomponents of the Hedgehog signaling pathway.Additionally, Sex-lethal responds to the presence ofHedgehog in a Patched-dependent manner. The latterprompted us to examine the role of Patched in signaling.We find that Cubitus interruptus, Sex-lethal, Patched and

Smoothened co-immunoprecipitate and co-fractionate,suggesting a large complex of both membrane andcytoplasmic components of the Hedgehog pathway. Theentire complex is present at the plasma membrane and theassociation of Patched changes depending on the activationstate of the pathway; it also is not female specific.Colocalization analyses suggest that Sex-lethal alters theendocytic cycling of the Hedgehog components and mayaugment the Hedgehog signal in females by decreasing theproteolytic cleavage of Cubitus interruptus, availing moreof it for activation.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/5/826/DC1

Key words: Hedgehog, Sex-lethal, Endocycling, Patched, Drosophila

Summary

A large complex containing Patched and Smoothenedinitiates Hedgehog signaling in DrosophilaSabrina L. Walthall1, Michelle Moses1 and Jamila I. Horabin2,*1Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA2Department of Biomedical Sciences, Florida State University, Tallahassee, FL 32306, USA*Author for correspondence (e-mail: jamila.horabin@med.fsu.edu)

Accepted 21 December 2006Journal of Cell Science 120, 826-837 Published by The Company of Biologists 2007doi:10.1242/jcs.03382

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immunoprecipitated from embryonic extracts. This associationcould be enhanced or disrupted, depending on the activation stateof Hh signaling. Immunoprecipitation and colocalization studiessuggest a large signaling complex at the plasma membrane thatincludes both membrane proteins and the cytoplasmiccomponents. This complex is endocytosed in a dynamin-dependent manner. Our data, together with that of others, suggestan altered model for passage of the Hh components through theendocytic pathway. Colocalization of Ptc, Smo, Ci and Sxl insalivary gland cells when signaling is on versus off suggests that,in females, Sxl may decrease the proteolytic cleavage of Ci byaltering its endocytic cycling with the Hh membranecomponents, thereby enhancing the Hh signal.

ResultsSxl associates with Smo in the Hh cytoplasmic complexAs Sxl co-immunoprecipitates with the cytoplasmic Hhcomponents Cos2, Fu and Ci, and studies show that Smophysically interacts with the Cos2-Fu complex via its C-terminal tail (Jia et al., 2003; Lum et al., 2003; Ogden et al.,2003; Ruel et al., 2003), we investigated whether Sxl wouldalso co-immunoprecipitate Smo. Wild-type embryonic extractsshow that Sxl immunoprecipitated Smo protein (~5.2%) andthe converse is also true; Smo immunoprecipitated Sxl (~2.7%;Fig. 1A). By comparison, full-length Ci (Ci-155)immunoprecipitated ~4.7% of Sxl and Smo; Cos2 ~5.1% ofSxl and ~3.5% of Smo. These immunoprecipitation (IP) values

are in the range of or are slightly moreefficient than those seen by others(Ogden et al., 2003) [see Fig. 5 of Jia etal. (Jia et al., 2003)] and indicate that Sxlis present in all the known Hhcytoplasmic complexes described to date.

Ptc is an integral member of the Hhsignaling complexAlthough Sxl resembles Ci in itsassociation with the Hh signalingcomponents, removing Smo activity hasno effect on Sxl nuclear entry in the wingdisc. It is Ptc which promotes nuclearentry of Sxl in a Hh-dependent manner(Horabin et al., 2003). This dependenceon Ptc suggested that an associationbetween Sxl and Ptc might exist. Toinvestigate this possibility, we determinedwhether Ptc was also present with the Hh

Fig. 1. A large complex involving Ptc, Smoand Sxl in Drosophila embryos. (A) Ci,Cos2, Ptc, Smo and Sxl immunoprecipitatesfrom wild-type 0- to 12-hour embryonicextracts probed for Sxl, Ptc and Smo. Inputlane (I) is 5% of extract used. The tablegives the percentage of proteinimmunoprecipitated from an average of twoor more experiments. (B) Fractionation ofcytoplasmic extract from wild-type embryosanalyzed by western blot using the antibodiesin A as well as anti-Fu. Arrows at top showthe elution position of the given size marker.Several of the Hh components arephosphorylated (asterisk) and migrate asdoublets (Cos2 and Fu); Smo is alsophosphorylated and does not migrate as adiscrete band. ‘F’ represents total extractfrom adult females used as a marker for eachprotein. Three arbitrary complex types ofchanging Hh components (Complex A-C)can be described. (C) Immunoprecipitationof fractions from each complex type withantibodies to known Hh components showthat Ptc and other Hh components, as well asSxl are associated. (D) Controls forimmunoprecipitates of Hh complexcomponents using antibodies to BicD, Dlgand Fz.

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signaling components in immunoprecipitates from wild-typeDrosophila embryo extracts. Sxl immunoprecipitated asignificant amount of Ptc (~10.7%, Fig. 1A) and inversely Ptcimmunoprecipitated ~3.1% of Sxl. As Sxl is in a complex withthe other Hh components, we determined whether Ptc waspresent in immunoprecipitates of full-length Ci, Smo and Cos2.We obtained IP values of ~6.8, ~2% and ~1.6%, respectively,for Ptc. Although the cause of the differences between theseproteins is not clear (there might be different complexes or thestability of each component within the complex may bedifferent), the fact that Sxl, Ci and Smo can co-immunoprecipitate Ptc suggests that Ptc is part of the Hhsignaling complex. As a negative control, we probedimmunoprecipitates with antibodies for proteins withsimilarities to those in the Hh signaling pathway (Fig. 1D).Bicuadal-D (Bic-D), a kinesin-like protein, Discs-large (Dlg), amember of the guanylate kinase family, Frizzled (Fz), the Wntreceptor a seven-pass transmembrane protein homologous toSmo, and evenskipped, an active repressor of transcription (datanot shown) were tested. The immunoprecipitates were positivefor the Hh component, but not for any of the controls.

To additionally show that Ptc is a part of the Hedgehogsignaling complex, we fractionated cytoplasmic extracts fromwild-type embryos (Fig. 1B). Three distinct populations can bedescribed. In population A we found that Sxl cofractionateswith Ptc, Cos2, and Ci (trace amounts of Fu were alsodetected). This population was relatively large (>700 kDa as

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noted by the size standards), suggesting the presence ofadditional proteins or from trimerization of Ptc (Lu et al.,2006). Population B, contained Ci in the first few fractions, andhad Ptc, Smo, Fu, Cos2 and Su(Fu) [Su(Fu) not shown] alongwith Sxl. Population B is also relatively large (centered around669 kDa) and suggests that the Hh signaling complex cancontain both Sxl and Ci, or only Sxl, as in population A. Inpopulation C, which had little Sxl, Ptc is still present alongwith the other Hh signaling components. These complexeswere smaller than 440 kDa, so they are unlikely to have severalof the Hh components together, and in the fractions of thesmallest size, probably exist as monomers.

Populations A, B and C were tested for co-IP of Sxl and Ptcwith Cos2, Ci, Sxl or Smo for which they were positive (Fig.1C), supporting the contention that Ptc and other Hh signalingmembers exist in a complex. Analysis by native gelelectrophoresis also supported this conclusion; at least twoextremely large complexes that contain the various Hhsignaling proteins, including Ptc and Smo, were detected (seesupplementary material Fig. S1A,B). These large complexesentered the stacking gel but failed to enter the resolving gel.The negative controls Eve (see supplementary material Fig.S1B) and Dlg (not shown) were in the resolving gel alone. Fzwas detected in the stacking as well as the resolving gel (notshown) but as demonstrated above it did not co-immunoprecipitate with the Hh components, suggesting that itexists in a large complex of its own.

Pathway activation changes associationof Ptc with Hh complex componentsWe next examined whether the activation stateof the pathway affected the association of Ptcwith the other Hh components. The pathwaywas activated using the Ptc1130X variant, whichhas the last 156 amino acids of the cytoplasmictail deleted. Ptc1130X is a dominant-negativeallele and leads to Ci activation independentlyof the Hh morphogen, inducing targets whichrequire high Hh levels for activation (Johnsonet al., 2000). Embryos expressing this and thevariants that follow were generated using theUAS-GAL4 system; homozygotes of theubiquitous daughterless-GAL4 (da-GAL4)driver were crossed to a line homozygous forthe UAS-Ptc construct. Western blots showedthat the prevailing Ptc is the variant form, whichfor Ptc1130X migrated a little faster than theendogenous protein.

Immunoprecipitates of Sxl, Ci, Cos2 andSmo were probed with antibodies for Sxl, Ptc

Fig. 2. Ptc association within the Hh complexresponds to activation state of the pathway. Ci,Cos2, Smo and Sxl immunoprecipitated fromembryos expressing a different Ptc variantfollowed by western blot analysis for Sxl, Ptc andSmo. Efficiency of IP is relative to the 5% extractin the input lane (I). (A) Embryos expressingPtc1130X. (B) Embryos expressing PtcD584N.(C) Embryos overexpressing wild-type Ptc.(D) Embryos expressing Ptc�Loop2.

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and Smo and each IP yielded ~5-6% of the tested proteins (Fig.2A). The exception was Smo which immunoprecipitated ~9%of Sxl. Compared with wild-type embryos, the amount of Ptcimmunoprecipitated increased regardless of the IP protein, andthe amount of Sxl in complexes that contain Smo alsoincreased from ~3% to ~9%. These data demonstrated that theactivity state of the pathway alters the association of Ptc withthe other Hh components. Removal of the last 156 amino acidsappears to not only hinder the ability of Ptc to inhibit Smo, italso ‘locked’ Ptc into the complex.

Intermediate activation of the pathway had a different effect.PtcD584N has an aspartic acid changed to asparagine at position584 in the sterol sensing domain (SSD), is also a dominantnegative and activates the pathway, although not as strongly asPtc1130X (Johnson et al., 2002). The amount of Ptcimmunoprecipitated (~1%) by Sxl and Ci appeared to decreasecompared with the wild type (Fig. 2B), Smo alsoimmunoprecipitated slightly less Ptc whereas Cos2immunoprecipitated ~4% of Ptc, elevated from the wild type(~2%). The amounts of Sxl and Smo that were associated withCi did not change substantially and remained close to ~5%.

These data suggest that complexes that contain full-lengthCi with Sxl or Smo remain unaltered when the pathway isactivated or ‘on’. However, Smo was able to more effectivelyimmunoprecipitate Sxl than in the wild-type condition (from~3% to ~5%), suggesting that the association of Sxl with Smowithin the complex may be enhanced while the association ofPtc is disrupted.

Although Ptc1130X and PtcD584N were overexpressed, eachdisplayed different interactions with the other components,suggesting that overexpression per se does not determineassociation. Both activate the Hh pathway; however, with respectto protein turnover, PtcD584N more closely resembles wild-typeprotein bound to ligand (Martin et al., 2001; Strutt et al., 2001;Lu et al., 2006). This suggests that the association of Ptc withinthe complex is disrupted when the pathway is turned on.

Association of Ptc with the Hh complex is enhancedwhen the pathway is offFor the ‘off’ state, wild-type Ptc or Ptc�Loop2 was expressed inembryos. Overexpression of wild-type Ptc switches the systemoff as there is not sufficient endogenous Hh. Ptc�Loop2 alsosuppresses pathway activation; it is unable to bind Hh as itlacks the extracellular loop between transmembrane segmentsseven and eight necessary for Hh binding. Embryosoverexpressing wild-type Ptc showed full-length Ci, Cos2,Smo as well as Sxl co-immunoprecipitated with significantamounts of Ptc (10% or greater, Fig. 2C). The Hh componentsalso immunoprecipitated significant amounts of Sxl (~8% orgreater), suggesting that Sxl remains a part of the complexwhen the pathway is off. Association of Ptc with Smo and thecytoplasmic Hh components appeared to be enhanced when thepathway is off. Compared with the wild type, all thecomponents tested showed substantial increases in the amountsof Ptc that co-immunoprecipitated with them. Similar resultswere obtained when the pathway was off through Ptc�Loop2

expression (Fig. 2D).The data taken together, suggest a large complex that

consists of Ptc, Smo and the cytoplasmic Hh components (andSxl in females). The similarity in the IP profile of wild-typeembryos to that of embryos expressing PtcD584N rather than

either variant that turns the system off, also suggests that wild-type embryos reflect primarily an activated state.

Presence of Ptc in the Hh complex is not dependent onSxlAs embryonic extracts do not distinguish between males andfemales, it could be argued that Sxl accounts for the IP of Ptcwith the other Hh components. Wild-type male and female fliesshowed that females do indeed give a greater yield of Ptc intheir Ci and Cos2 immunoprecipitates (Fig. 3G), however,males also showed Ptc, indicating that its presence is not sexspecific.

If the Hh cytoplasmic complex directly interacts with Ptc, itshould be able to do so in the absence of Smo. To demonstratethat Ci can associate with the plasma membrane when Ptc isthe lone Hh membrane component, we removed Smo in malesalivary gland cells using the SmoD16 deletion allele. As thelevels of Ci are greatly reduced under these conditions, amutation in PKA was introduced to increase the levels of Ci.Clones of cells mutant for SmoD16 and PKA showed that 15%of the Ptc and 14% of the Ci colocalize at the plasmamembrane and the vesicular network (Fig. 3A-F). Althoughthis was a little lower than we observed in the wild-typecondition (see below) and might suggest a more stable complexexists when both Hh membrane proteins are present, thecolocalization is consistent with the idea that the Hhcytoplasmic complex is associated with Ptc.

Overexpression of Ptc and its variants titrates Sxl out ofthe nucleusAs Sxl depends on Ptc to respond to Hh in wing discs, we

Fig. 3. Association of Ptc with the Hh complex is independent ofSxl. (A-F) Male salivary gland cells with the SmoD16 deletion allele(and PKAH2). Ci and Ptc colocalize at the plasma membrane andvesicular network (C and F colocalized pixels only). (G) Adult maleand female extracts treated with anti-Ci and Cos2 show that Cos2immunoprecipitates ~16% of the Ptc in males, ~22% in females; Ciimmunoprecipitates ~1% of the Ptc in males and ~16% in females.

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reasoned that Sxl might associate directly with Ptc. Thisprompted us to determine whether overexpression of Ptc wouldaffect the localization of Sxl (Fig. 4A-F), because, unlike mostof the other Hh components that reside in the cytoplasm, Sxlin embryos is primarily nuclear (Fig. 4A’).

Overexpression of Ptc1130X, PtcC (deletion of the N-terminusup to the seventh transmembrane region) and Ptc13 titrated Sxlout of the nucleus (Fig. 4D’,E’; Ptc13 not shown). Wild-typePtc and Ptc�Loop2 gave intermediate effects: Sxl was detectedin both cytoplasm and nucleus (Fig. 4B’,F’), whereas PtcD584N

did not significantly alter the nuclear localization of Sxl (Fig.4C’). Both dominant negatives, Ptc1130X and PtcD584N, result inthe activation of Ci; however, only Ptc1130X sequesters Sxl inembryos and facilitates its nuclear import in wing discs (ourunpublished results), correlating these two properties of Ptc.

The two Ptc dominant negatives also suggest that pathwayactivation is not responsible for the observed change in Sxllocalization. To confirm this, the state of Hh signaling wasdirectly assayed by staining embryos for full-length Ci andWingless (Wg; Fig. 4G-L). As predicted, the levels of Ci

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increase when Ptc1130X, PtcD584N or Smo isexpressed, and decrease when Ptc+ or Ptc�Loop2 isexpressed. The Wg signal was also altered in thesebackgrounds, reflecting its dependence on Hhsignaling. That transcriptional activation by Hhwas not involved in changing the subcellularlocalization of Sxl in embryos, is also supportedby the observation that overexpression of Hh itselfhad little effect, as did the overexpression of Smo(data not shown). Since Ptc variants that bothactivate or inhibit Hh signaling can sequester Sxl,and the sequestration does not show segmentalrepeats, it is more likely that the effect is causeddirectly by the overexpression of the Ptc proteins.

As PtcC can titrate Sxl out of the nucleus, thecarboxyl half of Ptc must provide the docking site. The 156 C-terminal residues do not appear to be involved, however, astheir removal in Ptc1130X did not severely compromisesequestering Sxl out of the nucleus. Between PtcC andPtc1130X, the cytoplasmic regions remaining are the loopbetween transmembrane segments 10 and 11 and the last ~27amino acids before the 1130X deletion point.

Ptc and Smo colocalize with Hh componentsDepending on the signaling condition, Ptc and Smo appearedto be in the same complex. To determine whether theycolocalize in vivo, we examined their distribution with full-length Ci in the large salivary gland cells (Fig. 5A-L); Ci servesas the marker of the cytoplasmic Hh complex. The state of Hhsignaling in wild-type salivary glands is normally off (Zhu etal., 2003). To activate it, Hh was expressed using a salivarygland GAL4 driver, sgs3 (Fig. 5M-X).

Optical sections were taken near the plasma membrane aswell as deeper within the cell to include a cross section of thenucleus. Sections near the plasma membrane sample vesicles

Fig. 4. Ptc can alter Sxl subcellular location.(A-F) Embryos expressing different Ptc variants stainedfor Sxl (green, A-F), and propidium iodide (PI; red)merged with Sxl (A’-F’). (A’) Sxl is primarily nuclearin wild-type (wt) embryos. Ptc+ (B’) and Ptc�Loop2 (F’)do not completely titrate Sxl out of the nucleus giving adiffuse image; the PI signal is yellow to orange.(C’) PtcD584N resembles the wild type and Sxl is moredistinctly nuclear. (D’,E’) Ptc1130X and the carboxylhalf of Ptc, PtcC, strongly titrate Sxl out of the nucleus;the PI signal is more red. (G-L) Effects of Ptc variantson Hh signaling in embryos reported by full-length Ci(red) and Wg (blue) expression. Relative to wt (G), Cilevels are increased by the expression of PtcD584N (I)and PtcC (K), decreased by Ptc+ (H) and Ptc�Loop2 (L).The increase caused by Ptc1130X (J) is very modest. Wgexpression is depressed by Ptc+ and Ptc�Loop2 (H’ andL’; arrowheads mark disrupted Wg stripe), elevated byPtcC (K’) and modestly elevated by PtcD584N (I’). Thefeedback between Ci and Wg (Lessing and Nusse,1998) appears most affected by Ptc1130X and Wg levelsare not strongly elevated (J’). This is more evident asthe embryos get older and the Wg levels drop. Embryosscanned at similar settings with a 40� objective. Bars,20 �m.

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more recently budded from the plasma membrane, whereasthose deeper in the cell sample later stages of the endocyticcycle. Colocalization of the two Hh membrane proteins withCi was compared between males and females, in the presenceand absence of Hh (Fig. 5, Fig. 7A1).

Several observations stand out. First, for both Ptc and Smothe plasma membrane as well as the underlying vesicularnetwork, appeared as sites of colocalization with Ci. Second,in the absence of Hh the degree of colocalization of the twoHh membrane proteins with Ci was generally a little lower infemales than males, both near the plasma membrane anddeeper within the cell. Third, in the presence of Hh, thesetrends were quite dramatically altered. Females showed moreCi colocalizing with Ptc at the plasma membrane than males,presumably reflecting a heightened response to signaling (Ptcis the receptor). They also showed a more dramatic decrease

of Ci colocalized with Ptc deeper within the cellsthan males. This enhanced separation of Ci from Ptcin females was mirrored by an increased level ofcolocalization of Ci with Smo within the cell.Combined, this suggests that in the presence of Hhfemale cells might recruit more Ci to receive thesignal at the plasma membrane, but as Ptc trafficstowards degradation deeper within the cells, femalessegregate more of their Ci away from Ptc. Changeswere also seen in the colocalization of Sxl with Ptc,Smo and Ci in the presence of Hh (Fig. 7A2); moreof the Sxl and Ptc signal overlapped at the plasmamembrane, and much of the Sxl appeared todissociate from these Hh components within the cell.

Although this colocalization approach is limited –the images are not a perfect sampling of specificsubcellular compartments – it did show Cicolocalization with Ptc, besides the expectedcolocalization of Ci with Smo. Additionally, thedifferences between the sexes are reliable as they aregenerated with similarly treated samples.

Blocking the first step in endocytosis increasescolocalization of Hh componentsThe wild-type data support the idea that Ptc, Smo andCi are together at the plasma membrane. SinceDynamin [Shibire (Shi)] is required for the pinchingof clathrin-coated pits from the plasma membrane(van der Bliek and Meyerowitz, 1991), reducing Shiactivity should inhibit the first step of endocytosis and‘freeze’ the Hh components there. Consistent with thisprediction, expressing the ShiK44A dominant negativeessentially doubled the colocalization of Ci with Ptcin sections near the plasma membrane in both sexes[from 20% to 41% in males, 15% to 33% in females(Fig. 6G-L, Fig. 7B1)]. Ci and Ptc colocalization isalso increased in sections deeper within the cell. Thecolocalization of Ci and Smo near the plasmamembrane did not change as dramatically, and wasaffected even less within the cell (Fig. 6A-F, Fig.7B1,C1). Sxl showed the same trends as Ci in itschanges in colocalization with Ptc and Smo,particularly at the plasma membrane (Fig. 7B1,B2).

Besides affecting the proteins at the plasmamembrane, inhibiting the first step of endocytosis

decreased the sexual dimorphism in the Ci and Ptccolocalization, and eliminated it for Ci and Smo. This suggeststhat the sexual difference is primarily a function of endocyticcycling, occurring after the events at the plasma membrane.

Sxl dissociates from Ptc early in endocytosisBlocking endocytosis at the first step appears to increase thecolocalization of Ci, and Sxl with Smo and Ptc. To confirm this,IPs using extracts from embryos expressing the dominantnegative ShiK44A were performed. Relative to the wild type, thedominant negative Shi increased the amount of Ptc complexedwith some of the Hh components. Ci and Sxlimmunoprecipitated ~8.4% and ~8.7% of the Ptc, which issimilar to the wild type. However, Smo and Cos2immunoprecipitated ~4.9 and 4% of the Ptc, an increase from~2% for both of them (Fig. 8B). The amount of Sxl

Fig. 5. Colocalization of Ptc and Smo, with Ci and Sxl. Wild-type female andmale salivary gland cells stained for Ci and Ptc or Ci and Smo (and Sxl infemales) in the presence and absence of Hh. Female glands in the absence ofHh stained for Sxl, Smo and Ci (A-C) or Sxl, Ptc and Ci (G-I). Male glands inthe absence of Hh signal stained for Smo and Ci (D,E) or Ptc and Ci (J,K).Female glands expressing Hh stained for Sxl, Smo and Ci (M-O) or Sxl, Ptcand Ci (S-U). Male glands expressing Hh stained for Smo and Ci (P,Q) or Ptcand Ci (V,W). ‘Col.’ panels (A’-C’,F,G’-I’,L,M’-O’,R,S’-U’,X) show only thepixels that are common between two proteins. Note the extensive colocalizationclose to the plasma membrane in most panels.

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immunoprecipitated by Smo and Ci was also elevated.This is in keeping with the idea that blocking the firststep of endocytosis traps Ptc and the entire Hhsignaling complex at the plasma membrane and thatPtc segregates from Smo and Cos2 as it progressesthrough the endocytic pathway.

From clathrin-coated pits, budding vesicles fusewith endosomes in a process that is mediated by thesmall Rab5 GTPase. Inhibiting the endocytic pathwayat this stage using a dominant negative Rab5S43N

variant (Stenmark et al., 1994; Entchev et al., 2000),showed the amount of Ptc immunoprecipitated by Sxlis considerably less (~3.9%, Fig. 8B) than for the Shidominant negative. The amount of Ptc in the Smo andCos2 IP appeared to elevate a little (from ~4 and 4.9%to ~7.2 and 7.3%, respectively), while Ci levelsappeared to drop (from ~8.4 to 5.2%). The co-IP ofSxl with Ci and Smo also decreased relative to the Shidominant negative condition. This suggests that theassociation of both Sxl and Ci with Ptc is weakened,while Ptc continues to progress with Smo and Cos2through the endocytic pathway. Sxl also appeared toreduce its association with Smo and Ci but itsassociation with Cos2 was not significantly altered.

Rab7 is a GTPase essential for transportingendocytic cargo from the early to late endosomes andlysosomes (Vitelli et al., 1997). Embryos expressing adominant gain-of-function Rab7 (Rab7Q67L) (Entchevet al., 2000) also showed lower Sxl and Ptc association(~2.9%, similar to the Rab5 dominant negative anddown from ~8.7% in the Shi dominant negative).However, the degree of association between Ptc withCi, Smo or Cos2 was not substantially different fromthe Shi dominant negative condition (Fig. 8B). TheRab7 gain of function increased the Ci and Ptcassociation compared with the Rab5 dominantnegative, as well as the interaction of Sxl with Smoand Ci (Fig. 8A). Consistent with this observedinhibition in sorting of Hh components,overexpression of wild-type Rab7, which increasesoverall Rab7 activity, also impairs the motility ofendosomes (Lebrand et al., 2002).

In summary, these data suggest that Sxl and Cibegin to segregate from Ptc relatively early, prior tothe stage requiring Rab5. Sxl also decreases itsassociation with Ci and Smo. The colocalization analyses (Fig.7A2) also suggest that within the cell, Sxl has low levels ofcolocalization with Ptc, Smo and Ci when signaling is on: thestate embryos more strongly reflect. Overactive Rab7 reversesthe segregation of Sxl from Ci and Smo, but not from Ptcsuggesting that Sxl and Ptc separate earlier in endocytosis. Ciappears to separate from Ptc a little later than Sxl, whereassegregation of Smo from Ptc must occur beyond the stageregulated by Rab5. Overactive Rab7 prevents Smo and Ptcsegregation, however, as they have opposite endocytic fatesthey would have to separate prior to sorting to the lysosome(Denef et al., 2000; Incardona et al., 2002; Zhu et al., 2003).

Detecting segregation of the Hh components and Sxlduring endocytosisThe sexually dimorphic colocalization of Ci with Ptc and Smo

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presumably reflects the effects of Sxl. Sxl appears to dissociatefrom some of the Hh components relatively early in theendocytic cycle. We therefore compared the colocalization ofPtc and Smo with Ci (and Sxl in females) in males and femalesexpressing a Rab5 dominant negative and a Rab7 gain-of-function variant.

Inhibiting the endocytic cycle at the Rab5 stage showed adrop in the level of Ci and Ptc that colocalized at the plasmamembrane in both sexes compared with the Shi dominantnegative (41% to 29% males, 33% to 22% in females; Fig. 6R’,Fig. 7B1). These values are still higher than the wild type, andsince the Shi dominant negative presumably reflects the stateof the components as they begin endocytosis at the plasmamembrane, suggest that sorting of the proteins is still inprogress. Colocalization of Ci with Smo showed a smallerdecrease relative to Shi, in both sexes (33% to 27% in males,

Fig. 6. Colocalization analyses of Hh components when endocytosis is blocked.Salivary gland cells expressing the dominant negative form of Shi, ShiK44A, orRab5, Rab5SN stained for Ci and Ptc or Ci and Smo (and Sxl in females).Female glands expressing ShiK44A stained for Sxl, Smo and Ci (A-C) or Sxl, Ptcand Ci (G-I). Male glands expressing ShiK44A stained for Smo and Ci (D,E) orPtc and Ci (J,K). ‘Col.’ panels (A’-C’,F,G’-I’,L) show only pixels common tothe indicated proteins. (M-O,S-U) Female glands expressing Rab5SN stained forSxl, Smo and Ci (M-O) or Sxl, Ptc and Ci (S-U). ‘Col.’ panels (M’-O’,P’-R’)show only pixels common to both proteins. All sections are from near theplasma membrane. Percentages of all colocalizations are given in Fig. 7.

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33% to 29% in females, Fig. 6O’, Fig. 7B2). Within the cells,these trends were upheld for Ptc, but not for Smo, whichremained almost unchanged. These data suggest that when thepathway is off (the ground state of salivary glands), Ptc andfull-length Ci segregate from each other by the Rab5 stage; butthe segregation of Ci and Smo does not change considerably.

Progression from early to late endosomes involvesdisplacement of Rab5 by Rab7 (Rink et al., 2005). Thedominant gain-of-function Rab7 (Rab7Q67L) (Entchev et al.,2000) might be predicted to accelerate Rab7 function andcompromise sorting of components in Rab5 positive vesicles.

We found that the colocalization values resembled the Shidominant negative and also the Rab5 dominant negativecondition (Fig. 7B1-2,C1-2, images not shown), but not thewild type suggesting a reversal in sorting events. In general,the changes in Smo and Ci colocalization were smaller thanfor Ptc with Ci. What stands out most clearly is that femalesshowed an elevation over males of Ci and Ptc colocalization atthe plasma membrane, and Ci and Smo colocalization withinthe cell. These differences indicate that the sorting effects onthe Hh proteins caused by the Rab7 gain of function do notoccur similarly in males and females.

Fig. 7. Colocalization percentages of Ci and Sxl with Ptc and Smo in both sexes. (A) Colocalization of full-length Ci with the two Hhmembrane components, Ptc and Smo (1), as well as Sxl (2), in wild-type salivary glands in the absence (–Hh) and presence of Hh (+Hh) at theplasma membrane (PM) and within the cell. Colocalization of the proteins at the plasma membrane (B) and within the cell (C), in salivaryglands of the wild type or animals expressing Shi, Rab5 or Rab7 mutant proteins. Males and females show differences in amounts. Plasmamembrane optical sections include region near the membrane. Sections taken within the cell include a cross section of the nucleus. Percentagesreflect the average of at least two, usually three separate optical sections.

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With respect to Sxl, altering endocytosis with the Rab5dominant negative and Rab7 gain-of-function variants,generally increased the amounts of Sxl that colocalized withall three Hh proteins relative to the wild type near the plasmamembrane. Sxl did show a drop in its colocalization levels withboth Ptc and Ci with the Rab5 dominant negative, suggestinga separation of Sxl from these two components around theRab5 stage. The Rab7 gain of function appeared to reverse thisdissociation. Within the cell, the colocalization of Sxl with Ptcwas enhanced by both Rab5 and Rab7 variants, particularly theRab7 gain-of-function variant; the association of Sxl with Smoand Ci was generally also slightly elevated.

These dynamics suggest a complex picture of componentsin different associations, depending on their stage within theendocytic cycle. They suggest that (1) in the wild-typecondition, the overall colocalization of Ptc and Smo with Ci islow, and perturbing the early stages of endocytosis tends toincrease their (and Sxl in females) colocalization levels; (2)females generally show lower protein colocalization levels thanmales and perturbing the early stages of endocytosis tends todecrease the difference; (3) males and females show variancesin their response to sorting perturbations, particularly by theRab7 gain-of-function variant, where females show greaterfluctuations; (4) changes in Ptc colocalization with Ci tend tobe greater than for Ci with Smo, with Smo showing greaterchanges near the plasma membrane than within the cell.

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DiscussionWe proposed that Sxl enhances the Hh signal to generate thelarger female body size (Horabin, 2005). Sxl is part of the Hhcytoplasmic signaling complex (Horabin et al., 2003) as wellas the complex attached to the Smo tail, suggesting that it isan integral signaling member. To elucidate how Sxl enhancesthe Hh signal, we attempted to quantify the associations of thevarious components under different signaling conditions. Wefound that Sxl influenced the interactions between thecomponents during endocytosis and, more importantly, that Ptcis also a member of the Hh signaling complex.

A large complex that contains both Hh membranecomponentsThe IP data showed Ptc with full-length Ci, Sxl and the otherHh signaling proteins in wild-type embryos; complexescontaining both Ptc and Smo suggest that this associationbecomes stronger when signaling is off. The complexcontaining Ptc is not sex specific, although female flies givegreater yields than males.

The idea that Drosophila Ptc and Smo are in a commoncomplex runs counter to prevailing thought. Experiments inmammalian cells suggested that Ptc might associate with Smo(Stone et al., 1996; Murone et al., 1999), but subsequentexperiments in S2 cells questioned whether theseobservations applied to the Drosophila proteins (Johnson etal., 2000). Ruel et al. (Ruel et al., 2003) also reported that Ptcdid not immunoprecipitate with Cos2 in cl-8 cells. This reportis the first using Drosophila embryonic extracts, and we alsofind a weak association of Ptc with both Cos2 and Smo.However, this result changes depending on the state of Hhsignaling.

The addition of Ptc to the Hh signaling complex was furthercorroborated by size fractionation experiments, which showPtc in large complexes that also contained other known Hhcomponents. It is not clear what proteins account for the verylarge complexes of population A in Fig. 1B, which contain Ptc,Cos2, Sxl, Ci and trace amounts of Fu. Ptc can trimerize (Luet al., 2006), and in vivo data suggest Ptc functions as a trimer(Casali and Struhl, 2004) requiring its co-receptors Ihog andBoi (Yao et al., 2006). Additionally, some of the other Hhcomponents besides Ptc may be present as multimers, or thecomplex may also contain unknown proteins.

Colocalization analyses also showed Ptc and Smo togetherwith Ci at the plasma membrane and in the network of theendocytic pathway. When endocytosis is blocked in embryosexpressing a dominant negative Shi variant, an enhancement inco-IP of Hh complex proteins with Ptc is observed, suggestinga large complex with all known signaling components at theplasma membrane. Images also show an increase in theamounts of Ptc and Smo that colocalize with Ci at the plasmamembrane, supporting this view.

Finally, the Ptc tail must play a key role in influencing theinteractions within the complex. Removal of the last 156 aminoacids not only hinders Ptc1130X from inhibiting Smo, it alsostabilizes most of the interactions within the complex. It alsoappears to affect the endocytic cycling and stability of Ptc. Asopposed to Ptc1130X being proteolyzed, which is the normal fatefor Ptc when Hh signaling is on, high levels of the dominantnegative protein are detected at the plasma membrane (Zhu etal., 2003; Lu et al., 2006).

Fig. 8. Ptc and Sxl are endocytosed with Hh signaling components.Ci, Cos2, Smo or Sxl IP of embryo (0- to 12-hour) extractsexpressing the dominant negative Shi (ShiK44A), Rab5 (Rab5SN) orgain-of-function Rab7 (Rab7QL) variant probed for Ptc and Sxl.Percentage of Sxl (A) and Ptc (B) immunoprecipitated for eachvariant compared with wild-type (wt) data of Fig. 1. The average andstandard error are the mean of two or more data sets. Error barsrepresent ±1 s.e. White bars, wild type; light gray bars, Shi;white/black dot bars, Rab5; dark gray bars, Rab7.

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Augmenting the Hh signalThe yields of Ptc with the Hh cytoplasmic components wasgreater in females than males, suggesting Sxl stabilizes Ptcwithin the complex. Sxl may bind directly to Ptc, asoverexpression of Ptc can titrate Sxl out of the nucleus.

How does Sxl enhance the Hh signal particularly as itstabilizes the association of Ptc, a negative component insignaling? We find that Sxl influences the segregation of theHh components during endocytosis. Sex-specific differences inthe colocalization of Ci with both Ptc and Smo are mostlyeliminated by blocking the first step of endocytosis (Fig. 7B1-2), suggesting that Sxl impacts signaling events at orimmediately after the plasma membrane. Additionally, sortingresponses when endocytosis is perturbed using a Rab5dominant negative or Rab7 gain-of-function variant, showeddifferences between males and females. Protein sorting muststill be in progress during these early steps in endocytosis asperturbing them increases the levels of colocalization of theproteins relative to the wild type (including Sxl).

When signaling is off, females show lower amounts of full-length Ci colocalizing with both Ptc and Smo. When signalingis on however, females show more Ci and Ptc colocalizing nearthe plasma membrane, and a concomitant increase in thecolocalization of Sxl with Ptc (Fig. 7A2). As Ptc is the Hhreceptor, this might reflect enhanced signaling or a delay inendocytosis, or both, relative to males. A delay at the plasmamembrane would not compromise signaling, but couldinfluence the interactions and subsequent segregation ofcomponents within the cell. The Hh complex is capable ofsignaling at peak levels when endocytosis is blocked by theloss of shi function (Han et al., 2004; Torroja et al., 2004). Thisindicates that although the Hh components are trapped at theplasma membrane, Hh alters the interactions within thecomplex and Smo is no longer inhibited by Ptc. In keeping withthe idea that Hh modifies interactions within the complex,alterations that affect Ptc activity also affect whether itimmunoprecipitates strongly or weakly with the othercomponents.

In the presence of Hh, females also show higher levels thanmales of Ci and Smo colocalization within cells, which should

favor Ci activation and signaling. Conversely, the associationof Ci with Ptc within cells is lower in females. The latter wouldimplicate a decrease in proteolysis of full-length Ci becausePtc is proteolyzed when Hh is present. This suggests that withboth membrane proteins, females favor production of full-length Ci more than males, sparing more of it from proteolysisas well as activating more. Consistent with this proposal, theamount of full-length Ci is higher in females and Hh signalingis enhanced (Horabin, 2005).

These effects during Hh signaling would appear to mostlyoccur near the plasma membrane, as the levels ofcolocalization of Sxl with the Hh components are relativelylow within the cell. Indeed, IP experiments suggested Sxlbegins to dissociate from the Hh cytoplasmic complexrelatively early, before the Rab5 stage (Fig. 8A). Theassociation of Sxl with Ptc appeared to be disrupted evenearlier, because unlike the cytoplasmic complex components,the Rab7 gain-of-function variant does not reverse itsdissociation from Ptc (Fig. 8B).

Model of Hh signaling and endocytosisObservations by others taken with the data described here,suggest a model for Hh signaling (Fig. 9). In the absence ofHh Ptc, Smo and the Hh cytoplasmic components areendocytosed. All the components, except Ptc, are degradedwhile Ci is proteolyzed to its repressor form. Most of the Ptcrecycles (cyloheximide slowly decreases Ptc levels in theabsence of Hh so low levels of Ptc are likely to also turn over)back to the plasma membrane (Denef et al., 2000; Incardonaet al., 2002; Zhu et al., 2003), permitting Ptc to repeat the cycleand regulate Smo levels in a ‘catalytic’ manner (Taipale et al.,2002).

In the presence of Hh the initial events are similar. Ptc isnow sorted for degradation while Smo is activated, and full-length Ci is generated. Membrane and microtubule associationof Cos2 and Fu decreases (Stegman et al., 2004) favoring theirearly release from Smo and vesicles. Activated Smo recyclesto the plasma membrane where it can activate more Ci.Activated Smo destabilizes Cos2 and Fu (Lum et al., 2003;Ruel et al., 2003), suggesting that once Ci is activated, Cos2

Fig. 9. Model of endocytosis and Hh signaling. Inthe absence of Hh Ptc, Smo and the cytoplasmiccomponents are endocytosed. All the componentsincluding some of the Ptc, are degraded; Ci isproteolyzed to its repressor form (Ci75). Most of thePtc recycles to the plasma membrane (broken arrow)permitting Ptc to repeat the cycle and regulate Smolevels in a ‘catalytic’ manner. In the presence of Hhthe initial events are similar. Ptc bound to Hh is nowsorted for degradation while Smo splits apart, isactivated and full-length Ci (Ci155) is generated.Membrane and microtubule association of Cos2 andFu decreases upon Hh signaling (Stegman et al.,2004) favoring their early release from Smo andvesicles. Activated Smo recycles to the plasmamembrane where it activates more Ci; Cos2 and Fuare destabilized (Lum et al., 2003; Ruel et al., 2003),suggesting that once Ci is activated, Cos2 and Fu aredegraded. Shi, Rab5 and Rab7 denote where thesecomponents function within the endocytic cycle.

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and Fu are degraded. The fate of Ci (and associated Hhcytoplasmic components) thus depends on whether Ptc or Smoreturns to the cell surface by progression through the endocyticcycle.

Ptc has been described as cycling to and from the membraneindependently of Hh (Strutt et al., 2001). Cells that are notexposed to Hh maintain Ptc at the cell surface and inunidentified internal stores, while Smo colocalizes with lateendosomes and lysosomal markers. Vertebrate cells transfectedwith the Hh membrane proteins show that in the presence ofShh (the vertebrate Hh homolog), Ptc and Smo segregate in thelate endosomal compartment. Smo recycles back to themembrane while Ptc is fated for lysosomal degradation(Incardona et al., 2002). Endocytosis has thus been proposedto segregate Ptc from Smo, relieving Smo of its repressionwhile inducing the degradation of the Hh ligand (reviewed byPiddini and Vincent, 2003; Torroja et al., 2005).

Hh signaling elevates Smo levels and induces itsphosphorylation at the plasma membrane, while lowering thelevels of Ptc (Denef et al., 2000; Zhu et al., 2003; Jia et al.,2004; Zhang et al., 2004). The oncogenic Smo proteins,which signal constitutively, do not cycle with Ptc and remainat the plasma membrane (Incardona et al., 2002; Zhu et al.,2003). These observations are accommodated by the abovemodel.

The homology of Ptc to bacterial transporter proteins takenwith the findings that small molecules such as cyclopamine caninhibit mammalian Smo, and Ptc acts catalytically in itsinhibition of Smo, led to the suggestion that a small moleculetransported into the cell by Ptc serves to inhibit Smo (Taipaleet al., 2002). This obviated the need for a close interaction.[Note that Casali and Struhl (Casali and Struhl, 2004) suggestthat in vivo, the degree of the ‘catalytic effect’ of Ptc on Smomay be smaller than proposed.] Our model does not precludePtc from inhibiting Smo through a small molecule. Indeed,their proposed proximity would enhance the efficacy, elevatingthe local concentration. Additionally, within small endocyticvesicles, Ptc would more rapidly affect Smo as it would requirefewer molecules to alter the concentration.

The association of Ptc with Smo need not be direct. Cellsincapable of giving a Hh response show very highcolocalization of the two human proteins, but the two proteinsdo not co-immunoprecipitate (Incardona et al., 2002). A directinteraction between the two membrane components may notexist or it may be unstable. Alternatively, the data of Incardonaet al. (Incardona et al., 2002) may suggest a requirement for(some of) the cytoplasmic components to bridge the two Hhmembrane proteins. Future analysis will determine which is thecase.

Materials and MethodsFly stocks and clone generationFlies were raised at 25°C; OreR was the wild-type control. Immunoprecipitationsand embryo analysis used the following strains: UAS-ptc1130X, UAS-ptcC, UAS-ptc+ (Johnson et al., 2000), UAS-ptcD584N (Johnson et al., 2002), UAS-ptc�Loop2(Page, 2002), UAS-shiDN (Moline et al., 1999), UAS-rab5S43N, UAS-rab7Q67L

(Entchev et al., 2000), UAS-hh (Azpiazu et al., 1996), UAS-ptc13 (Strutt et al.,2001). Transgenes were expressed using the UAS-Gal4 system (Brand andPerrimon, 1993). da-Gal4 (Wodarz et al., 1995), or the salivary gland driver sgs3-gal4 were used for expression. Description of genes can be found in FlyBase(http://www.flybase.org/).

To generate clones SmoD16, DCOH2, FRT40/CyO were mated to y, w, hs70-FLPflies and first instars heat shocked for 30 minutes. Salivary glands from third instarswere used for immunostaining.

Immunoprecipitation, western blots and immunofluorescenceExcept for the wild type which used 50 �l, immunoprecipitations used 75 �l of 0-12 hour embryo extracts. Western blots, immunoprecipitations and whole mountstains were done as described (Vied and Horabin, 2001) varying only the lysis andwash buffers: lysis buffer 100 mM Tris-HCl pH 8.0, 300 mM NaCl, 1% NP40, 2mM EGTA plus protease inhibitors; wash buffer 50 mM Tris-HCl pH 8.0, 300 mMNaCl, 0.5% NP40, 1 mM EGTA. Antibody dilutions were as described (Horabin etal., 2003) except for anti-Sxl (1:600; western blot), anti-Ptc (1:400; from P. Ingham,Johns Hopkins University, Baltimore, MD), anti-Smo (immunoprecipitation 1:3,from P. Beachy, University of Sheffield, Sheffield, UK; western blot 1:100, from J.Hooper, University of Colorado Health Science Center, Boulder, CO), anti-Cos2(immunoprecipitation 1:3, from P. Beachy; western blot 1:250, from D. Robbins,Dartmouth Medical School, Hanover, NH) and Fu rabbit polyclonal serum (1:100).The anti-Fu serum was made as described (Robbins et al., 1997). Quantification ofimmunoprecipitates used IP lab from Scanalytics, taking the average of two or moreexperiments, except for the Ptc variants where amounts are for samples shown.Whole mount stains used anti-Ci at 1:2.5, anti-Ptc 1:300, anti-Smo 1:100, anti-Sxl1:250 and anti-Wg at 1:10.

Image capture and analysisImages were obtained on a Leica TCS_NT or Zeiss LSM 510 Laser Scanningconfocal microscope. For colocalization analysis, salivary gland cell images werecaptured with a 63� objective lens and a zoom of 1-2X. Plasma membrane sectionscapture the region near the membrane; within cell sections included cross sectionsof the nucleus. Colocalization percentages used IP Lab colocalization software(Scanalytics). For each image, the threshold value to eliminate background first useda visual guide. The threshold exclusion value was then raised or lowered todetermine whether the percentage of signal pixels was significantly altered. The cutoff was set as the value that did not significantly alter the percentage of signal pixelsincluded when it was increased (indicating inclusion of significant signal), but didchange significantly if it was lowered by two or more increments (indicatinginclusion of noise as signal). For a given antibody most images had relatively similarthreshold values. The overlap percentage for two probes was generated by thesoftware. Two or more different images were averaged for each genotype andsection type.

ChromatographyFractionations were done as described (Gay et al., 1988).

We are grateful to D. Page, G. Struhl, I. Guerrero, J. Jiang, M.Gonzales-Gaitan, K. Ho and M. Scott, for fly stocks. Thanks also toR. Holmgren, J. Hooper, D. Robbins, P. Ingham for antibodies, R.Huijbregts and I. Chesnokov for help with the fractionationexperiment, and G. Marques and J. Engler for input on the manuscript.The anti-Smo and Cos2 antibodies developed by P. Beachy, anti-Wgdeveloped by S. M. Cohen, were from the Developmental StudiesHybridoma Bank maintained by the University of Iowa, Departmentof Biological Sciences, Iowa City, IA 52242. We also thank AlbertTousson from the UAB imaging facility. This work was supported bya grant from NIH to J.I.H. S.L.W. was supported by a fellowship fromthe Comprehensive Minority Faculty Student Development Programat UAB.

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