apical and lateral cell protrusions interconnect ... · apical and lateral cell protrusions...

11
RESEARCH ARTICLE Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and Christian Dahmann * Communication among cells by means of the exchange of signaling cues is important for tissue and organ development. Recent reports indicate that one way that signaling cues can be delivered is by movement along cellular protrusions interconnecting cells. Here, by using confocal laser scanning microscopy and three-dimensional rendering, we describe in Drosophila melanogaster wing imaginal discs lateral protrusions interconnecting cells of the columnar epithelium. Moreover, we identified protrusions of the apical surface of columnar cells that reached and apparently contacted cells of the overlying squamous epithelium. Both apical and lateral protrusions could be visualized by expression of Tkv-GFP, a green fluorescent protein (GFP) -tagged version of a receptor of the Dpp/BMP4 signaling molecule, and the endosome marker GFP-Rab5. Our results demonstrate a previously unexpected richness of cellular protrusions within wing imaginal discs and support the view that cellular protrusions may provide a means for exchanging signaling cues between cells. Developmental Dynamics 236:3408 –3418, 2007. © 2007 Wiley-Liss, Inc. Key words: Drosophila; cell protrusion; microvilli; Prominin-like; Tkv; Rab5 Accepted 10 August 2007 INTRODUCTION Cell protrusions are outward exten- sions of the cell plasma membrane made by many different cell types and organisms. Important cellular pro- cesses involve cell protrusions, includ- ing nutrient resorption (Louvard et al., 1992), mechanosensing (Frolenkov et al., 2004), photosensing (Corbeil et al., 2001; Pellikka et al., 2002), estab- lishment of cell adhesion (Vasioukhin et al., 2000), cell migration (Fulga and Rorth, 2002), fusion of epithelial sheets (Jacinto et al., 2000; Martin- Blanco et al., 2000), wound healing (Wood et al., 2002), axon guidance (Ritzenthaler et al., 2000), and cell-to- cell communication (reviewed in Rorth, 2003). Intercellular communi- cation is important for the growth and patterning of tissues and organs and cell protrusions may mediate this communication by one of several mechanisms. Signaling mediated by cell protrusions can occur by means of transport and local release of signal- ling molecules, shedding of vesicles (Marzesco et al., 2005), display of re- ceptors (Tomlinson et al., 1987; Hsi- ung et al., 2005) and membrane-teth- ered ligands on the protrusion (De Joussineau et al., 2003), or by estab- lishing direct organelle exchange be- tween connected cells (Rustom et al., 2004; reviewed in Demontis, 2004). The wing imaginal discs of Drosoph- ila melanogaster, which will give rise to the wings and parts of the body wall of adult flies, provide an attractive system for studying cell protrusions within a developing tissue. In wing imaginal discs, a monolayer of colum- nar and squamous epithelial cells is arranged in a sac-like structure with the apical membranes facing an inter- Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany The Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmat Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: DA586/4-1. Dr. Demontis’ present address is Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, Boston, MA 02115. *Correspondence to: Christian Dahmann, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. E-mail: [email protected] DOI 10.1002/dvdy.21324 Published online 13 September 2007 in Wiley InterScience (www.interscience.wiley.com). DEVELOPMENTAL DYNAMICS 236:3408 –3418, 2007 © 2007 Wiley-Liss, Inc.

Upload: others

Post on 02-Nov-2019

14 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

RESEARCH ARTICLE

Apical and Lateral Cell ProtrusionsInterconnect Epithelial Cells in LiveDrosophila Wing Imaginal DiscsFabio Demontis and Christian Dahmann*

Communication among cells by means of the exchange of signaling cues is important for tissue and organdevelopment. Recent reports indicate that one way that signaling cues can be delivered is by movementalong cellular protrusions interconnecting cells. Here, by using confocal laser scanning microscopy andthree-dimensional rendering, we describe in Drosophila melanogaster wing imaginal discs lateralprotrusions interconnecting cells of the columnar epithelium. Moreover, we identified protrusions of theapical surface of columnar cells that reached and apparently contacted cells of the overlying squamousepithelium. Both apical and lateral protrusions could be visualized by expression of Tkv-GFP, a greenfluorescent protein (GFP) -tagged version of a receptor of the Dpp/BMP4 signaling molecule, and theendosome marker GFP-Rab5. Our results demonstrate a previously unexpected richness of cellularprotrusions within wing imaginal discs and support the view that cellular protrusions may provide a meansfor exchanging signaling cues between cells. Developmental Dynamics 236:3408–3418, 2007.© 2007 Wiley-Liss, Inc.

Key words: Drosophila; cell protrusion; microvilli; Prominin-like; Tkv; Rab5

Accepted 10 August 2007

INTRODUCTIONCell protrusions are outward exten-sions of the cell plasma membranemade by many different cell types andorganisms. Important cellular pro-cesses involve cell protrusions, includ-ing nutrient resorption (Louvard etal., 1992), mechanosensing (Frolenkovet al., 2004), photosensing (Corbeil etal., 2001; Pellikka et al., 2002), estab-lishment of cell adhesion (Vasioukhinet al., 2000), cell migration (Fulga andRorth, 2002), fusion of epithelialsheets (Jacinto et al., 2000; Martin-Blanco et al., 2000), wound healing

(Wood et al., 2002), axon guidance(Ritzenthaler et al., 2000), and cell-to-cell communication (reviewed inRorth, 2003). Intercellular communi-cation is important for the growth andpatterning of tissues and organs andcell protrusions may mediate thiscommunication by one of severalmechanisms. Signaling mediated bycell protrusions can occur by means oftransport and local release of signal-ling molecules, shedding of vesicles(Marzesco et al., 2005), display of re-ceptors (Tomlinson et al., 1987; Hsi-ung et al., 2005) and membrane-teth-

ered ligands on the protrusion (DeJoussineau et al., 2003), or by estab-lishing direct organelle exchange be-tween connected cells (Rustom et al.,2004; reviewed in Demontis, 2004).

The wing imaginal discs of Drosoph-ila melanogaster, which will give riseto the wings and parts of the body wallof adult flies, provide an attractivesystem for studying cell protrusionswithin a developing tissue. In wingimaginal discs, a monolayer of colum-nar and squamous epithelial cells isarranged in a sac-like structure withthe apical membranes facing an inter-

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, GermanyThe Supplementary Material referred to in this article can be found at http://www.interscience.wiley.com/jpages/1058-8388/suppmatGrant sponsor: Deutsche Forschungsgemeinschaft; Grant number: DA586/4-1.Dr. Demontis’ present address is Harvard Medical School, Department of Genetics, 77 Avenue Louis Pasteur, Boston, MA 02115.*Correspondence to: Christian Dahmann, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse108, 01307 Dresden, Germany. E-mail: [email protected]

DOI 10.1002/dvdy.21324Published online 13 September 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:3408–3418, 2007

© 2007 Wiley-Liss, Inc.

Page 2: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

nal lumen (Fig. 1A; reviewed in Co-hen, 1993). Signaling among colum-nar cells as well as between columnar

and squamous cells is important forgrowth and patterning of wing imagi-nal discs (reviewed in Ramirez-Weber

and Kornberg, 2000; Gibson andSchubiger, 2001). Several protrusionshave been identified in imaginal discs.The apical side of imaginal disc cells isdecorated with microvilli (Poodry andSchneiderman, 1970; Ursprung, 1972)and filopodia have been described attheir basal side (Eaton et al., 1995). Inaddition, three more kinds of protru-sions have been observed, all of whichhave been implicated in exchangingsignaling molecules important for thegrowth and patterning of wing imagi-nal disc cells. Cells at the periphery ofthe columnar cell sheet have long, pla-nar filopodia-like protrusions, termedcytonemes, which connect to cellswithin the center of the columnar cellsheet (Ramirez-Weber and Kornberg,1999). Columnar cells also displayapical cell protrusions that are a fewcell diameters long and that are in-volved in the process of lateral inhibi-tion (De Joussineau et al., 2003).Furthermore, a subpopulation ofsquamous cells forms microtubule-containing protrusions that extendthrough the lumen toward columnarcells (Cho et al., 2000; Gibson andSchubiger, 2000).

A comprehensive analysis of thetypes of cell protrusions present in thewing imaginal disc has not been re-ported. It is therefore conceivable thatwe are currently underestimating thenumber and sort of cell protrusionspresent in this tissue. The identifica-tion and characterization of additionalcell protrusions could be hampered byfixation of the tissue, which is knownto result, at least in some cases, in theprofound alteration or destruction ofcell protrusions (Ramirez-Weber andKornberg, 1999).

Here, we have identified cellularprotrusions of wing imaginal discs byexpressing a membrane-bound form ofgreen fluorescent protein (GFP) in co-lumnar cells. Live, unfixed imaginaldiscs were then analyzed by laserscanning microscopy and three-di-mensional (3D) rendering. We haveidentified and characterized two kindsof previously unreported protrusions.Protrusions of the apical plasmamembrane that extended through theimaginal disc lumen and apparentlycontacted the squamous cells, and pro-trusions of the lateral plasma mem-brane that extended in betweenneighboring columnar cells. Both

Fig. 1. Apical cell protrusions connect the columnar epithelium to the squamous epithelium in wingimaginal discs. A: Scheme of the Drosophila wing imaginal disc and location of apical cell protrusions.When viewed onto the plane of the epithelium (XY), cells of the squamous epithelium (s.e.) have a largercircumference than those of the columnar epithelium (c.e.). In cross-section (XZ), cells of the squamousand columnar epithelia are apposed with their apical membranes facing a lumen. Apical cell protrusions(Pr.) arise from the columnar epithelium and are directed toward the squamous epithelium. B,B!: XZview of a three-dimensional (3D) rendered, living wing imaginal disc expressing CD8–green fluorescentprotein (GFP; green) in the dorsal compartment (ap-GAL4, UAS-CD8-GFP) and stained with thelipophilic dye FM4-64 (red). In B!, only the CD8-GFP channel is shown. Apical cell protrusions arise fromthe columnar epithelium (bottom) and extend toward the squamous epithelium (top). C,D: Apical viewsonto the columnar epithelium of the 3D-rendered tissue show that these apical protrusions areabundant and present over the entire tissue with no apparent regional preference. E–H!: The 3Drendering of a living wing imaginal disc expressing CD8-GFP (green) in cell clones (act5c"GAL4,UAS-CD8-GFP) and stained with the lipophilic dye FM4-64 (red). Different views of the tissue areprovided. E,E!: An XY view of the columnar epithelium from the basal side shows cell clones belongingto the columnar epithelium that express CD8-GFP. F,F!: An XY view of the squamous epithelium andthe underlying columnar epithelium shows apical protrusions extending from the columnar cells. G–H!:Tilting of the 3D-rendered tissue shows that the apical protrusions of columnar cells extend to the levelof the squamous epithelium, detected by the presence of a squamous cell expressing CD8-GFP(asterisk). I,I!: A higher magnification XY view of apical protrusions at the level of the squamousepithelium is shown. Apical protrusions display enlarged terminal tips (arrowhead), local bulges (arrows),and are sometimes branched (asterisk). In addition, some apical protrusions were bent in their terminaltract when contacting the squamous epithelium. Scale bar # 10 $m in B–D,E–H!, 5 $m in I,I!.

PROTRUSIONS INTERCONNECT EPITHELIAL CELLS 3409

Page 3: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

kinds of protrusions could be identi-fied with GFP-actin, a GFP-taggedversion of the Dpp receptor Thick-veins (Tkv), and the endosomalmarker GFP-Rab5. Implications forsignaling among wing imaginal disccells are discussed.

RESULTSApical Protrusions of theColumnar EpitheliumContact the ApposingSquamous Epithelium inLive Drosophila WingImaginal DiscsTo identify and analyze cellular pro-trusions of columnar wing imaginaldisc cells, we used the ap-GAL4 driverto express CD8-GFP, a transmem-brane protein routinely used to markplasma membranes (Lee and Luo,1999), in columnar cells of the dorsalcompartment of wing imaginal discs.ap-GAL4 is not active in the squa-mous wing imaginal disc cells, limit-ing CD8-GFP expression to the colum-nar cells. Third instar larvae weredissected, transferred to culture me-dium containing the lipophilic dyeFM4-64 to stain plasma membranes ofall wing imaginal discs cells, and im-aged by confocal microscopy. Stacks ofXY confocal sections were collectedand rendered in three dimensions (seethe Experimental Procedures section).We have identified protrusions ex-tending from the apical plasma mem-brane and protrusions extending fromthe lateral plasma membrane of co-lumnar cells. We will first describe theapical protrusions and in subsequentparagraphs the lateral protrusions.

The apical surface of the columnarepithelium displayed many cell pro-trusions, ranging in length from 1 to10 $m (Fig. 1B–D and SupplementaryMovie S1, which can be viewed athttp://www.interscience.wiley.com/jpages/1058-8388/suppmat). Theseapical protrusions appeared to be acommon attribute of all wing imaginaldisc columnar cells, with an averagedensity of 2.4 % 1.0 (S.D.) apical pro-trusions per cell (n # 48). Apical pro-trusions projected through the lumenof the wing imaginal disc toward thesquamous epithelium (scheme in Fig.1A).

To investigate in more detail the

morphology of these apical protru-sions, we expressed CD8-GFP inclones of cells. The 3D renderingshowed that these cell protrusionsarose from CD8-GFP–expressing cellsin the columnar epithelium (Fig.1E,E!), passed through the lumen thatseparated the columnar from thesquamous epithelium (Fig. 1G,G!),and reached and apparently contactedthe squamous epithelium (Fig.1F,F!,H,H! and Supplementary Mov-ies S2,3). Most of these protrusionswere linear, elongated structures;however, some protrusions werebranched, especially in the terminaltracts. Sometimes, conversely, twoprotrusions initially distinct came inclose contact with one another and ap-parently coalesced to form a singlestructure (Fig. 1I). Remarkably, mostof these protrusions had an enlargedand roughly spherical terminal tip(Fig. 1F!,I,I!) whose diameter of ap-proximately 1 $m exceeded the nor-mal protrusion diameter of less than0.2 $m. Sometimes this enlarged ter-minal tip was the only part of the pro-trusion that contacted the squamousepithelium; however, in other cases,the terminal tract of the protrusion,preceding the tip, also seemed to con-tact the squamous epithelium (Fig.1I,I!).

Some apical protrusions had localbulges or widenings, which may havebeen due to vesicles inside the protru-sion (Fig. 1I,I!), as previously reportedfor other types of cell protrusions(Ramirez-Weber and Kornberg, 1999;Rustom et al., 2004; Hsiung et al.,2005). We conclude that wing imagi-nal disc columnar cells display apicalprotrusions that contact the squa-mous epithelium and that have spe-cific morphological features.

Apical Protrusions ContainActinMany cellular protrusions contain anactin-based core (reviewed in Rorth,2003). To test whether apical protru-sions of columnar cells also containactin, we used ap-GAL4 to express aGFP-actin fusion protein in the dorsalcompartment of wing imaginal discs.Confocal micrographs through the co-lumnar epithelium detected GFP-ac-tin in the cortical area of dorsal colum-nar cells, as expected (Fig. 2B–B&).

Confocal micrographs taken at thelevel of the squamous epithelium oflive wing imaginal discs showed GFP-actin in dot-like structures in closeproximity to the squamous epithelialcells (Fig. 2A–A&), similar to the CD8-GFP labeled tips of apical protrusions(Fig. 1F,F!). The 3D rendering of thecollected micrographs identified GFP-actin on apical protrusions of colum-nar cells that extended across the lu-men and that came in close contactwith the apposing squamous epithe-lium (Fig. 2C–E, SupplementaryMovie S4), resembling CD8-GFP–la-beled apical protrusions. We concludethat the apical protrusions of colum-nar cells contain actin.

Drosophila Prominin-likeGFP Appears to Be Presenton Apical Protrusions ofColumnar CellsMicrovilli are protrusions of the apicalmembrane of polarized epithelialcells, including wing imaginal disccells, containing an actin-based core.To test whether the CD8-GFP–labeledprotrusions may correspond to mi-crovilli, we investigated whether amarker for microvilli would identifyCD8-GFP–like apical protrusions.Members of the Prominin family ofpentaspan transmembrane proteinsare among the most specific markersof microvilli (Corbeil et al., 2001). Inmammalian cells, endogenous Promi-nin-1 and a Prominin-2-GFP fusionprotein selectively localize to mi-crovilli (Weigmann et al., 1997; Far-geas et al., 2003). To date, two mem-bers of the prominin family, Promininand Prominin-like, have been identi-fied in the Drosophila melanogastergenome (Fargeas et al., 2003; Zelhof etal., 2006). To test whether Prominin-like localizes to apical protrusions, wefused Drosophila Prominin-like toGFP and expressed the fusion proteinusing the GAL4/UAS system in a sub-set of columnar wing imaginal disccells. By analyzing live wing imaginaldiscs expressing the Prominin-like-GFP fusion protein, we noticed, how-ever, that the GFP fluorescence wasundetectable (data not shown), possi-bly because the proper folding of theGFP molecule was impaired in thecontext of the fusion protein, as de-scribed for other GFP-fusion proteins

3410 DEMONTIS AND DAHMANN

Page 4: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

(Pedelacq et al., 2006; and referencestherein). We therefore tested whetherProminin-like-GFP localized to apicalprotrusions of wing imaginal disc cellsby immunostaining of fixed wingimaginal discs.

To this purpose, we expressed ei-ther CD8-GFP or Prominin-like-GFPin the dorsal compartment of wingimaginal discs and compared the lo-calization of these two transmem-brane proteins by immunostainingagainst GFP. Zonula adherens, whichlocalize to the apical region of the lat-eral plasma membrane, and the morebasal region of the lateral plasmamembrane were identified using anti-bodies against E-cadherin and Fasci-clin III, respectively. Consistent withour analysis of live wing imaginaldiscs expressing CD8-GFP, opticalcross-sections (XZ) showed that CD8-GFP localized to the apical and lateralmembrane of expressing cells and thatCD8-GFP was enriched apical to thezonula adherens (Fig. 3A,A!). By con-trast to live wing imaginal discs, thelumen between the squamous epithe-lium and columnar epithelium couldnot be clearly resolved by confocal mi-croscopy in the fixed tissue. We there-fore analyzed XY confocal sectionsthat, because they were slightly tiltedrelative to the plane of the squamousand columnar epithelia, displayed thesquamous cells, the apical surface ofthe columnar cells, and the lumen inbetween the two epithelia (Fig. 3G). Inthese images, the larger circumfer-ence distinguishes squamous cellsfrom columnar cells. We detectedCD8-GFP in dot-like structures at theinterface between the squamous epi-thelium and the columnar epitheliumin the dorsal compartment, but not inthe non–CD8-GFP-expressing ventralcompartment (Fig. 3B,B!,C). Thesedot-like structures presumably corre-sponded to cross-sections of the apicalprotrusions that we have identified inlive wing imaginal discs (Fig. 1).

To test whether Prominin-like-GFPlocalizes to apical cell protrusions infixed wing imaginal discs, we com-pared its localization with that ofCD8-GFP expressing, fixed wingimaginal discs. In XZ confocal micro-graphs, Prominin-like-GFP localizedto the plasma membrane apical to thezonula adherens and was mainly ex-cluded from the lateral plasma mem-

brane (Fig. 3D,D!). A weaker but re-producible staining of Prominin-like-GFP was also detected on the basalplasma membrane (SupplementaryFigure S1). In apical XY confocal mi-crographs, we detected Prominin-like-GFP in dot-like structures at theinterface between the squamous epi-thelium and the columnar epithelium

in the dorsal compartment (Fig.3E,E!,F), similar to CD8-GFP in fixedwing imaginal discs (Fig. 3B,B!,C). Nosuch dot-like structures were detectedin the ventral compartment. The lo-calization of Prominin-like-GFP isconsistent with CD8-GFP and Promi-nin-like-GFP identifying the sameapical protrusions. Because members

Fig. 2. Green fluorescent protein (GFP) -actin localizes to apical protrusions of the wing disccolumnar epithelium. A–E: Different views of live wing imaginal disc expressing GFP-actin (green)in the dorsal compartment (ap-GAL4, UAS-GFP-actin) and stained with the lipophilic dye FM4-64(red). A–B&: Micrographs of single confocal XY sections through the squamous (A–A&) and columnar(B–B&) epithelium are shown. The dorsal compartment is to the top. Dot-like GFP-actin–containingstructures are detected in the squamous cells overlying the dorsal compartment of the columnarepithelium (A!,A&). These structures presumably correspond to the cell protrusions arising fromcolumnar epithelial cells (B–B&) and presumably contact the squamous epithelial cells (red, A,A!).C–E: Different views of the three-dimensional (3D) rendered tissue are shown. In C,C!, a XZ view isdepicted with the dorsal compartment to the right. GFP-actin–labeled protrusions project from thecolumnar cells (bottom) toward squamous epithelium (top). In C!, only the GFP-actin channel isshown. D–E: Apical views onto the columnar epithelium of the 3D-rendered tissue show that theseapical protrusions are abundant and present over the entire tissue with no apparent regionalpreference. Scale bars # 10 $m.

PROTRUSIONS INTERCONNECT EPITHELIAL CELLS 3411

Page 5: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

of the Prominin family are markersfor microvilli, this finding indicatesthat apical protrusions may corre-spond to microvilli.

Lateral Protrusions of theColumnar Epithelium Extendin Between Cells Within theSame Epithelium

In addition to apical protrusions, ex-pression of CD8-GFP in clones of cellsalso identified previously uncharac-terized protrusions that extendedfrom the lateral plasma membrane ofcolumnar cells (Fig. 4A–A&). These lat-eral protrusions extended in betweenneighboring cells, usually one or twocells apart from the expressing cells.The lateral protrusions had a diame-ter of less than 0.2 $m and appearedto be randomly distributed along thelateral side of the cell. On average,approximately 0.5 % 0.2 (S.D.) lateralprotrusions per cell (n # 77) were de-tected on the apical most 8 $m of thelateral plasma membrane. Of interest,we noticed that some of the lateralprotrusions displayed an enlarged ter-minal tip (Fig. 4A&), similar to the api-cal protrusions. Prominin-like-GFPwas not detected on the lateral mem-brane (Supplementary Figure S1),suggesting that Prominin-GFP is ex-cluded from lateral protrusions. Thisfinding indicates that lateral protru-sions might have a distinct naturefrom the apical protrusions.

By 3D-rendering of micrographs se-rially acquired along the Z-axis (seethe Experimental Procedures section),we observed that some lateral protru-sions extended in between neighbor-ing cells parallel to the plane of theepithelium (XY) as well as perpendic-ular to the plane of the epithelium(XZ), that is parallel to the lateralmembrane of columnar cells (Fig. 4B–B!,D–E!; see Fig. 4C for a scheme). Totest whether these lateral protrusionscontained, like the apical protrusions,actin, we expressed a GFP-actin fu-sion protein in clones of few cells. Asshown in Figure 4F,F!, GFP-actinidentified lateral cell protrusions of co-lumnar epithelial cells that closely re-sembled the CD8-GFP–labeled lateralprotrusions, indicating that the lat-eral protrusions contained actin.

Tkv-GFP Identifies Apicaland Basal Protrusions ofColumnar Wing ImaginalDisc CellsThe growth and patterning of wingimaginal discs requires the communi-cation between cells through the ex-change of secreted signaling mole-cules. The location and connectivity ofapical protrusions (columnar cells tosquamous cells) and lateral protru-sions (columnar cells to columnarcells) makes them well suited to play arole in the exchange of such signalingmolecules. Dpp is a member of thetransforming growth factor-' familyof secreted molecules that is impor-tant for the growth and patterning ofwing imaginal discs (Spencer et al.,1982; Padgett et al., 1987; Capdevilaand Guerrero, 1994; Zecca et al., 1995)and that has been shown to be presentin the lumen between columnar cellsand squamous cells (Gibson et al.,2002). To test whether apical or lat-eral protrusions could mediate thecommunication between cells by dis-playing a receptor for Dpp, we ex-pressed a GFP-tagged version of theDpp receptor Thickveins (Tkv; Hsiunget al., 2005) in the dorsal compart-ment of columnar wing imaginal disccells. XY confocal sections through thelumen of live wing imaginal discs wereanalyzed. Most of the Tkv-GFP fluo-rescence was detected on the nonpro-truding plasma membrane of express-ing cells. In addition, Tkv-GFP wasalso present on dot-like structures.These dot-like structures werecostained with the lipophilic dyeFM4-64 and presumably representcross-sections of apical protrusions(Fig. 5A–B&). To test this further, werendered the tissue in three dimen-sions. As shown in Figure 5C, Tkv-GFP–labeled protrusions of the apicalplasma membrane projected towardthe squamous epithelium, indicatingthat Tkv-GFP was present on apicalprotrusions (see also SupplementaryMovies S5 and S6).

To test whether Tkv-GFP was alsopresent on lateral protrusions, we ex-pressed Tkv-GFP in clones of a fewcells. As shown in Figure 5D–D&, Tkv-GFP identified protrusions of thelateral membrane that resembledCD8-GFP–labeled lateral protrusions.Thus, we detected Tkv-GFP on both

apical and lateral protrusions of wingdisc columnar cells. The presence ofTkv-GFP on apical and lateral protru-sions is consistent with the notionthat these cell protrusions could me-diate communication between cells.

GFP-Rab5 CompartmentsAre Detected Inside BothApical and LateralProtrusionsOur analysis of CD8-GFP–labeled api-cal protrusions of live wing imaginaldisc cells revealed bulges that ex-ceeded the normal protrusion diame-ter of 0.2 $m (Fig. 1I,I!), suggestingthat vesicles may be transported in-side these cell protrusions. To test thishypothesis, we expressed GFP-Rab5(Wucherpfennig et al., 2003), amarker of early endosomes (Chavrieret al., 1990), in clones of a few cellsand analyzed its localization in livewing imaginal discs. GFP-Rab5 com-partments were detected in XY confo-cal micrographs corresponding to theluminal space or to the squamousepithelium overlying the express-ing cells (Fig. 6A–A&). FM4-64–labeled plasma membrane was encir-cling GFP-Rab5 compartments (Fig.6A–A&), suggesting that GFP-Rab5compartments were inside apical pro-trusions. In addition, by analyzing XYmicrographs corresponding to the lat-eral membrane of columnar cells, weobserved the presence of distinct GFP-Rab5 compartments in lateral protru-sions (Fig. 6B–F&). Thus, both apicaland lateral protrusions contain GFP-Rab5 compartments.

DISCUSSIONCell protrusions are a common at-tribute of most cell types and play fun-damental roles in development anddisease. Here, we identify to-date un-characterized protrusions of the apicaland lateral plasma membrane in theDrosophila melanogaster wing imagi-nal disc columnar epithelium. We pro-vide evidence that both apical and lat-eral protrusions contain actin and,furthermore, that apical protrusionsmight be microvilli. We find a receptorfor the Dpp signaling molecule, Tkv-GFP, as well as the endosomal markerRab5-GFP, on lateral and apical pro-trusions, consistent with the view that

3412 DEMONTIS AND DAHMANN

Page 6: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

these protrusions are involved in cell-to-cell communication.

Apical Protrusions ConnectColumnar Cells WithSquamous CellsMicrovilli are a paradigm for apicalcell protrusions of epithelia (e.g.,Heintzelman and Mooseker, 1992).Several electron microscopic studieshave described microvilli on the apicalsurface of columnar wing imaginaldisc cells (Schlichting et al., 2006; Ur-

Fig. 3.

Fig. 4.

Fig. 3. CD8-green fluorescent protein (GFP)and Prominin-like-GFP localize to apical punc-tuate structures of the columnar epithelium infixed wing imaginal discs. A–F: Immunostainingof wing imaginal disc expressing CD8-GFP(A–C, ap-GAL4, UAS-CD8-GFP) or a Prominin-like-GFP fusion protein (D–F, ap-GAL4, UAS-prominin-like-GFP) in the dorsal compartmentstained for GFP (green), DE-Cadherin (DE-Cad,blue), and Fasciclin III (FasIII, red). A,A!,D,D!: XZviews of the dorsal compartment (apical to thetop) show that both CD8-GFP and Prominin-like-GFP are enriched on the apical side of co-lumnar epithelial cells. B,B!,E,E!: Apical XY sec-tions (dorsal compartment to the top) areshown. C and F are higher magnifications of theboxed areas shown in B! and E!. CD8-GFP andProminin-like-GFP localize to apical punctuatestructures of the wing disc columnar epithe-lium. Because the confocal sections are slightlytilted, both columnar and squamous epithelialcells are depicted in different parts of the mi-crographs. G: A cartoon depicting the positionof the XY sections shown in B,B!,E,E!. Scalebars # 20 $m in A–B!,D–E!; 5 $m in C,F.

Fig. 4. Lateral protrusions of columnar epithelialcells extend in between neighboring cells andcontain green fluorescent protein (GFP) -actin.A–A": XY micrographs of a live wing imaginaldisc expressing CD8-GFP (green) in clones ofcolumnar cells (act5c"GAL4, UAS-CD8-GFP)and stained with the lipophilic dye FM4-64(red). Protrusions (A&, asterisks) arising from thelateral membrane of columnar cells extend inbetween neighboring cells. B,B!: Three-dimen-sional (3D) rendering of the columnar epitheliumcontaining CD8-GFP expressing clones. Onlythe GFP channel (white) is shown. Basolateral(B) and basal (B!) views of the 3D-renderedtissue are depicted. Yellow arrows indicate theX-axis and the Y-axis and red arrows the Z-axis. Lateral protrusions arise from the lateralmembrane and can extend along the Z-axis. C:Scheme of protrusions arising from the lateralmembrane of wing disc columnar cells. D–E!:Higher magnifications of the 3D-rendered tis-sue shown in B. F–F": XY-micrographs of alive wing imaginal disc expressing GFP-actin (green) in clones of columnar cells(act5c"GAL4, UAS-GFP-actin) and stainedwith the lipophilic dye FM4-64 (red). Protru-sions arising from the lateral membrane ofcolumnar cells extend in between neighbor-ing cells. Scale bars # 5 $m in A–A&,F–F&, 10$m in B,B!.

PROTRUSIONS INTERCONNECT EPITHELIAL CELLS 3413

Page 7: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

Fig. 5. A green fluorescent protein (GFP) -tagged form of the Dppreceptor Thickveins (Tkv) localizes to both apical and lateral protrusionsof the wing disc columnar epithelium. A–C!: Different views of a live wingimaginal disc expressing Tkv-GFP (green) in the dorsal compartment(ap-GAL4, UAS-tkv-GFP) and stained with the lipophilic dye FM4-64(red). A–B": Micrographs of single confocal XY sections through thelumen (A–A&) and columnar epithelium (B–B&) are shown. The dorsalcompartment is to the top. FM4-64 labels within the lumen dots thatcorrespond to cross-sections of apical protrusions (A). Tkv-GFP colo-calizes with some of these dots (A!). C,C!: A XZ view of the three-dimensional (3D) -rendered tissue is depicted with the dorsal compart-ment to the front. Tkv-GFP–labeled protrusions project from thecolumnar cells (bottom) toward the squamous epithelium (top). In C!,only the Tkv-GFP channel is shown. Tkv-GFP–expressing cells areshorter and the lumen between columnar epithelium and squamousepithelium wider presumably as a consequence of the role of Dppsignaling in maintaining proper columnar architecture (Gibson and Per-rimon, 2005; Shen and Dahmann, 2005). Under this experimental con-dition, FM4-64 staining identifies within the dorsal wing disc pouchprotrusions of the squamous epithelium projecting toward the columnarepithelium (red, see also Supplementary Figure S2). D–D": XY micro-graphs of live wing imaginal discs expressing Tkv-GFP (green) in clonesof columnar cells (act5c"GAL4, UAS-tkv-GFP) and stained with thelipophilic dye FM4-64 (red). Tkv-GFP–labeled protrusions arising fromthe lateral membrane of columnar cells extend in between neighboringcells. Scale bars # 10 $m in A–C!, 2 $m in D–D&.

Fig. 6. Rab5–green fluorescent protein (GFP) vesicles are detected onapical and lateral protrusions. A–F&: XY micrographs of live wing imaginaldiscs expressing GFP-Rab5 (green) in clones of columnar cells(act5c"GAL4, UAS-GFP-Rab5) and stained with the lipophilic dye FM4-64(red). A–A": A micrograph of an apical protrusion contacting the squamousepithelium, identified by FM4-64 staining (red). GFP-Rab5 compartmentsare detected at the tip of apical protrusions. B–B": Micrograph of thecolumnar cell clone from which the apical protrusion shown in A arises. Alateral protrusion extends in between neighboring cells and contains aGFP-Rab5 compartment. C–F": Serial XY sections along the Z-axis. GFP-Rab5 is only detected on cellular extensions in some sections, but notothers, indicating that these structures are cell protrusions. Numbers to theright indicate the distance from the squamous epithelium along the Z-axis.Scale bars # 2.5 $m in A–B&, 5 $m in C–F&.

3414 DEMONTIS AND DAHMANN

Page 8: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

sprung, 1972; Poodry and Schneider-man, 1970). As seen in electron micro-scopic preparations, microvilli arenumerous, short (on average approxi-mately 500 nm long) protrusions ex-tending into the lumen of wing imag-inal discs. Using CD8-GFP, we haveidentified protrusions that extendfrom the apical surface of columnarwing imaginal disc cells. Unlike themicrovilli identified by electron mi-croscopy, these protrusions are long(up to 10 $m), extend through the en-tire lumen getting in close contact tothe overlaying squamous cells, anddisplay enlarged terminal tips (Fig. 1).Despite these apparent differences,several observations suggest that api-cal CD8-GFP–labeled protrusions andmicrovilli are one and the same struc-ture. First, both kinds of protrusionsare extensions of the apical plasmamembrane. Second, both kinds of pro-trusions contain actin. Third, the twokinds of protrusions have a compara-ble diameter of less than 0.2 $m. Fi-nally, our data are consistent with thenotion that apical protrusions containthe microvilli marker Prominin-like.It is therefore conceivable that themorphological differences between mi-crovilli, as seen in electron micro-scopic preparations, and CD8-GFP–labeled protrusions, as seen in livepreparations, are a consequence of thetechnique used to visualize the protru-sions. If CD8-GFP–labeled protru-sions were microvilli, this would indi-cate that microvilli of columnar cellsextended through the entire lumenand got in close contact to the overly-ing squamous epithelium.

Analysis of the Drosophila follicularepithelium has provided evidence thatalso in this epithelium microvilli cancontact overlying cells, in this case theoocyte (Mahowald, 1972; Mahowaldand Kambysellis, 1980). Microvilli offollicle cells projecting toward the oo-cyte have also been described in am-phibians and mammals (Dantzer,1985; Villecco et al., 2002; Makabe etal., 2006), suggesting that follicle cellmicrovilli might establish connectionswith oocytes in many animal species.Considering that, over the past de-cades, microvilli have been identifiedin a plethora of epithelia and organ-isms, we speculate that microvillimight connect apposed epithelia dur-ing different developmental stages in

several organisms. This raises there-fore the intriguing possibility that mi-crovilli, in addition to increasing thesurface area of cells, also serve addi-tional functions, for example, to medi-ate the communication or cohesion ofcells from apposed cell layers.

What is the function of apical pro-trusions? Cells of the squamous andcolumnar epithelium exchange sig-nals that are important for the growthand patterning of wing imaginal discs(Cho et al., 2000; Gibson and Schu-biger, 2000; Gibson et al., 2002; Pal-lavi and Shashidhara, 2003, 2005).Cells of the squamous epithelium dis-play long microtubule-based protru-sions directed toward the columnarepithelium (Cho et al., 2000; Gibsonand Schubiger, 2000). It has been pro-posed that these protrusions couldmediate the exchange of instructivesignals between the two apposed epi-thelia that are important for prolifer-ation and patterning of imaginal discs(Cho et al., 2000; Gibson and Schu-biger, 2000). However, these protru-sions were detected mainly in the pro-spective hinge and notum regions, butnot in the pouch region of the wingimaginal disc that gives rise to theadult wing (Gibson and Schubiger,2000). It is therefore uncertainwhether squamous wing imaginal disccells could also exchange signals withthe underlying columnar pouch cellsthrough protrusions. Our results nowopen up the possibility that the apicalprotrusions of columnar cells, whichare present on all columnar cells, ful-fill the role in transmitting signals be-tween the two apposed epithelia.Other possible functions for these pro-trusions include the provision of phys-ical links between the two epithelia,which might be important for main-taining the disc-like shape of the wingimaginal disc. Tools to specifically ab-late these protrusions will be ulti-mately required to address the func-tion of these apical protrusions.

Lateral ProtrusionsInterconnect Cells Withinthe Columnar EpitheliumThe development of the wing imaginaldisc and other epithelia relies onshort-range cell interactions amongcolumnar cells (e.g., Diaz-Benjumeaand Cohen, 1995; Rulifson and Blair,

1995; Zecca et al., 1995; Neumann andCohen, 1996; Milan et al., 2001, 2002).Short-range cell interactions could bemediated by the spread of signalingmolecules through the extracellularspace in between cells (reviewed inTeleman et al., 2001; Entchev andGonzalez-Gaitan, 2002; Vincent andDubois, 2002). In addition, short-range cell interactions could be medi-ated by specialized cellular struc-tures. Gap junctions are presentbetween neighboring cells of the co-lumnar epithelium of imaginal discs(Agrell, 1968; Poodry and Schneider-man, 1970). However, exchange of sig-nals by means of gap junctions is lim-ited to molecules of small size (( 1kDa; Loewenstein, 1981). Electron mi-croscopy studies have further identi-fied cytoplasmic bridges in the colum-nar epithelium of imaginal discs(Poodry and Schneiderman, 1970).Cytoplasmatic bridges are short cellu-lar protrusions of the basolateralmembrane that connect neighboringcells and establish direct membraneand cytoplasmic continuity betweenconnected cells, indicating that localexchange of information could occurby means of these cellular structures(Poodry and Schneiderman, 1970).However, cytoplasmic bridges aresimilar to remnants of cytokinesis(Poodry and Schneiderman, 1970),suggesting that they only connect sis-ter cells and would, therefore, not beof major importance for pattern for-mation in imaginal discs, which hasbeen shown to be independent of celllineage (Bryant and Schneiderman,1969; Garcia-Bellido and Merriam,1969). Finally, recent reports suggestthat short-range communication inthe wing imaginal disc can occur bymeans of cellular protrusions (DeJoussineau et al., 2003).

We observed in live wing imaginaldiscs that columnar cells have protru-sions of the lateral membrane that ex-tend in between the lateral plasmamembranes of neighboring cells. Incommon with the apical protrusions,these lateral protrusions also dis-played enlarged terminal tips (Fig.4D–D&), suggesting that this might bea common morphological feature ofcell protrusions in live tissue. The lo-calization and direction of these pro-trusions is consistent with a role inmediating some of the short-range sig-

PROTRUSIONS INTERCONNECT EPITHELIAL CELLS 3415

Page 9: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

naling that has been observed in wingimaginal discs. In principle, connec-tions established by lateral protru-sions could result in a global interac-tion network facilitating the exchangeof survival information and positionalcues. Moreover, the lateral protru-sions may also contribute to long-range signaling between cells, for ex-ample, by increasing the rate oftransport of signaling molecules.

The apical and lateral protrusionsdescribed here differ from cytonemesand protrusions emanating from sen-sory organ precursor cells, two kindsof cellular protrusions that were re-cently described in wing imaginaldiscs (Ramirez-Weber and Kornberg,1999; Hsiung et al., 2005; DeJoussineau et al., 2003). Apical pro-trusions project toward the overlyingsquamous epithelium, whereas cy-tonemes and sensory organ precursorcell protrusions project toward colum-nar cells. Similar to the lateral protru-sions described here, sensory organprecursor cell protrusions are shortprotrusions interconnecting cells inthe columnar epithelium. However,sensory organ precursor cell protru-sions were mainly detected at thebasal side of cells or apical to thezonula adherens (De Joussineau et al.,2003). In contrast, the lateral protru-sions observed here mainly emanatefrom below the zonula adherens. Thelateral protrusions also differ from cy-tonemes present in the wing imaginaldisc pouch in that they are shorter(3–5 $m instead of 20 $m in length)and that they apparently emanate inall directions and are not, as cy-tonemes, directed toward the antero-posterior or dorsoventral compart-ment boundaries.

Apical and LateralProtrusions and IntercellularSignalingSeveral recent studies have identifiedsignaling molecules or their receptorsassociated with various cellular pro-trusions, providing evidence thatthese cellular protrusions are impor-tant for mediating intercellular sig-naling. Molecules associated with pro-trusions include Sevenless, a receptorfor the Boss ligand (Tomlinson et al.,1987); Delta, a Notch ligand (DeJoussineau et al., 2003); Scabrous, a

signaling molecule (Chou and Chien,2002); and Tkv, a receptor for Dpp(Hsiung et al., 2005) on cellular pro-trusions in Drosophila and membersof the erbB/HER family, receptors forEGF and related ligands, on exten-sions in mammalian cells (Lidke et al.,2004). We extend these findings byshowing that Tkv-GFP can also local-ize to apical and lateral cell protru-sions. We note, however, that we donot find Tkv-GFP enriched on theseprotrusions compared with the non-protruding plasma membrane, nor dowe find Tkv-GFP in these protrusionson motile punctae, as previously de-scribed for cytonemes (Hsiung et al.,2005).

A further indication that apical andlateral protrusions might be involvedin mediating signals between cells isour finding that Rab5-GFP localizes tothese protrusions. One way by whichcellular protrusions convey instruc-tive cues is by endocytosis and traf-ficking of signaling complexes on en-dosomes. The early endosomal proteinRab5a localizes, for example, to axonsand dendrites of neurons (de Hoop etal., 1994) and is important for cou-pling clathrin-dependent endocytosisto axonal retrograde transport (Dein-hardt et al., 2006). Furthermore, sev-eral signaling molecules are associ-ated with endosomes (reviewed inMiaczynska et al., 2004). It will there-fore be interesting to learn whetherendocytosis takes place from theplasma membrane of apical and lat-eral protrusions and whether endo-somes in these protrusions associatewith signaling complexes.

EXPERIMENTALPROCEDURESMolecular Cloning and FlyStocksTo generate the UAS-prominin-like-GFP transgene, the enhanced GFP(EGFP) coding sequence was polymer-ase chain reaction (PCR) amplifiedwith primers 5!-CGGCTCGAGATG-GTGAGCAAGGGCGAGGAGCTG-3!and 5!-CTAGTCTAGATTACTTGTA-CAGCTCGTCCATGCC-3! (the under-lined sequences are XhoI and XbaI re-striction sites used for cloning) usingpEGFP-N1 (Clontech) as template (nn679-1398), and cloned in the pUAST

vector (Brand and Perrimon, 1993).Drosophila melanogaster prominin-like (CG7740) coding sequence (nucle-otides 158-3199 of AF197345) and 5!-UTR (Fargeas et al., 2003) were PCRamplified from cDNA clone LD16666(kindly provided by Denis Corbeil)with primers: 5!-CCGCTCGAGTCG-ATCGGATAAATTTGGAATAGAAAA-GGC-3! and 5!-CCGCTCGAGATCCT-GCTCGGAGGCACCCGGATAG-3! (theunderlined sequences are XhoI restric-tion sites used for cloning). The PCRproduct was digested with XhoI andcloned in frame in the pUAST-EGFPvector. The correct nucleotide se-quences of the cloned PCR productswere confirmed by sequencing before in-jection into y w embryos to obtain trans-genic flies.

Additional fly stocks used in thiswork include ap-GAL4 (Calleja et al.,1996), act5c"CD2"GAL4 (Pignoniand Zipursky, 1997), UAS-CD8-GFP(Lee and Luo, 1999), UAS-tkv-GFP(Hsiung et al., 2005), UAS-GFP-Rab5(Wucherpfennig et al., 2003), andUAS-GFP-actin (Verkhusha et al.,1999). Marked clones composed of fewcells were generated by Flp-mediatedmitotic recombination (Golic andLindquist, 1989) subjecting third-in-star larvae to a 34°C heat-shock for 30min at 20 hr before dissection.

ImmunohistochemistryLate third-instar larvae were dis-sected in ice-cold Ringer’s solution andfixed for 40 min at room temperaturein PEM solution (0.1 M PIPES, 2 mMMgSO4, 1 mM EGTA) with 4% form-aldehyde and 0.1% Triton X-100. Lar-val carcasses were washed in PBT(phosphate buffered saline, 0.1% bo-vine serum albumin, 0.1% TritonX-100) and incubated with appropri-ate primary antibodies for 1 hr atroom temperature. Subsequently, lar-val carcasses were washed in PBT,blocked with PBT containing 5% heat-inactivated goat serum, and incubatedwith fluorophore-conjugated second-ary antibodies for 1 hr at room tem-perature. Wing imaginal discs werethen washed, dissected out of the car-casses, and mounted in 50% glycerol,0.1 M sodium carbonate pH 9, andPPDA (p-phenylene diamine, Sigma,Taufkirchen). Stained tissues wereobserved with a ZEISS Laser Scan-

3416 DEMONTIS AND DAHMANN

Page 10: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

ning Microscope 510 Meta (Carl ZeissAG, Jena). Primary antibodies used inthis study were mouse anti-FasciclinIII 7G10 (1:100, Developmental Stud-ies Hybridoma Bank), rabbit anti-GFP (1:2,000, Clontech 8372-1), andrat anti–DE-Cad (DCAD1 andDCAD2, 1:100; (Oda et al., 1994). Sec-ondary antibodies used were Alexa-488-, Alexa-594- (Molecular Probes,Eugene, OR), and Cy5- (Jackson Im-munoResearch, West Grove, PA) con-jugated anti-rabbit, anti-mouse, oranti-rat IgG (1:200).

Laser Scanning Microscopyof Live Wing Imaginal Discsand 3D RenderingWing imaginal discs were dissected inRinger’s solution from third-instarlarvae and processed as previously de-scribed (Greco et al., 2001). Briefly,live wing imaginal discs were trans-ferred to a microscope glass-slide witha chamber delimited by double-sidedadhesive tape containing eitherShields and Sang’s M3 culture me-dium (Shields and Sang, 1970) orRinger’s solution with 9 $M FM4-64(Molecular Probes). Wing imaginaldiscs were oriented with the squa-mous epithelium facing the coverslip.The wing imaginal discs were ob-served immediately using a ZEISSLSM 510 laser scanning confocal mi-croscope (Carl Zeiss AG), usually us-ing a water immersion 63) objective.Stacks of closely spaced (0.05 $m) sec-tions were taken starting from thesquamous epithelium progressing to-ward the basolateral side of the co-lumnar epithelial cells. Confocal sec-tions were then exported as TIF filesand assembled in a library for process-ing using the 3D rendering softwareVolocity v2.5 (Improvision, Lexington,MA). Quicktime movies and singlesnapshots were then obtained fromthe 3D rendering of the tissue. Cy-tonemes were not observed in ourpreparations as their identification re-quires a slight flattening of imaginaldiscs (Ramirez-Weber and Kornberg,1999; Hsiung et al., 2005). All imagesshown here refer to the pouch regionof wing imaginal discs.

ACKNOWLEDGMENTSWe thank Konrad Basler, MarcosGonzalez-Gaitan, Suzanne Eaton,

Tom Kornberg, and the BloomingtonDrosophila Stock Center for fly stocks;Tadashi Uemura and the Develop-mental Studies Hybridoma Bank forantibodies; and Denis Corbeil for thecDNA clone LD16666. We also thankSuzanne Eaton and Andrew Oates forcritical comments on the manuscript.This work was supported by the MaxPlanck Society and a grant fromthe Deutsche Forschungsgemein-schaft (C.D.).

REFERENCES

Agrell IPS. 1968. Differentiation of themembrane systems in the cells of imagi-nal disks. Z Zellforsch 88:365–369.

Brand AH, Perrimon N. 1993. Targetedgene expression as a means of alteringcell fates and generating dominant phe-notypes. Development 118:401–415.

Bryant PJ, Schneiderman HA. 1969. Celllineage, growth, and determination inthe imaginal leg discs of Drosophilamelanogaster. Dev Biol 20:263–290.

Calleja M, Moreno E, Pelaz S, Morata G.1996. Visualization of gene expression inliving adult Drosophila. Science 274:252–255.

Capdevila J, Guerrero I. 1994. Targetedexpression of the signaling molecule de-capentaplegic induces pattern duplica-tions and growth alterations in Drosoph-ila wings. EMBO J 13:4459–4468.

Chavrier P, Parton RG, Hauri HP, SimonsK, Zerial M. 1990. Localization of lowmolecular weight GTP binding proteinsto exocytic and endocytic compartments.Cell 62:317–329.

Cho KO, Chern J, Izaddoost S, Choi KW.2000. Novel signaling from the peripo-dial membrane is essential for eye discpatterning in Drosophila. Cell 103:331–342.

Chou YH, Chien CT. 2002. Scabrous con-trols ommatidial rotation in the Dro-sophila compound eye. Dev Cell 3:839–850.

Cohen SM. 1993. Imaginal disc develop-ment. In: Bate M, Martinez Arias A, ed-itors. The development of Drosophilamelanogaster. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory Press.

Corbeil D, Roper K, Fargeas CA, Joester A,Huttner WB. 2001. Prominin: a story ofcholesterol, plasma membrane protru-sions and human pathology. Traffic 2:82–91.

Dantzer V. 1985. Electron microscopy ofthe initial stages of placentation in thepig. Anat Embryol (Berl) 172:281–293.

de Hoop MJ, Huber LA, Stenmark H, Wil-liamson E, Zerial M, Parton RG, DottiCG. 1994. The involvement of the smallGTP-binding protein Rab5a in neuronalendocytosis. Neuron 13:11–22.

De Joussineau C, Soule J, Martin M, An-guille C, Montcourrier P, Alexandre D.2003. Delta-promoted filopodia mediate

long-range lateral inhibition in Drosoph-ila. Nature 426:555–559.

Deinhardt K, Salinas S, Verastegui C,Watson R, Worth D, Hanrahan S, BucciC, Schiavo G. 2006. Rab5 and Rab7 con-trol endocytic sorting along the axonalretrograde transport pathway. Neuron52:293–305.

Demontis F. 2004. Nanotubes make big sci-ence. PLoS Biol 2:E215.

Diaz-Benjumea FJ, Cohen SM. 1995. Ser-rate signals through Notch to establish aWingless-dependent organizer at thedorsal/ventral compartment boundary ofthe Drosophila wing. Development 121:4215–4225.

Eaton S, Auvinen P, Luo L, Jan YN, Si-mons K. 1995. CDC42 and Rac1 controldifferent actin-dependent processes inthe Drosophila wing disc epithelium.J Cell Biol 131:151–164.

Entchev EV, Gonzalez-Gaitan MA. 2002.Morphogen gradient formation and ve-sicular trafficking. Traffic 3:98–109.

Fargeas CA, Florek M, Huttner WB, Cor-beil D. 2003. Characterization of promi-nin-2, a new member of the promininfamily of pentaspan membrane glycopro-teins. J Biol Chem 278:8586–8596.

Frolenkov GI, Belyantseva IA, FriedmanTB, Griffith AJ. 2004. Genetic insightsinto the morphogenesis of inner ear haircells. Nat Rev Genet 5:489–498.

Fulga TA, Rorth P. 2002. Invasive cell mi-gration is initiated by guided growth oflong cellular extensions. Nat Cell Biol4:715–719.

Garcia-Bellido A, Merriam JR. 1969. Celllineage of the imaginal discs in Drosoph-ila gynandromorphs. J Exp Zool 170:61–75.

Gibson MC, Perrimon N. 2005. Extrusionand death of DPP/BMP-compromised ep-ithelial cells in the developing Drosoph-ila wing. Science 307:1785–1789.

Gibson MC, Schubiger G. 2000. Peripodialcells regulate proliferation and pattern-ing of Drosophila imaginal discs. Cell 103:343–350.

Gibson MC, Schubiger G. 2001. Drosophilaperipodial cells, more than meets theeye? Bioessays 23:691–697.

Gibson MC, Lehman DA, Schubiger G.2002. Lumenal transmission of decapen-taplegic in Drosophila imaginal discs.Dev Cell 3:451–460.

Golic KG, Lindquist S. 1989. The FLP re-combinase of yeast catalyzes site-specificrecombination in the Drosophila ge-nome. Cell 59:499–509.

Greco V, Hannus M, Eaton S. 2001. Argo-somes: a potential vehicle for the spreadofmorphogensthroughepithelia.Cell106:633–645.

Heintzelman MB, Mooseker MS. 1992. As-sembly of the intestinal brush border cy-toskeleton. Curr Top Dev Biol 26:93–122.

Hsiung F, Ramirez-Weber FA, Iwaki DD,Kornberg TB. 2005. Dependence of Dro-sophila wing imaginal disc cytonemes onDecapentaplegic. Nature 437:560–563.

Jacinto A, Wood W, Balayo T, Turmaine M,Martinez-Arias A, Martin P. 2000. Dy-

PROTRUSIONS INTERCONNECT EPITHELIAL CELLS 3417

Page 11: Apical and lateral cell protrusions interconnect ... · Apical and Lateral Cell Protrusions Interconnect Epithelial Cells in Live Drosophila Wing Imaginal Discs Fabio Demontis and

namic actin-based epithelial adhesionand cell matching during Drosophiladorsal closure. Curr Biol 10:1420–1426.

Lee T, Luo L. 1999. Mosaic analysis with arepressible cell marker for studies ofgene function in neuronal morphogene-sis. Neuron 22:451–461.

Lidke DS, Nagy P, Heintzmann R, Arndt-Jovin DJ, Post JN, Grecco HE, Jares-Erijman EA, Jovin TM. 2004. Quantumdot ligands provide new insights intoerbB/HER receptor-mediated signaltransduction. Nat Biotechnol 22:198–203.

Loewenstein WR. 1981. Junctional inter-cellular communication: the cell-to-cellmembrane channel. Physiol Rev 61:829–913.

Louvard D, Kedinger M, Hauri HP. 1992.The differentiating intestinal epithelialcell: establishment and maintenance offunctions through interactions betweencellular structures. Annu Rev Cell Biol8:157–195.

Mahowald AP. 1972. Ultrastructural ob-servations on oogenesis in Drosophila. JMorphol 137:29–48.

Mahowald AP, Kambysellis MP. 1980. Oo-genesis. In: Ashburner M, Wright TRF,editors. The genetics and biology of Dro-sophila. New York: Academic Press. p141–224.

Makabe S, Naguro T, Stallone T. 2006. Oo-cyte-follicle cell interactions during ovar-ian follicle development, as seen by highresolution scanning and transmissionelectron microscopy in humans. MicroscRes Tech 69:436–449.

Martin-Blanco E, Pastor-Pareja JC, Gar-cia-Bellido A. 2000. JNK and decapen-taplegic signaling control adhesivenessand cytoskeleton dynamics during tho-rax closure in Drosophila. Proc NatlAcad Sci U S A 97:7888–7893.

Marzesco AM, Janich P, Wilsch-Braun-inger M, Dubreuil V, Langenfeld K, Cor-beil D, Huttner WB. 2005. Release ofextracellular membrane particles carry-ing the stem cell marker prominin-1(CD133) from neural progenitors andother epithelial cells. J Cell Sci 118:2849–2858.

Miaczynska M, Pelkmans L, Zerial M.2004. Not just a sink: endosomes in con-trol of signal transduction. Curr OpinCell Biol 16:400–406.

Milan M, Weihe U, Perez L, Cohen SM.2001. The LRR proteins capricious andTartan mediate cell interactions duringDV boundary formation in the Drosoph-ila wing. Cell 106:785–794.

Milan M, Perez L, Cohen SM. 2002. Short-range cell interactions and cell survivalin the Drosophila wing. Dev Cell 2:797–805.

Neumann CJ, Cohen SM. 1996. A hierar-chy of cross-regulation involving Notch,wingless, vestigial and cut organizes the

dorsal/ventral axis of the Drosophilawing. Development 122:3477–3485.

Oda H, Uemura T, Harada Y, Iwai Y,Takeichi M. 1994. A Drosophila homologof cadherin associated with armadilloand essential for embryonic cell–cell ad-hesion. Dev Biol 165:716–726.

Padgett RW, St Johnston RD, Gelbart WM.1987. A transcript from a Drosophilapattern gene predicts a protein homolo-gous to the transforming growth factor-beta family. Nature 325:81–84.

Pallavi SK, Shashidhara LS. 2003. Egfr/Ras pathway mediates interactions be-tween peripodial and disc proper cells inDrosophila wing discs. Development 130:4931–4941.

Pallavi SK, Shashidhara LS. 2005. Signal-ing interactions between squamous andcolumnar epithelia of the Drosophilawing disc. J Cell Sci 118:3363–3370.

Pedelacq JD, Cabantous S, Tran T, Terwil-liger TC, Waldo GS. 2006. Engineeringand characterization of a superfoldergreen fluorescent protein. Nat Biotech-nol 24:79–88.

Pellikka M, Tanentzapf G, Pinto M, SmithC, McGlade CJ, Ready DF, Tepass U.2002. Crumbs, the Drosophila homo-logue of human CRB1/RP12, is essentialfor photoreceptor morphogenesis. Na-ture 416:143–149.

Pignoni F, Zipursky SL. 1997. Induction ofDrosophila eye development by decapen-taplegic. Development 124:271–278.

Poodry CA, Schneiderman HA. 1970. Theultrastructure of the developing leg ofDrosophila melanogaster. Wilhelm RouxArch 166:1–44.

Ramirez-Weber FA, Kornberg TB. 1999.Cytonemes: cellular processes thatproject to the principal signaling centerinDrosophila imaginaldiscs.Cell97:599–607.

Ramirez-Weber FA, Kornberg TB. 2000.Signaling reaches to new dimensions inDrosophila imaginal discs. Cell 103:189–192.

Ritzenthaler S, Suzuki E, Chiba A. 2000.Postsynaptic filopodia in muscle cells in-teract with innervating motoneuron ax-ons. Nat Neurosci 3:1012–1017.

Rorth P. 2003. Communication by touch:role of cellular extensions in complex an-imals. Cell 112:595–598.

Rulifson EJ, Blair SS. 1995. Notch regu-lates wingless expression and is not re-quired for reception of the paracrinewingless signal during wing margin neu-rogenesis in Drosophila. Development121:2813–2824.

Rustom A, Saffrich R, Markovic I, WaltherP, Gerdes HH. 2004. Nanotubular high-ways for intercellular organelle trans-port. Science 303:1007–1010.

Schlichting K, Wilsch-Brauninger M, De-montis F, Dahmann C. 2006. CadherinCad99C is required for normal microvilli

morphology in Drosophila follicle cells.J Cell Sci 119:1184–1195.

Shen J, Dahmann C. 2005. Extrusion ofcells with inappropriate Dpp signalingfrom Drosophila wing disc epithelia. Sci-ence 307:1789–1790.

Shields G, Sang JH. 1970. Characteristicsof five cell types appearing during invitro culture of embryonic material fromDrosophila melanogaster. J Embryol ExpMorphol 23:53–69.

Spencer FA, Hoffmann FM, Gelbart WM.1982. Decapentaplegic: a gene complexaffecting morphogenesis in Drosophilamelanogaster. Cell 28:451–461.

Teleman AA, Strigini M, Cohen SM. 2001.Shaping morphogen gradients. Cell 105:559–562.

Tomlinson A, Bowtell DD, Hafen E, RubinGM. 1987. Localization of the sevenlessprotein, a putative receptor for positionalinformation, in the eye imaginal disc ofDrosophila. Cell 51:143–150.

Ursprung H. 1972. The fine structure ofimaginal disks. In: The biology of imagi-nal disks. New York: Springer Verlag.

Vasioukhin V, Bauer C, Yin M, Fuchs E.2000. Directed actin polymerization isthe driving force for epithelial cell–celladhesion. Cell 100:209–219.

Verkhusha VV, Tsukita S, Oda H. 1999.Actin dynamics in lamellipodia of mi-grating border cells in the Drosophilaovary revealed by a GFP-actin fusionprotein. FEBS Lett 445:395–401.

Villecco EI, Genta SB, Sanchez Riera AN,Sanchez SS. 2002. Ultrastructural char-acteristics of the follicle cell-oocyte inter-face in the oogenesis of Ceratophryscranwelli. Zygote 10:163–173.

Vincent JP, Dubois L. 2002. Morphogentransport along epithelia, an integratedtrafficking problem. Dev Cell 3:615–623.

Weigmann A, Corbeil D, Hellwig A, Hutt-ner WB. 1997. Prominin, a novel mi-crovilli-specific polytopic membrane pro-tein of the apical surface of epithelialcells, is targeted to plasmalemmal pro-trusions of non-epithelial cells. Proc NatlAcad Sci U S A 94:12425–12430.

Wood W, Jacinto A, Grose R, Woolner S,Gale J, Wilson C, Martin P. 2002. Woundhealing recapitulates morphogenesis inDrosophila embryos. Nat Cell Biol 4:907–912.

Wucherpfennig T, Wilsch-Brauninger M,Gonzalez-Gaitan M. 2003. Role of Dro-sophila Rab5 during endosomal traffick-ing at the synapse and evoked neuro-transmitter release. J Cell Biol 161:609–624.

Zecca M, Basler K, Struhl G. 1995. Sequen-tial organizing activities of engrailed,hedgehog and decapentaplegic in theDrosophilawing.Development121:2265–2278.

Zelhof AC, Hardy RW, Becker A, Zuker CS.2006. Transforming the architecture ofcompound eyes. Nature 443:696–699.

3418 DEMONTIS AND DAHMANN