Received 1 August 2001; accepted 11 January 2002.

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Supplementary Information accompanies the paper on Nature’s website

(http://www.nature.com).

AcknowledgementsWe thank our colleagues in Cambridge and Toronto for comments on this work, andM. Sokolowski for providing support during its completion. M.L.S was funded by a LuisVelez Scholarship from the Venezuelan National Academy of Sciences at Clare College,Cambridge. M.B. is a Royal Society Research Professor. This work was supported by agrant from the Wellcome Trust.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to M.L.S.

(e-mail: msuster@credit.erin.utoronto.ca).

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Drosophila Crumbs is a positionalcue in photoreceptor adherensjunctions and rhabdomeresShayan Izaddoost*, Sang-Chul Nam*, Manzoor A. Bhat†,Hugo J. Bellen*‡§ & Kwang-Wook Choi*‡k

* Department of Molecular and Cellular Biology; ‡ Program in DevelopmentalBiology; § Department of Molecular and Human Genetics, Howard HughesMedical Institute; and kDepartment of Ophthalmology; Baylor College ofMedicine, One Baylor Plaza, Houston, Texas 77030, USA† Cardiovascular Research Institute, Department of Medicine, Department ofMolecular Cell and Developmental Biology, Mount Sinai School of Medicine,New York, New York 10029, USA.............................................................................................................................................................................

Drosophila Crumbs (Crb) is required for apical – basal polarityand is an apical determinant in embryonic epithelia1,2. Here, wedescribe properties of Crb that control the position and integrityof the photoreceptor adherens junction and photosensitiveorgan, or rhabdomere3. In contrast to normal photoreceptoradherens junctions and rhabdomeres, which span the depth ofthe retina, adherens junctions and rhabdomeres of Crb-deficientphotoreceptors initially accumulate at the top of the retina andfail to maintain their integrity as they stretch to the retinal floor.We show that Crb controls localization of the adherens junctionthrough its intracellular domain containing a putative bindingsite for a protein 4.1 superfamily protein (FERM)4,5. Althoughloss of Crb or overexpression of the FERM binding domaincauses mislocalization of adherens junctions, they do not resultin a significant loss of photoreceptor polarity. Mutations inCRB1, a human homologue of crb, are associated with photo-receptor degeneration in retinitis pigmentosa 12 (RP12) andLeber congenital amaurosis (LCA)6 – 10. The intracellular domainof CRB1 behaves similarly to its Drosophila counterpart whenoverexpressed in the fly eye. Our studies may provide clues formechanisms of photoreceptor degeneration in RP12 and LCA.

The phototransduction machinery of both vertebrate and Droso-phila photoreceptors is housed in a morphologically unique andconserved apical domain specialization called the rod/cone outersegment and rhabdomere, respectively. Both of these structures arisefrom the explosive growth of the photoreceptor apical domain,giving rise to stacks of membrane packed with the photopigmentrhodopsin11 – 14. The role of apical proteins in rhabdomere forma-tion is poorly understood.

To address this matter, we studied Crb function in the eye ofDrosophila melanogaster. The transmembrane protein Crb and itsinteracting partner, Discs lost (Dlt)15, co-localize to the apicaldomain of undifferentiated cells and differentiating photoreceptorsthroughout eye development (Fig. 1a–c). During the pupal stage,Crb, Dlt and adherens junctions (AJs) involute with the photo-receptor apical domain. AJs anchor photoreceptors to the surfaceand floor of the retina. Thus, the axes of the photoreceptor apicaldomains and AJs rotate 908 and are perpendicular to the surface ofthe retina (Fig. 1a–c).

In the third-instar and pupal stages before rhabdomere for-mation, Dlt and Crb are juxtaposed to AJs marked with anti-Arm(Fig. 1d, e). As the actin-rich rhabdomeres form, Crb and Dltlocalization is confined to the region referred to as the rhabdomerestalk between the rhabdomere and AJ (Fig. 1f–h). To examine thefunction of Crb in eye morphogenesis, we generated eye-specificcrb2 mosaic clones using the ey-Flp/FRT system16,17 and twoseparate alleles of crb, crbD88-3 (a hypomorph) and crb11A22

(null)1. Neither type of clone showed any detectable level of Crbprotein, although crb11A22 clones show a slightly more severe

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phenotype. Longitudinal sections of the crb2 clones show lack ofrhabdomere elongation (compare arrows to arrowheads, Fig. 2a).The rhabdomeres remain at the top of the retina, and appear thickerthan wild-type rhabdomeres. Thus, loss of Crb results in failure ofrhabdomere elongation.

To determine the initial defect causing short rhabdomeres, crb2

mid-pupal-stage eyes were dissected and stained for Crb and Arm. Alongitudinal three-dimensional reconstruction of photoreceptorAJs in a photoreceptor cluster is shown in Fig. 2b and c, with theapical domain of the photoreceptors pointing towards the centre ofthe cluster (modelled in Fig. 1a–c). Wild-type clusters containsmooth and well-defined AJs spanning the length of the retina fromthe surface to the floor (Fig. 2b). The crb2 cluster shows anaccumulation of Arm staining at the top of the cluster (brackets,

Fig. 2c), forming thick and highly irregular junctions. A top view ofwild-type ommatidia shows that AJs form a tight circle (arrowhead,Fig. 2d; see also Fig. 1e) surrounding the apical domain of thephotoreceptor cluster (asterisk, Fig. 2d). However, AJs in crb2

photoreceptors (arrow, Fig. 2d) stretch further basolaterally, leadingto the appearance of disorganized patches of Arm staining through-out the lateral membrane. The AJ defects appear before the

Third instarApical

Crb Dlt

AJ

Basal

Bas

al

e

Distal

Ap

ical

Proximal

Distal

Proximal

Rhabdomere

Rhabdomere stalkCrb Dit

d e f

g h

DltArm

DltCrbArm

PhlDltArm

f–h

37% p.d. 67% p.d.a b c

AJ

R

37%

Figure 1 Dlt and Crb are localized to the rhabdomere stalk. The colour of each protein is

marked at the bottom of all fluorescence images. a, Third-instar eye disc. Apical domains

of photoreceptors (green) face the retinal surface and are held together by the AJ (red).

b, 37% pupal development (p.d.). Apical domains involute and face each other, without

breaking their AJ. c, 67% p.d. The distal apical domain is anchored by AJs to the retinal

surface while the proximal apical domain is bound by AJs to the retinal floor.

d, Longitudinal view of a third-instar eye disc stained for Dlt and Arm. e, Tangential

section of a photoreceptor cluster at 37% p.d. stained for Dlt, Crb and Arm. The position of

the section is shown in b. White dots, photoreceptor (R) basolateral membrane.

f, g, Transmission electron micrographs of a tangential section of an adult photoreceptor

cluster (f), and the same at higher magnification (g). h, Confocal section of a 55% p.d. eye

stained for Dlt, Arm and phalloidin (Phl). g, h, Asterisk, photoreceptor; arrowhead,

rhabdomere; arrow, rhabdomere stalk; double arrow, AJ. Scale bar, 1 mm.

a

b c

d

Distal

Proximal

CrbArm

CrbArm

CrbArm

crb–

43% p.d.

37% p.d.

Figure 2 crb2 photoreceptors fail to extend rhabdomeres and accumulate Arm distally.

a, Longitudinal section of a crbD88-3 adult eye clone containing wild-type (w+, arrowhead)

and crb2 (w2, arrows) photoreceptors; bracket, relatively intact w2 retinal floor. Distal is

to the top. b–d, 43% p.d. (b, c) and 37% p.d. (d) eyes containing crb11A22 clones marked

by Crb and Arm. b, c, Longitudinal three-dimensional (3D) reconstruction of a wild-type

(b) and a crb2 photoreceptor (c) in the same eye. Arrowheads, AJ; brackets, distal apical

accumulation of Arm. d, Top view of a 3D reconstruction of a wild-type (left) and a crb2

(right) pupal photoreceptor. Black asterisk, apical Crb staining; arrowhead, AJ; arrow, Arm

staining in crb2 cluster.

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formation of the rhabdomere and localization of Crb and Dlt to therhabdomere stalk.

To understand why crb2 rhabdomeres do not extend, we exam-ined initial stages of rhabdomere formation and elongation. Cross-sections of the distal apical domain of crb2 photoreceptors depictrhabdomeres that are larger than the wild type (Fig. 3a, b; 95% of 80photoreceptors scored). However, the rhabdomeres are very small atthe proximal apical domain (Fig. 3a, d), even though cell mem-branes are present as marked by Dlt (Fig. 3a, c, e). Longitudinal

views of near-adult or adult stages show that individual crb2

photoreceptors extend down to different levels in the retina(Fig. 3f–h). AJs of crb2 photoreceptors are variable in size at thedistal portion of the retina and discontinuous and thin in theproximal retina (arrows, Fig. 3g, h). The AJs attaching the photo-receptors to the floor of the retina remain relatively intact (Fig. 3g,h). Therefore, crb2 photoreceptor rhabdomeres and AJs areenlarged basolaterally in the distal domain of the photoreceptorand fail to extend proximally (Fig. 3a).

Transmission electron microscopy (TEM) was used to directlyexamine the structure of the crb2 photoreceptor rhabdomeres andAJs in adult eyes. Because crb2 photoreceptors contain rhabdo-meres and AJs of variable length, tangential sections at the mid-retina level revealed both bulky rhabdomeres and small or norhabdomeres (Fig. 3a, j and k, respectively). In addition, crb2

photoreceptors contain AJs of various sizes (Fig. 3a, m–o), rangingfrom large AJs (Fig. 3m) to no AJs (Fig. 3o). Rhabdomeres and AJsof wild-type clusters at the same level of the same mosaic eye areuniform in size and are properly positioned (Fig. 3a, i, l). crb2

photoreceptors retain apicobasal cell polarity because their rhab-domeres localize to the apex of the apical domain and their AJs arepositioned laterally to the rhabdomeres. Enlarged rhabdomeresrarely overlap with AJs either by TEM (Fig. 3j–o) or by fluorescentimaging (Fig. 3g, h). Furthermore, although Dlt is not concentratedin the rhabdomere stalk, a significant amount remains apical,suggesting that the cell retains cell polarity. Therefore, Crb providesthe positional cues that allow for extension of AJs and rhabdomeresalong the growing proximal apical domain of the photoreceptor,independently of its role in determining apicobasal polarity.Phal

Arm

Distal Distal

Proximal Proximal

CrbPhal

CrbPhal

CrbDlt

CrbDlt

CrbPhal

PhalArm

90% p.d.WT crb– crb–

WT crb– crb–crb–

crb–crb+

AJ AJProximal

Distal

b,c

i–o

d,e

a b c

d e

f g h

i j k

l m n o

PhalArm

Figure 3 crb2 photoreceptor AJs and rhabdomeres are mispositioned without loss of

polarity. a, Diagram of crb+ (left) and crb2 (right) photoreceptors depicting the position of

tangential sections in this figure along the proximal–distal axis. Distal is to the top. crb2

photoreceptors accumulate rhabdomeres and AJs distally. b–e, Tangential section of

crb11A22 photoreceptors at 65% p.d. stained with either rhodamine–phalloidin (b and d)

or for Dlt (c and e) and Crb. b and c are the same section, showing expression of different

markers, and are distal portion of the same clone in d and e. b, Distal portion of crb2

(arrows) and crb+ (arrowhead) photoreceptors. c, Thick arrow, peripheral Dlt; thin arrow,

apical Dlt; arrowhead, wild-type Dlt localization. d, Small (arrows), missing (asterisk), and

wild-type (arrowhead) rhabdomeres. e, Asterisks, Dlt in proximal membrane. f–h, crbD88-

3 clone at 90% p.d. f and g are images from the same clone; h is a different clone. f, Crb

marks wild-type ommatidia left of the white outline. Arrows, enlarged rhabdomeres.

g, Arrows, discontinuous AJs; arrowheads, wild-type AJs; black arrows, retinal floor

attachment. h, crb2 clone, right of the white outline (Crb staining not shown). Thick arrow,

distal AJ; arrows, no proximal AJ. i–o, Transmission electron micrograph of wild-type

(i and l) and crb2 photoreceptors (j, k and m–o). All panels are sections from the same

eye at the mid-retinal level to show crb2 photoreceptors (see a). crb2 clones were located

and oriented to section crb2 photoreceptors. j–o, Arrowheads, rhabdomere; arrows, AJ.

j, Small arrows with asterisk mark photoreceptors with multiple rhabdomeres. k, Asterisk

marks an unidentified cell. Scale bars, 1 mm.

a b c

d

e

37% p.d.

WT UAS–crbMyc-intra UAS–CRB1intraDltArm

DltArmArm

JM PBM

DrosophilaHuman RP12 locusHuman chr. 9CeCrb 1CeCrb 2ConsensusIdentical

SP TM JM PBMCrbWT

CrbMyc-intra

CrbMyc-JM

CrbMyc-PBM

CrbMyc-∆PBM/∆JM

G8Y10E16

G8Y10E16

G8Y10E16

G8A10A16

G8A10A16

Myc

Figure 4 Overexpression of CrbMyc – intra and human CRB1intra mislocalizes AJs and Dlt,

leading to loss of cell polarity. a–c, Tangential section of 37% p.d. wild-type (WT, a) and

pGMR–GAL4/UAS–crbMyc – intra (b) eyes grown at 18 8C, and pGMR–GAL4/UAS–

CRB1intra (c) grown at 25 8C. Arrows, AJs (ectopic in b and c); arrowheads, Dlt

(mislocalized in b and c). Section location is shown in the schematic diagram. d, Alignment

of conserved amino acids in the intracellular domain of Crb and homologues from humans

and the nematode Caenorhabditis elegans (CeCrb 1 and 2). Identical amino acids

between the Drosophila and other homologues are denoted in red. Purple, residues

identical in three of five members; green, residues identical in all five members. JM,

juxtamembrane (red) region; PBM, PDZ binding motif (yellow). The JM contains R1-G8-

T9-Y10-E16, similar to the 12-amino-acid FERM binding site of glycophorin C (R1-G8-

T9-T10-E13), b-neurexins (R1-G8-S9-T10-E13) and syndecans (K1-G8-S9-T10-

E13)22,23. e, Scheme of different Crb proteins used in this study. SP, signal peptide; TM,

transmembrane domain; Myc, Myc-epitope tag. Mutant constructs are described in text.

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Because loss of Crb caused the mislocalization of AJs, over-expression of Crb throughout the cells might lead to sequestrationof AJs. Overexpression of full-length Crb (CrbWT)2 causes themislocalization of Arm, Dlt and Dlg, leading to loss of cell polarityin the third-instar eye disc, followed by pupal lethality, with escaperslacking eyes (data not shown). To find the region of Crb that mightsequester AJs, the Crb intracellular domain (CrbMyc – intra), which isidentical to wild-type Crb in its ability to rescue crb2 embryos, wasoverexpressed in the eye2. Overexpression of CrbMyc – intra in the eyecaused a slightly milder phenotype than CrbWT, as the adult eye ispresent but rough. Two phenotypes were observed in pupal eyes.First, 97% of the photoreceptors contained multiple ectopic AJcomplexes of variable length (n = 324; Fig. 4b). Second, Dlt proteinwas mislocalized to other areas in the membrane (Fig. 4b), insteadof its normal localization to the apical domain (Fig. 4a). Therefore,overexpression of CrbMyc – intra causes mislocalization of bothphotoreceptor AJs and Dlt throughout the cell membrane, resultingin loss of polarity.

Mutations in a human homologue of crb, CRB1, result inphotoreceptor degeneration in RP12 and LCA6 – 8. We and others(ref. 7, and M. Pinto and U. Tepass, personal communication) haveidentified sequences in the CRB1 locus homologous to the intra-cellular domain of Drosophila Crb (Fig. 4d) (these sequence datawere produced by the Sanger Centre and can be obtained at http://www.sanger.ac.uk and from GenBank). To determine whether theproperties of the Crb intracellular domain are conserved acrossspecies, we overexpressed the CRB1 intracellular domain(CRB1intra) in the eye. Overexpression of human CRB1intra resultedin a phenotype weaker but similar to CrbMyc – intra, suggesting that

the function of the Crb intracellular domain is conserved acrossspecies (Fig. 4c). The intracellular domain of Crb contains twoconserved regions: a juxtamembrane (JM) region similar to theband 4.1 binding site on glycophorin C, and a PDZ binding motif(PBM) at amino acids 34–37 at the carboxy terminus (Fig. 4d, e)4,15.To define the portion of the Crb protein responsible for AJpositioning, we used versions of CrbMyc – intra containing the JMregion but lacking the PDZ binding motif (UAS–crbMyc – JM), orcontaining mutations replacing the conserved Y10 and E16 of theJM region with alanine but retaining a wild-type PDZ binding motif(UAS–crbMyc – PBM) (Methods, Fig. 4d, e)4. As a control, we usedUAS–crbMyc –DPBM/DJM, in which all three mutations were present4.Flies overexpressing CrbMyc –DPBM/DJM in the eye had no significantphenotype (Fig. 5a, d, g), indicating that these two regions, JM andPBM, are responsible for the phenotypes associated with the over-expression of the Crb intracellular domain.

To determine which region of Crb was responsible for the ectopicAJ phenotype, we overexpressed CrbMyc – JM. This causes mislocali-zation of AJs similar to that observed with CrbMyc – intra (comparearrows in Figs 5b and 4b). CrbMyc – JM pupal eyes exhibit lengthenedor ectopic AJs in 70% of photoreceptors scored (n = 280; seeMethods). However, Dlt protein localization was unaltered: Dltlocalized properly to the centre of the cluster (Fig. 5b). Over-expression of CrbMyc – PBM, which carries a wild-type PBM butmutations in the JM region, did not result in ectopic AJs (arrow,Fig. 5c), despite mislocalization of Dlt (arrowhead, Fig. 5c). Some89% of photoreceptors contained punctate and single AJs (n = 624;arrow, Fig. 5c). A significant amount of Dlt remains at the apicalpole of the cell, presumably bound to the endogenous Crb (Fig. 5c).

UAS–crb∆PBM/∆JM UAS–crbJM UAS–crbPBM

∆PBM/∆JMUAS–crb UAS–crbJM UAS–crbPBM

UAS–crbPBMUAS–crbJM∆PBM/∆JMUAS–crb

DltArm

DltArm

DltArm

DltDE-cad

DltDE-cad

DltDE-cad

DE-cad DE-cad DE-cad

AJ

a b c

d e f

g h i

Distal

Proximal

j

Figure 5 The juxtamembrane region of Crb is suffcient to recruit AJs independently of Dlt

or polarity defects. In all panels, arrows indicate AJs and arrowheads mark Dlt localization.

Panels a–c are tangential sections of an eye at 37% p.d. labelled for Dlt and Arm.

a, pGMR–GAL4/UAS–crbMyc –DPBM/DJM. b, pGMR–GAL4/UAS–crbMyc – JM. c, pGMR–

GAL4/UAS–crbMyc – PBM. d– i, Three-dimensional reconstruction of longitudinal sections

of photoreceptors (37% p.d.). d, g, pGMR–GAL4/UAS–crbMyc –DPBM/DJM. e, h, pGMR–

GAL4/UAS–crbMyc – JM. f, i, pGMR–GAL4/UAS–crbMyc – PBM. j, Model of Crb action at the

growing proximal apical domain of the developing photoreceptor. Rhabdomeres, red;

rhabdomere stalk, green; AJs, blue. As the retina deepens proximally (top), it pulls (thick

blue arrow) the apical domain resulting in addition of membrane, AJ (blue) and

rhabdomere (red). Crb proteins (bottom; grey cylinders) restrict the rhabdomere apically

and force it to stretch proximally. Simultaneously, proximal Crb organizes AJ material

(blue circles) into a continuous AJ through its FERM binding site (red box in Crb) that binds

a putative FERM protein (pink boxes denoted by 4.1). The PBM of Crb (yellow box in Crb

cylinder) binds Dlt (grey fuzzy molecule), which homomultimerizes15 to localize Crb to the

rhabdomere stalk (green).

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To determine whether CrbMyc – JM overexpression affects AJs, andnot just Arm localization, we stained eyes overexpressing the threedifferent CrbMyc – intra proteins for Dlt and Drosophila E-cadherin(DE-cad). Eyes overexpressing CrbMyc –DPBM/DJM depict wild-typeDlt, localized to the apical domain (Fig. 5d), and DE-cad staining,marking the AJs, which smoothly span the apical domain (Fig. 5g).Overexpression of CrbMyc – JM results in mislocalization of DE-cadthroughout the cell membrane (arrows, Fig. 5h) while Dlt isretained at the most apical part of the photoreceptor cells (arrow-head, Fig. 5e). The retention of Dlt at the apical domain of thephotoreceptors suggests that the cells retain apicobasal polarity.Conversely, overexpression of CrbMyc – PBM mislocalizes Dltthroughout the cell membrane (arrowheads in Fig. 5f) butDE-cad remains localized at the normal position of the AJ(Fig. 5i). Importantly, nearly all of the ommatidia in eyes over-expressing CrbMyc – PBM (97%, n = 1,273) and CrbMyc – JM (96%,n = 830) contain photoreceptors that retained cell polarity.Mislocalization of the Dlt throughout the membrane using theCrbMyc – PBM construct did not result in ectopic AJ formation,suggesting that the AJ defects seen in crb2 photoreceptors did notresult from mislocalization of Dlt outside the rhabdomere stalk(Fig. 3). Thus, our data indicate that overexpression of the JMregion of Crb recruits AJs to ectopic sites of the cell membranewithout causing loss of apicobasal polarity.

Crb regulates cell polarity and AJ formation in the Drosophilaembryo and follicular epithlium, but whether Crb controls AJs byregulating cell polarity or by an independent pathway is notknown1,18,19. Our work reveals a role for Crb in localizing photo-receptor AJs and rhabdomeres, independent of apicobasal polarity.Furthermore, we show that the putative FERM binding site of Crbcontrols AJ localization, which may act either through a FERMprotein or spectrins (modelled in Fig. 5j).

Various mutations in the human homologue of crb, CRB1, causetwo severe, early-onset photoreceptor-degeneration diseases, RP12and LCA6 – 8. LCA patients seem to carry more severe mutations atthe CRB1 locus and are blind from early infancy9,10. The photo-receptors in these patients, although present, have short or absentinner and outer segments9,10, analogous to the short crb2 photo-receptors in Drosophila. Furthermore, the CRB1 intracellulardomain seems functionally conserved in fly eyes (Fig. 4c). Thus,studies of Crb in Drosophila eyes may help to unravel the patho-genesis of related human diseases. A

MethodsFly strainsThe isolation of crbD88-3 and crb11A22 has been previously described1 as have othermutant strains20. ey-FLP (provided by B. Dickson) and FRT82B were used for FRT/FLPmosaic analysis16,17. The UAS/GAL4 system was used for overexpression experiments21.Crb transgenic lines including UAS–crbWT, UAS–crbMyc – intra (ref. 2).UAS–crbMyc – intraDPBM/DJM, UAS–crbMyc – JM and UAS–crbMyc – PBM (also known asUAS–crbMyc – intraY10A/E16A/DERLI, UAS–crbMyc– intraY10A/E16A and UAS–crbMyc – intraDERLI inref. 4), and were donated by E. Knust. pGMR–GAL4 was supplied by M. Freeman.

Induction of mosaic clones and ectopic CrbClones of crbD88-3 and crb11A22 were generated using ey-Flp;FRT82B crbD88-3/FRT82B andey-FLP;FRT82B crb11A22/FRT82B, respectively. For adult dissections, clones wereinduced in a w2 background. The C-terminal part, including the intracellular andtransmembrane domain, of human CRB1 was amplified by polymerase chain reaction(PCR) from a human brain complementary DNA library (Clontech) using theprimers 5 0 -TTCGAGATCTTTCACCACTATTGGCTCAGTGAC-3 0 and5 0 -TGTGCTCGAGCATCTCGAAGGGACACATGCTC-3 0 . This fragment was fused withthe signal peptide sequence of fly crb, and inserted into pUAST, using a previouslydescribed method2. Different mutant forms of Crb protein were overexpressed by usingpGMR–GAL4, which is expressed in differentiating retinal cells. CrbMyc – intra wasoverexpressed in flies kept at 18 8C and CRB1intra in flies kept at 25 8C, whereas all otherconstructs were overexpressed in flies kept at room temperature. At least fiveindependent eyes were used to score for defective photoreceptor AJ in bothpGMR–GAL4/UAS–crbMyc – JM and pGMR–GAL4/UAS–crbMyc – PBM, whereas threeindependent eyes were used to score abnormal contacts between photoreceptors.

Histology, immunohistochemistry and confocal microscopyTEM was done as previously described13, except 1.25% glutaraldehyde and 1%paraformaldehyde in 0.1 M sodium phosphate buffer was used as fixative. Eyes from pupaeaged at room temperature were prepared as previously described13 and fixed in 2%paraformaldehyde-lysine-periodate (PLP). DE-cad staining was carried out in solutionscontaining 1 mM CaCl2. Late-pupal-stage eyes stained for Crb and phalloidin were fixed asabove and then placed in ice-cold acetone for 10 min. All primary antibodies used wererabbit anti-Dlt (1:500), mouse anti-Dlt (1:500), rat anti-Crb (1:400), mouse anti-Arm(1:200; from M. Peifer), guinea pig anti-Dlg (1:1000; from P. Bryant), rabbit anti-Dlg(1:500; from K.-O. Cho), and rat anti-DE-cad (1:50; from M. Takeichi). Fluoresceinisothiocyanate (FITC)-conjugated phalloidin was from Sigma, whereas fluorescentsecondary antibodies were from Jackson Immunochemicals. Images were scanned using aZeiss LSM laser-scanning confocal microscope.

Received 30 November 2001; accepted 22 January 2002.

Published online 17 February 2002, DOI 10.1038/nature720.

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AcknowledgementsWe especially thank K.-O. Cho for her contribution to experimental design and for hersupport. We thank E. Knust for reagents and U. Tepass for discussion of unpublishedresults. We thank J. Chern and J. Lim for comments and discussion, B. Kehl and Z. H. Chenfor technical assistance. We thank the Indiana Stock Center for providing flies. We thankH. Adams for technical assistance with TEM. Confocal microscopy was supported by agrant from the National Institutes of Health to D. B. Jones. S.I. and S.-C.N. arepostdoctoral fellows. H.J.B. is a Howard Hughes Medical Institute Investigator. M.A.B. issupported by a Howard Temin Career Development Award from the National CancerInstitute. M.A.B., H.J.B., and K.-W.C. are supported by the NIH and K.-W.C. by the RetinaResearch Foundation.

Competing interests statement

The authors declare that they have no competing financial interests.

Correspondence and requests for materials should be addressed to K.-W.C.

(e-mail: kchoi@bcm.tmc.edu).

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