PRVELOPMENTAL BIOLOGY 171, 415-433 1995) Integrins and the Development of Three-Dimensional Structure in the Drosophila Compound Eye Robert L, Longley, Jr., and Donald F. Ready Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 A stereotyped, three-dimensional network of cell-cell contacts mediated by adherens junctions and cell~extracelhular matrix contacts mediated by focal adhesions defines the architecture of the Drasophifa ommatidium. Developmental reconstruction shows that this network is builtin an incremental and generally conservative sequence, contacts established early in eye development typically persist into adulthood. Reconstructions show that photoreceptor apical surfaces are involuted into the retinal epithelium and are subsequently elaborated to form the photosensitive rhabiomeres. Rhabdomeres become {0 the ommatidial optical axis via their anchorage to the retinal floor at the cone cell plate, a specialized nexus of cell and cell-extracellular matrix contacts, Parallel reconstructions of retinal development in integrin mutants show that several eve phenotypes trace their origin to the structural failure of the cone cell plate. ©109 Acis Ps INTRODUCTION 1995}, Patten formation in the retina takes place in the plane defined by these junctions. Beginning along the mor- ‘The Drosophila compound eye poses a simple example ‘ofa general problem in morphogenesis: how does three dimensional structure arise within a two-dimensional epi- thelium? Although devived from an epithelial monolayer, the cells of each unit eye, oF ommatidium, ate intercon: neeted ina three dimensional network of cell-cell and cell extracellular matrix (ECM) contacts. Functionally ime portant features of ommatidial architecture, for example the alignment of the thabdomeres and the photosensitive membranes of the photoreceptors with the optical axis of the ommatidium, arc decermined by chese contacts. The regular, repetitive cellular landscape of the developing fly cyeand its access to manipulation using genetic and moles: ular methods presents an opporcunity to examine how cell~ cel and cell-ECM junctions are regulated and cooperste to produce a complex tissue ‘Typical of polarized epithelia, cell-cell contacts in the developing Drosophila compound eye are mediated by ad- herens junctions, multimolecular linkages which couple the microfilament eytoskelctons of adjacent cell (reviewed in Fristrom, 1986; Luna and Hitt, 1992; Kemles, 1993). Ad- hherens junctions contribuce to the mechanical stability of the epithelium, demareate functionally and structurally dis tinct plasma membrane domains, and have been incteas- ingly implicated in signal ansduction and the coordination fof cell shape changes (reviewed in Kiekpateik and Deiter, cor. eogps 1009, Gert ey enon phogenetic furrow, the leading edge of retinal differentia on, ommatidial precursors are assembled through the step- wise recruitment of cells into stereotyped clusters (re viewed in Wolff and Ready, 1993}, As cells join a growing cluster, the adherens junctions they share with previously recruited cells become stabilized, Although subsequent de- velopment radically reshapes ommatidial cells, the june- tions formed between them during recruitment persist into the adult retina Cell-ECM contacts in the fly eye build a specialized ret nal floor, the fenestrated membrane (Cagan and Ready, 1989}. ‘The basal endfeet of the retinal pigment cells, the cells which make the compound eye's honeycomb of ommatidial cham- bers, flatcen into plate-like tiles which pave the floor in a regular hexagonal patter. Each ommatidial bundle of eight photoreceptor axons projects through the floor at specialized holes or ports. These axon exit ports are ringed by focal adhe: sions which anchor a planar network of stress fibers that sive the pigment cell feet their plate-like morphology. Focal adhesions are commonly encountered at the cell-substrate ‘contacts of cultured fibroblasts, where they mediate stress fiber linkages to the substrate [reviewed in Burridge, 1986, ‘Burridge etal, 1988, Burridge and Fath, 1989}. Like adherens junctions, focal adhesions are increasingly recognized as sites specialized for both mechanical coupling and cell-ECM sig: naling {reviewed in Juliano and Haskill, 1993} as3D Structure in Drosophila Compound Eye Integrins, 2 family of heterodimeric receptors, provide the transmembrane link of focal adhesions by binding ECM proteins with their extracellular domains and connecting to stress bers at their cytoplasmic domains through a com plex of cytaskeleton-associated proteins {reviewed in Hynes, 1987; Albelda and Buck, 1990; Hynes, 1992}. Dro- sophia integrins were first identified in monoclonal anti body screens designed to detect spatially restricted proteins in imaginal dises and were therefore designated position- specific {PS| proteins Wilcox et al, 1981; Brower et al., 1984}, These observations led to the identification of two Integrin receptor complexes having distinct « subunits and sharing a common subunit, aysiBos and aps, (Wilcox et al, 1984, Bogaert et al., 1987; Leptin et al, 1987). Subse- quent analysis of the ars: gene, inflated (if, the ars gene, multiple edematous wings (mew, and the Acs gene, my- ‘spheroid (mys), shows that the proteins have strong simi larity with their vertebrate counterparts {MacKrell et al., 1988, Wilcox er al_, 1989; Wehrli et al, 1993; Brower et al. 1995}, Recent observations have added a new f subunit, B, (Yee and Hynes, 1993} and a new a subunit, ans, (Gotwals et al, 1994} co the fly integrin family. Genetic analyses of integrins during Drosophila develop. ment indicate that they funetion in cell attachment {re- viewed! in Brown, 1993). mys mutants were first identified ‘as embryonic lethals in which somatic muscles pull free from their epidermal attachment sites {Wrighe, 1960; New- ‘man and Wright, 1981). Similarly, clones homozygous for null alleles of either mys or if show blisters in the wing ‘where the apposed epithelia which form the wing blade 417 have separated (Brower and Jaffe, 1989, Zusman et al., 1990; Brabant and Brower, 1993). Retinal development is also de fective in integrin mutants. Clones of retinal tissue homo- _2ygous for the null allele ofthe rs subunit, mys*°*, exhibit defects in thabdomere number (less than seven ‘thabdo- ‘meres in a given plane}, etzaction of rhabdomeres from the retinal floor, and a general disruption of the retinal floor (Zusman et al., 1990, 1993), In the present study, developmental reconstructions were used to trace the reorganization of adherens junetions, cell shape changes, and the formation of the fenestrated met brane during the pupal stage of retinal morphogenesis. We find that the adherens junctions are instrumental in orient- ing the apical surfaces ofthe photoreceptors onto the optical axis of the ommatidia, coordinating their elongation within the epithelium and their attachment to the cone cell plate, a specialized nexus of cell and cell-ECM contacts which anchors the photoreceptor apical surfaces to the floor. In addition, we have used these reconstructions to extend the observations made in integrin mutants and show that the retinal phenotypes trace their origin to the structural failure of the eone cell plate and the focal adhesions of the pigment cells MATERIALS AND METHOD! Drosophila Stocks All stocks were raised at 20°C on standard com meal agar medium. Since autofluorescence obscures detail in pig: HIG.1. Photoreceptor apical surfaces are involuted during development {A,B,C} Schematic side views. (D, E,F} Schematic apical views showing the outlines of adherens junctions between photoreceptors and cone cells {A) Ina thied instar eve dise, approximately nine rows bbchind the furrow, photoreceptor apical surlaces are exposed on the apical surface of the epithelium. [8 In « papal eye at 37% pad. (pupal development) the apical tips ofthe photoreceptors have becn involuted into the epithelium by the closure uf the cone cells: Cone cell processes have met mid-center atthe floor ofthe oramatidium to ereate the cone cell plate. {C} In a topologically mature ommatidium, a 67% pa, expansion of photoreceptor apical surfaces has brought them into contact withthe cone eel plate. Adherens janetions anchor the inner, proximal end of photoreceptor apical surfaces to the cone eell plate. Tor clarity of the diagram, RY has been swing #0 the anterior and the connection of R7 withthe cone cell plate is not dawn. (D) Adhereas junctions between photoreceptors and cone cells Tie in stereoeyped, planar configuration nine rows behind the furrow. Note that Riis not connected to RB, Compare ta (A) [F, oss ‘eyed pai, E’ strcoseope par} R cell apical surfaces form a simple pocket below the cone cells untl 37% pa. Nove that RB forms the floor of the pocket and R¢ does not share an adherens junction with RB. The sweeping arcs closest co the viewer ae pactial outlines of the cone cells which are exposed on the surface ofthe epithelium. Compare to (Bl, (FF) By 67% pa, the cone cell plate has become the floor of the IRS. RS has swing into ite mature basal and antetior position. Fr clarity ofthe diagram, the contact between R7 and RB is snot drawn. However, this junction is maintained and drawn “in front of” Ri's apical face, This movement establishes the orthogonal ‘meeting of the RY and RS thabdomeres on the central axis ofthe mature ommatidium. bl, basal lamina; 2-2, zonula adherens junctions, 4, cone cells, IRS, interhabdomeral space; 1°, 2, and 3, primary, secondary, and tertiary pigment cells; si, stress fibers; fa, focal adhesions; cp. cone eel plate FIG. 2. Developing rhabomeres are bounded by 2.2. junctions. {A} In third instar eye discs, photoreceptors (R] expose a microvllar caxcamb a the apical surface ofthe epithelium, Adherens junctions jz. form an epicolatcral belt which join adjacent cells and separate the apical and basolateral domains. {B| At 37% pad, photoreceptor apical surfaces face the tapped apical cavity, the IRS, formed by the Closure of the overlying cone cals (CC). Adherens junctions surround the microvillus photoreceptor apical membrane. (C| By 55% p.d, photoreceptor apical surfaces have elongated and anchored to the cone cell feet (CCF) the outer, distal ends ofthe photoreceptors are anchored to the overlying cone cells (CC), The forming shabdomere [ss bracketed by Za. junctions (D} Cross section ofan adult wi ‘ype ommatidium displaying the shabdometes of photoreceptors RLR7 in an opes, eapezoidsl pattern. The rhabdomereof RS lies hel ‘that of RY. Rhabomeres and the stalks which heat them face the interthabdoryeral space TRS}, Adherens junctions connect phoworeceptars to their neighbors and clase the IRS, Seale bars, ym,Longley and Ready3D Structure in Drosophila Compound Eve eel dei (nixon) £ ace of pupal developent ae j | : 4 Ba Tay sti ce of pp deepen FIG. 3. (A) Wild-type retinal depth at 20°C over the course of pupal development. During the early stages of retinal morpho: genesis, the epithelial thickness remains relatively the same. After the photoreceptor apical sustaces have anchored them- solves to the cone cell plate at $5% p.d, there isa rapid increase sm retinal dopth, Adult retinas reach a final depth of about 100 um. (8) Wile-eype rhabdomere depth at 20°C over the course of pupal development. Changes indepth after 56% pl. parallel the deepening of the epithelium, 419 mented eyes, w! was used to establish the baseline of nor mal retinal’ development. mys#/EM7_ [Costello and Thomas, 1981; De LaPompa et al, 1989; Wilcox et al., 1989) and y D/(1}w67¢2;pCHSPSP flies were « generous gilt from Dr. Mare Brabant and Dr. Danny Brower, University of Ari zona, mys‘*/EM7 |Wieschaus et al,, 1984; Digan et al. 1986, Leptin et al, 1989) flies were a gift from Dr. Susan Zusman and Dr. Richard Hynes, MIT. Transmission Electron Microscopy Flies were prepared for transmission electron microscopy ‘essentially a5 described in Baumann and Walz (1989) with modifications. Staged pupac and adult flies were affixed to Alouble stick tape dorsal side up. Animals were impaled trough the thorax with a micropipette and injected with a mixed aldehyde fixative (1% formaldehyde, 0.88% slutor aldehyde, and 0.1 M sodium cacodylate, pH 7.4) As much hemolymph as possible was finshed out of the animals in order to allow penetration of the fixative. Pupae were then overlaid completely witha large dropo fixative and allowed. to incubate for 1 hr. In the case of adult flies, the heads ‘were severed after injection, placed on the tape, overlaid with a large drop of fixative, bisected, and allowed to incu bute for 1 hr, Revinas were then dissected and placed into 1 ml of fresh fixative for 4 hr, Samples were then incubated dvemight in 1 ml of 1% tannic acid in fixative, Retinas were next washed in 0.1 M sodium cacodylate butter for 30 min, incubated for 2 hrin 2% osmic acid and 0.1 M sodium cacodylate, given 3x 10-min washes in distilled water, and then incubated overnight in 2% uranyl acetate. The samples were then dehydrated by Semin washes in 50, 75, 9, and 100% ethanol, washed for 30 min in 100% propylene oxide, incubated in 50/50 mixture of propylene oxide and Epox 812 for 4h, and then allowed to incubate in 100% Epox 812 overnight. Samples were embedded in 100% Epox 812 and sectioned using a Reichert ultsa microtome. Staining of Whole-Mount Eyes with Rhodamine- Conjugated Phalloidin Pupae were affixed onto double stick tape, removed from their pupal case, and placed dorsal side up onto the tape, Retinas were dissected at room temperature into Ringers solution and transferred immediately to a solution of 4% formaldehyde in PBS for 30 min. For adult retinas, the head ‘was severed, stuck onto tape, and bisected in a 4% formalde- hyde fix; the brain was removed immediately to allow pene. tration of the fixative, Because the comes interferes with confocal micsoscopy, tungsten needles were used to remove the comea during fixation. Eyes were then washed 3 15 min in PBT {1 PBS and 0.3% Triton X-100}. The samples ‘were then incubaced overnight at 4°C in thodemine-coniu- gated phalloidin (Sigma Chemical Co, St, Louis, MO| at a final concentration of 2 pg/ml in PBT and 5% horse serurn, Retinas were washed 3x 15 min in PBT at room tempera: ture. Before mounting, samples were held for 15 min in a ennih © 1995 by Andes Fag In Al tf eps yfFIG. 4. Rhabdomere momphogencsis in wildcype retinas, (A) A eross section from a 37% pu ommatidium showing the thabdomeres of photoreceptors R1-RY.[B! Rhsbdomeres at 55% p.d. The apical surfaces now possess distinct microvillar and stalk domains. Note the plane of separation thet lies between neighboring microvils. (C] Rhabdomeres at 67% pad. The microvilli have elongated and are more tightly packaged. The IRS is bepinning to open up duc to the withdrawal ofthe photoreceptors from the center of the ommatiduin. ;D) habdomeres at 78% p.d. Microvili at the sides of the thabdomere ate shorter telative 10 those in the canter and the rhabdomere has taken on a more oval profile, Seale bats, 1 ym. pri ©1985 by Ace rs Ash pin nan for eer3D Structure in Drosophila Compound Eye small drop of the mounting medium (0.25% n-propyl gal: late, 50% glycerol, and 1x PBS, pH 8.6), Retinas were mounted onto slides using coverslip risers to prevent the tissue from being crushed by the coverslip. Retinas were mounted basal side up for viewing ofthe retinal floor, sess fibers, and cone cell plate retinas were mounted right side up for analysis of phocoreceptors. Samples were examined and photographed using a Bio-Rad MRC-600 confocal mi croscope. Immunolocalization Mouse monoclonal antibodies directed against the arse subunit (MAb CF2C7, Brower etal, 1984) andthe fy, sub unit [MAb CF6GIL; Brower et al, 1984) were provided by the laboratory of Dr. Michael Wilcox. The mouse mono. clonal antibody directed against the ayay subunit (MAb DMIA3, Wileox et al, 1981) was provided by Dr. Richard Smith, MRC, Cambridge. Retinas from both pupae and adult animals were dissected, fixed, and permeabilized ac cording to the procedure described for phalloidin staining Eyes were incubated overnight (2 minimum of 12 hr) at 4°C in primary antibody diluted in PBT plus 5% horse serum. Samples were washed 3x 15 min in PBT plus 5% horse serum and incubated fora minimum of7 hr at 00m temper ature in biotinylated anti-mouse antibody (Vector Labora: tories} diluted 1.250 in PBT plus 5% horse serom. After 3x 15-min washes in PBT, retinas were incubated for a ‘minimum of 2 hr at room temperature in a 1:500 dilution of thodamine-conjugated avidin in PBT. Samples were mounted as described following 3x 1S-min washes in PBT. ‘Measurement of Retinal Floor Area Retinal floor areas were calculated from phalloidin: stained whole mounts using the Comos image analysis soft ware of the Bio-Rad confocal microscope. For each time point measured, ewenty retinas from different animals were examined. A defined unit area of the retinal floor, alhexagon formed by six grommets, was measured, Means were caleu lated from these measurements. Three potential sources of error were considered, Fist, fixation can lead to shrinkage of tissue samples, meaning the areas measured could be smaller than the actual in vivo area. To determine the extent of this decrease, retinas from upac aged 10 38% of pupal life were dissected into Ringers and mounted for video microscope examination, and a tac. ing was made of a selected hexagon. A 4% formaldehyde solution was then infused under one end of the coverslip while the Ringers was drawn off at the other end, Thirty minutes after fixation, a second tracing of the same hexagon wwas taken, A comparison of the two hexagon areas showed an average decrease of 4%. Second, in order to make comparisons of ares from differ ent retinas, area measurements must be made from the same region of the eye and the same plane of section for each eye. The plane of section in each ease was made such 421 that the cone cell plate was visible in all six of the hexagon grommets; the cone cell plate is about 12 um thick. Mea surements were made only from the center of each retina For several retinas, after a measurement was made, the mi croscope was focused away from the measured plane and then refocused. The distances between the original and the second focal plane were found to be within 0.5 am, showing that the plane of section is well defined. In addition, areas taken from 2 um either above or below the optical plane differed on average by only 19 yan’, demonstrating that there is not a sharp difference in area on either side of the plane of section which could substantially affect the mea surements, ‘Third, since the center of the grommet was determined by eye when measuring, itis possible that the vertexes of the hexagon would be off center and the measurement dis: torted. The areas for 10 measurements were therefore caleu- lated twice. It was found that the two readings differed on average by 0.1%. ‘An analysis of variance (ANOVA was used to evaluate the effect of age and left or right eye on the cross-sectional ‘rea measurements. The analysis was used to determine if the areas differed in the rate of contraction aver develop. ment, The ANOVA reveals a significant age effect, Fi, 45} = 798, P< 0.0001, meaning that the hexagon areas differ among the age groups. Further analysis with a Newman- Keuls’ post hoc test indicate that all age groups differ with the exception of 80% of pupal life and 7-day adults. The ANOVA also indicates that there is no difference between right and left eye measurements, Fl, 45) = 2.84, P > 0.08, ‘and that there is no significant age x eye interaction, M4, 45) = 0.5, P > 0.7, meaning there is not a time during development when the left and right eyes differ in hexagon ‘A variation of the above procedure was used to calculate the hexagon areas of mys mutants. In many instances, mu tant retinas have ommatidia in which the cone cell plate contacts are disrupted and the cone cell feet have detached from the floor. Because the cone cell plates could no longer be used to identify the proper plane of section, the stress fiber network of the secondary and tertiary pigment cells was used as a reference point instead. This optical plane Ties about 2 pm below that of the cone cell plate; however, as noted above, the area of the hexagons at this lower level dhffers litte from that of the cone cell plate level of section. ‘Measurement of Retinal Depth To determine retinal depth, phalloidin-stained retinal whole mounts were mounted distal side up with risers. The distal-most optical section was used as a reference point and the microscope focused through che sample in T-4m steps until the floor of the retina was reached, The floor ‘was defined as the point beyond which the feet of the see ondary and tertiary pigment cell feet are no longer visible in profile; this plane of section is about 2 um helow the cone cell plate. Measurements from retinas that are ewo- Ccopmah ©1995 Aelemi Pr, Allg epi yn ed422 Longley and Ready3D Structure in Drosophila Compound Eye 423 FIG. 6. Patteming of the retinal floor during pupation. |A) A cross section of 2 20% p.. retina stained with rhodamine-conjugated ‘haloidin, The floor ofthe vetina is unparterned a this time due to-an excess of cells (8) By 37% pd. surplus cells have been eliminated and the floor cakes on 2 petal pattem With the secondary and tertiary pigment cells and bristle complexes forming the petals and the photoreceptor axon bundle forming the ircula center. Secondary and tetaty cells are anchored to two and ehree grommets, respectively. Bristle complexes and tertiary pigment cells occupy alternating positions. (C} By 67% pu, a stress fiber network has been constructed in the feet ofthe secondary and ttiary pigment cells. (D} Inn adult retina, the feet ofthe secondary and tetany pigment cells have drawn igo small profiles dominated by stress fibers. Scale bars, 10 urn FIG.5. Cone cell plate formation and the grommet ECM in wild-type retinas. (A| Ommatidial eross section showing the positioning of the cone cell fet {CC) before 14% p.d. The feet lie to the sides of the axon (ax) bundle. In this section it cannot be determined which of the fixe profiles surzounding the axon bundle is not a cone cell and accordingly five are labeled as cones. (B) At 14% pd, the anterior [ACC] and posterior (PCC) cone cells extend flanges that meet mid-center and divide the photoreceptor axons into two groups. The equatorial (ECC) and polar (PoCC| cone cells remain at the periphery, (C] At 37% pa, the equatorial and polar cone cells extend! anges mid-center to complete plate construction. Axons aze broken into individual profiles, (D) Between 37 and 55% pad, the plate increases in size and, withthe anchorage of the photoreceptor apical surtaces, reaches its mavure state. The photoreceptor ‘axons pass around the periphery ofthe plate. (£) The grommet, the ring of ECM 10 which the base] endfect of the pigment eclls (FC) are anchored, is indicated inthis 55% pad. cross section. The ECM is perforated by the exiting photoreceptor axons. [F} Photoreceptor ‘axons gather into 4 bundle below the perforated dise of ECM seen in E. Note the apposition ofthe pigment cell feet to the grommet ECM, Sesle bars, 1 pam. Cheyne © 198 sd Pey Ali pedsin nay fr ene424 ag (qe mice) FIG.7. Longley and Ready Toe Sse GIN — ore Toy aT Perce of pupa evelopment Changes in retinal Moor area over the course of pupal development, (A}A defined unit ofthe retinal floors hexagon of photoreceptor axon bundles (AB), was measured and used to examine fluctuations in sloor ares during morphogenesis. (B) Wildcype retinal oor area at 20°C increases about twofold between the beginning of pupation and S5% p.d, Between 85 and 67% the floor decreases twofold, by the ‘due stage, the floor has undergone a fourfold reduction thirds of pupal life or later in age do not include the depth of the comes due to the necessity of its removal. Rhabdomere lengths were measured in a similar manner. RESULTS Photoreceptor Apical Surfaces Are Involuted into the Retinal Epithelium and Become Anchored to the Retinal Floor Approximately 9 rows behind the morphogenetic furrow, developing ommatidia have recruited theit adult comple: ment of 8 photoreceptors (R cells) and 4 cone cells (Figs. 1A and ID), RB occupies the center of the cluster and possesses adherens junctions with all of the ocher photoreceptors ex- cept for Ra [Tomlinson, 1985}. The cone cells surround the R cells and are joined to them by adherens junctions. Duc tw its central position, RS does not share adherens junctions with any of the cone cells. The apical membranes of all 12 cells remain exposed on the surlace of the epithelium at this stage (Fig, 2A). Berween 18 and 22 rows bebind the furrow, the 4 cone cells close above the apical tps ofthe photoreceptor, elfac- {ng them from the retinal surface (Figs. 1B, LE, and 28) Like the closure of a drawstring pouch, photoreceptor apices are snvoluted into the epithelium, Adherens junctions berween photoreceptors are maintained during involution and photo- receptor apices are turned to face each other in a trapped apical cavity, the fiture interchabdomeral space (IRS). At this stage, the apical face of RB forms the floor of the IRS. and newly formed adherens junctions between the cone cells scal the roof, Following their involution, photorecep: tor apical surfaces remain ditectly beneath the cone cells until about 37% of pupal life Between 37 and 55% of pupal life, photoreceptor apical surfaces enlarge and extend downward until they reach the cone cell plate a structure atthe base of the ommatidium formed by the meeting ofthe cone cel ee see below) (Figs 1C and IF}. As the photoreceptor apical surfaces reach the plate, they connect to it through newly formed adherens junctions. Junctions beeween R8/R3, RB/RS, and RA/RS are lost as nev ones are established with the plate; the cone cell plate replaces RB asthe floor of che IRS. Since che cone cell plate is attached to the retinal basal larming, the apical surfaces of the photoreceptors, the farure chabdomeres, De- comme anchored inditecty tothe retinal floor and aligned to the optical axis of the ommatidium (Fig. 2C) ‘Aer the photoreceptor apical surfaces become anchored to the cone cell plate, the entire retina undergoes a substan tial increase in depth (Fig, 34), Between 85 and 67% of pupal life, the retina deepens from 31 to $5 um. Adult retinas reach a final depth of about 100 pm. Photoreceptor topology is conserved during the enormous expansion of the apical surfaces which accompanies retinal deepening Fig. 38}. The adherens junctions between photoreceptors in mature om- matidia, although deformed and elongated, are the con served descendants of the subapical circumferential junc: tions of eye dise cells (Fig. 2D) Rhabdomere Morphogenesis Prior to the onset of chabdomere moxphogenesis, the api- cal surfaces of the photoreceptors are thrown into irregular Spy 985 by Acres eA epi nay fr esBD Structure in D Integrins atc localized t0 the grommet/cone cell plate complex. [A| At 31% pad, anti-B staining i not detcetable at the retinal (B) At 37% pd, anticBps antibody stains strongly around the grommet {compare to Fig. 5E} and more weakly along the borders between the secondary aad tertiary pigment cell feet. C) A closeup of the grominet labeling seen in B. The central staining corresponds ‘The outer ring comesponds to the ends of secondary and tertiary pigment cell feet that converge on the grommet ECM. (D! A plane of section } jam below that seen in C shoves the staining ofthe pigment cells azound the grommet (F) Labeling of the ccone cell plate snd pigment cells with an antibody against the cs subunit (F} Labeling of the cone cell plate and pigment eels with an antibody again ne Alege nay fr re426 Longley and Ready3D Structure in Drosophila Compound Eye ‘membrane projections (Fig. 4A}. At about 55%, two distinet apical domains become apparent: a central fringe of short ‘microvilli facing the futute IRS and a flanking stalk domain that extends smoothly to the adherens junctions (Fig. 4B) Each rhabdomere is built against a stereotyped combination of stalks and thabdomeres of apposed photoreceptors in 2 pattern which prefigures the trapezoid of che adult omma- tidium, For example, R3, whose rhabdomere lies atthe peak of the adult trapezoid, builds its microvillar fringe against the stalks of R2 and R4, R4 builds its rhabdomere against the rhakdomeres of R2 and R7. A clear plane of separation marking the future IRS lies between apposed rhabdomeres. By 67% of pupal life, shabdomeral microvilli have deep- ened and become more organized (Fig, 4C). Stalk and rhab domere distinctions are especially pronounced in R2, Ré, and R7. The IRS has begun to open at this stage. At 78%, the rhabdomere begins to take on a more elliptical profile as microvilli become graded in length from side to side (Fig, 4D}. Continued elongation of microvilli and withdrawal of the outer thabdomeres from the ommatidial center bring the photoreceptors to their adult form. Cone Cell Plate Formation From their recruitment in the thisd instar eye dise until 14% of pupal life, the cone cells lie entirely to the sides of the photoreceptors the basal feet ofthe cone cells surround the axons as they exit the retina, (Figs. 1A and 5A). At 14%, the anterior and posterior cone cells extend flanges which meet mid-center of the ommatidia, partitioning the eight pho: toreceptors into two groups of four [RI, R6, RZ, RB and RO, RS, RA, RS) (Fig. $8), The busal feet ofthe cone cells remain attached to the basement membrane during the extension, ‘AL 37% of pupal development, the equatorial and polar cone cells extend similar flanges between RI and R7 and 13 and RA, respectively. These join with the basal feet of the anterior and posterior cone cells to form the cone cell plate Fig. 8). The plate subsequently enlarges in size and bbecomes consolidated in shape until 55%, when phocore- ceptor apical surfaces become anchored to it. Photoreceptor axons pass individually around the periphery of the cone cal feet (Fig, SD). A disc of ECM, perforated by individually exiting axons, lies below the cone cell feet [Fig SE}. It is linked to the retinal floor at the grommet, the sleeve of ECM that sur rounds photoreceptor axons which group into bundle be 427 low the cone cell plate (Fig. SF). The grommet is continuous ‘with the basement membrane ECM that extends below the basal endfeet of the retinal pigment cells. Retinal Floor Development Cell substrate contacts in the developing eye remain rel atively unspecialized during pattern formation. Behind the furrow, the fenestrated membrane, a new basal lamina ‘which will become the floor of the adult retina, develops ina plane just above and parallel to the original basal lamina of the epithelium. Photoreceptor axons pass through the fenestrated membrane, while the basal endfeet of other cells are linked to it at simple contacts. ‘The retinal floor is unpattemed early in pupal life (Fig. 6A), At this stage, the retina contains a surplus of cells from which the pigment cells and bristle-forming cells will be recruited. By 37% of pupal life, cell death has eliminated excess cells and the floor now exhibits a regular pattern, with the secondary and tertiary pigment cel feet converging, ‘on the grommets [Fig. 6B). By 55%, dense adhesion plaques, typical of focal adhesions, have developed along the cyto: plasmic face of the pigment cell membrane apposed to the grommet {Fig. SF) A stress fiber network is established in he feet of che pigment cells between 61 and 67% of pupal life (Fig. 6). Stress fibers are anchored at the focal adhesions apposed to the grommet and run in a planar network parallel to the retinal floor. Formation of the network coincides bath with a reduction in retinal floor area (Fig, 7|and with the deepen {ng of the retinal epithelium (Fig. 3}, suggesting that it may provide a tensile force that helps to maintain floor integrity uring times of stress. The retinal floor reaches its fall ex- tent of contraction by eclosion (Fig. 6D) Integrins Localize to the Cone Cell Plate/Grommet Complex Integrins localize tothe cone cell plate/grommet complex beginning with the resolution of the mature floor pattem 2° 87% of pupal life, immunostaining using an sntiePy ati body highlights the focal adhesions which join the pigment cell fet to the grommet ECM (Figs. 8A and 8B), Lighter Staining ss also seen along the contacts between pigment cll fect (Fig. 88). Contacts between the conc eel feet and the underlying plate of ECM also stain with antnfs (Fig FIG. 9. Defects during mys" retinal momphogencsis.{A) A cross section from a 55% pd. wild-type retins showing the positioning of the R8 photoreceptor. The cell body and nucleus. IRE are anchored between photorcceptoys Rl and R2. (BA cross section from a 55% pd. mys™® recina. Although RS maintsns its za, contacts with RI and R, the cell hody and nucleus of RB have fallen thtough the retinal floor. (C} The movement of R8's cell body shifts R8 into the middle of the ommatidium and interrupts cone eel plate contacts {D) The nucleus of RS comes co zest in the axon hundle underneath the cone cell plate (E] A cross section from a 67% pd. ys” retina By this point im development, the pigment cell feet have pulled away from the grommet ECM, opening holes around the cone eell plate [compare with C).[F| A longitudinal schematic demonstrating the positioning of the RB cell ody in wild.aype and mys™® retinas, The tose hatched cizcula proSile is the nucleus, Seale bars, 1 ym. Contig © 1955 by Andie Peni: Al ht epiton ny frm ered428 8C}, Below this place, in the region of bundled axons, anti- {rs shows a simple ring of the pigment cell endeet |Fig. 8D), Identical staining patterns were detected using antibodies against the integrin ays; and as subunits (Figs. 8E and $F} ‘The staining patterns developed at 37% persist throughout the remainder of pupal development and in adults. Integrins Are Required for Floor Integrity ‘The distribution of integrins in the developing eye sug: gests that they may play a role in maintsining retinal floor incegrity and organization by mediating the attachment of the pigment cell feet and cone cell feet to the retinal ECM. Retinal development of the mys" mutant, a temperature hypomomph for the firs subunit, supports this suggestion. Eatly stages of pupal eye morphogenesis including the formation of the adherens junctions between the photore: ceptors and the cone cell plate, the spatial organization of photoreceptors RI-R7, the extension of the photoreceptor apical surfaces to the cone cell plate, and the formation of the focal contacts between the pigment cell feet and grom: met ECM are unaffected in mys"® animals (not shown) Matant retinal development begins to break down at 55% of pupal life, At chis point in wild-type development, R8's cell body has moved anteriorly between RI and R2 {Fig 9A}. In approximately one-half of similarly staged mys” ‘ommatidia, R8's ecll body has fallen through the retinal floor, coming to lie underneath the cone cell plate (Figs. 9B and 9D), Conelated with this aberrant movement of R8 is a disruption of the normal contacts between the cone cell feet (Fig, 9C, compare to Fig, SD]. Note that the pigment cell feet have not pulled away from the axons at this stage; floor contraction has not begun at this stage and presumably these contacts are not under increased stress, By 67% of development, all mys" ommatidia contain a displaced RS cell body and a disrupted cone cell plate. In addition, the pigment cel fect have now pulled away from theiranchorage sites with the grommet ECM, opening holes in the retinal floor and allowing intrusion of the basement membrane ECM [Fig, 9E), Disruption of the anchorage sites oes not affect stress fber construction or interfere with retinal floor contraction. The cone cells maintain theit at- tachment to the grommet ECM. By the adult stage, the retinal floor is completely dis rupted. The pigment cell fect have drawn up to small pro- files that are no longer in contact with each other (data not shown}. The resulting holes produced in the floor are mainly filed by an enlargement of the photoreceptor axon bundles Also, the cone cell feet have pulled free from the retinel floor and have come to lie above it. Since the shebdomeres are anchored to the floor via the cone cell feet, the thabdo- meres also lose their foothold on the floor, This loss of normal anchorage allows them to buckle along their length [Fig, 10A) and in cross section can give the appearance of 2 photoreceptor possessing an intemal rhabdomere (Fig, 10B) Longley and Ready Tongitudinal section from a 7-day-old mys" retina stained with rhodsmine-conjugated phalloidin displaying photorecep tor rhabdomeres, individual thabdomeres appear as bands that extend the depth ofthe retina. The arrow points to asite where 2 thabdomere buckles in on itself and intrudes into the cell hy. [B}A cross section from a 7-day-old mys™” retina showing buckled thabdomere, giving the appearance af a receptor pos- sessing an internal shablomere, A plane of section correspond ing to the arrow shawn in A gives such an image. Scale bat, Vy Compnaht © 1995 by Adem Fes, nell es ees yf se3D Structure in Drosophila Compound Eye HG.11 Diginene cell feet have drawn Biabdometes da not extend ta ye retinal floor ( Normal Integrin Function Is Required during Cone Cell Plate Formation mys is temperature-sensitive, allowing the temporal requirement for normal integrin activity to be examined, mys pupae raised at 30°C from the white prepupal stage to 67% of pupal life exhibit more severe defects than those seen in pupae raised at 20°C (compare Fig. LIC with Fig, 12A). The pigment cell feet pull free from their anchorage sites with the grommet ECM and draw up into small pro files. Stress fibers persist alshough phalloidin staining along their length is sometimes reduced in intensity. In addition, the rhabdameres and cone cell fect pull free from the floor (Fig. LID), 2 phenotype not scen until eclosion in flies raised at 20°C. Mechanical stresses appear to underlie these de- fects. mys animals raised at 30°C until 85%, before the onset of floor contraction and retinal deepening, do not show floor disruption and rhaldomere detackment {aot £21995 AenlemPe,In Al Wild-type |A, Bl and mays" (C, D) retinal development at 37°C, Phalloidin-sained eyes at 67% pa. (A grommets in a willstype retinal floor. (B] Rhabsomeres extend to the floor {arzow/ in wildtype ommatidi. (C} lingo small profiles, opening large gape. Suess hers ae stil present in ehe pigment eel feet. (D| my iw. Note that thabomeres in 429 res fibers link tight ‘mys retinas, the ferent ommatidia extend to diferent lengshs. shown). Wild-type pupse raised at 30°C showed ncither floor disruption [Fig. TA) nor thabomere detachment fFig. 118} Although floor and thabsiomere defects become manifest only under latedeveloping sess, normal integrin activity is required beginning a 37%, che onset of integrin accumu lation a he grommet, to prevent these defects. mys" white prepupae raised at SO°C until 87% and then shifted 10 20°C vant 67% (Fig. 128) show a patéern of stress Aber formation and retinal floor organization comparable to mu: tant pupae raised at 20°C (Fig. 12) However, ifthe animals ate held at 30°C until 44% belore being retumed to 20°C, pigment cell detachment from the axon bundles is dis: rupted, resulting n large gaps (Fig. 12C]. A pulse of reste Live temperature between 37 and 44% produces similar dis ruptions {Fig 12D}. A continuing requirement for normal integrin activity i indicated by the observation that shifts ro 40°C between 55 and 67% prodsice phenotypes equiva430 FIG. 12. The semperature-ser animal raised from the white pr ‘oor organic ve pe Longley and Ready w1 of mys" {A) A crass section showing the organization of the retinal floor from a mys I stage to 67% pd. at 20°C. The eye is stained with showamine-conjugated phalloidin, (8) Retinal fora 4 mys" aninal raised from a white prepupae t0 37% pa. st 80°C and then allowed to develop to 67% at 20°C, {C) Retinal floor organization from a mys" animal raised from a white prepupac (© 48% pad. at 30°C ang then allowed to develop 19 {57% at 20°C. Noe how faethe pigment cell feet have pulled away from axon bundles. (D} Retinal Floor organization feom a mys" snimal raised from 8 white prepupae 037% at 20°C, rized ffom 37 to 44% at 30°C, and then allowed to develop to 67% at 20°C, Compare with the floor presented in C Jent to maintaining the animals ata restrictive temperature from the beginning of pupal life (not shown) Integrin-mediated attachment of the shabdomere to the retinal floor appears necessary for the elongation of the thabdomere which accompanies retinal deepening (Fig. 13). Heterozygotes of mys*** and the null allele mys" show the same spectrum of retinal phenotypes as mys homozygotes, but with increased severity. When znys*"/ myst heterozygotes are raised to 55% at 30°C, photorecep: tor apical surfaces reach the retinal floor normally, but by 667%, all chabdomeres have detached and lie, om average, 19 um above the floor, a distance close to the 24.jm increase in depth that normally occurs between 55 and 67% (Fig. 138). When returned t0 20°C, the heterozygote retina deep- ens approximately normally but the thabdomeres do not increase in depth; they remain at about the depth reached at 35%. Tension provided by anchorage to the floor may contribute to normal rhabdomere clongation, A mys Transgene Rescues the mys"** Phenotype ‘To confirm chat the defects in mys™? development are due to the mutation in the mys gene, the phenotype was rescued by using a fll length mys eD)NA under the control of the Tisp-70 heat shock promoter. Males from a wans- formant line homozygous fora P insert containing the heat shock construct (provided by Dr. Mare Brabant and Dr. Dan- {el Brower} were crossed to wimys™ virgins. Progeny were eamiahs © 55 by Aden From, eA igh eosin yf xeSD Structure in Drosophila Compound Eye erent of pupa development FIG. 13. Rhabdomere depth ftom mys" animals raised to 67% ped at 30°C and then allowed to develop to adults at 20°C, Compare ‘with the depths shoven in Fig, 3B. collected as white prepupae, raised at 20°C to eclosion, and eyes from 20 wmys"™; +/Hsp-70.mys males were exam: ined using confocal microscopy. All 20 showed intact reti- nal floors and wild-type photoreceptor organization (data rot shown}, In contrast, 20 wimys'™, +/+ males from a cross with w' flies ll exhibited a lack of floor integrity and disrupted photoreceptor organization, DISCUSSION ‘The sequential recruitment of cells during pattern forma: ‘ion establishes the foundation for morphogenesis in che developing Drosophila eye. Most of the cell-cell contacts of the adult eye trace their origin co the time at which the cell is recruited. For example, the adherens junctions between photoreceptors in an adult ommatidium are a sub- set of the junctions of the symmetrical 8-cell cluster. The ‘most pronounced reorganization of adherens junctions oc: ccurs with the interpolation of the cone cell plate into the floor of the IRS. The simple eye cup-like inpocketing of the photoreceptor apical surfaces is transformed into mature ‘ommatidial form as 8's junctions with R3, RS, and R6 are ‘exchanged for newly formed junctions with the cone cell plate. The closure of the cone cells “above” and “below” the photoreceptor apical surfaces forms a cage into which shabdomeres project. The continuity of the adherens june 431 tion belt encircling a chabdomere is maintained at both the inner and outer ends of the retina by junctions with cone cells, The mechanisms which govern adherens junction be havior in the eye remains to be determined, but they ate ‘well-positioned to integrate a variety of molecular and me- chanieal influences ‘The observation thet the rhabdomere is a subdomain of the apical plasma membrane complements Baumann et al’s (1994) immunolocalization of the blow fly photoreceptor NaK-ATPase to the nonIRS facing membrane plasma membrane; Na,K-ATPase is typically concentrated in the basolateral plasma membrane. The conserved apical topol- ogy of the rhabdomere also reinforces the perallel between the thabdomere and the brush border of the intestinal epi thelial cell (Arikawa et al, 1990}, Myosin and spectrin, both components of the brush border terminal web, localize to the base ofthe microvilli during the first two-thirds of pupal development (R. Longley, unpublished results}, however, we have not observed, even in the best preserved thin-sec- tioned material, a dense filamentous plexus beneath the rhabdomere which would correspond to the terminal web. Itis likely that adhesion between the closely packed micro villi of thabdomere provides a stractural support not avail- able to the free-standing microvilli of the brush border. Intcgrin-mediated cell-ECM contacts participate in Sev: cral mechanical aspects of retinal morphogenesis. By an- cchoring the fect of the pigment cells to the grommet ECM, they establish a retinal floor which limits the downward ‘migration of R8 during apical surface elongation to the cone cell plate and later withstands the forces generated during floor contraction, Integrin-mediated attachment of cone cell plate to the retinal ECM is essential for the phase of rhab- domere elongation which accompanies retinal despenin, Since both a5, re and cos, Ars integrins show identical distributions and mys mutations compromise both recep tors, i was not possible to assess the contribution made by each using mys alleles. Although both the viable ars hypomorph if and iP if" heterozygotes {if2” is an if ull, allele) show wing blisters (Brower and Jaffe, 1989, ncither exhibits retinal defects (R. Longley, unpublished results|, suggesting either that only low levels are required or that it may be redundant in the eye. These obscrvations are con- sistent with the those of Brower etal, (1995) that homozy ‘gous if null clones do not disrupt retinal structure, In con- trast, homozygous mew null elones disrupt rhabdomere or- ganization (Brower et al., 1995], suggesting that the PSI integrins play a dominant role in eye morphogenesis. ‘The ligand for retinal integrins remains to be determined. Laminin has been shown to be @ ligand for the as: rs receptor in cell-binding assays (Gotwals et al., 1994) and immunolocalization using an anti-laminin antibody (Fes- sler et al, 1987} indicates that the protein is present in the grommet ECM (R. Longley, unpublished observation], suggesting that rs, Brs could anchor the pigment cells and cone cell feet to laminin. However, severe hypomorphs for laminin A {Jamal exhibit roughened eyes and fused orama tidia, a distinet phenotype from mys" mutants (Hench. opti © 1995 by ead Pein Al of epic ay fen rel432, cliffe tal, 1993), suagesting that residual laminin suffices for grommet integrity or that integrins bind to other ECM components In vitro studies of cultured cells suggest thas integrins anchor stress fibers via cytoplasmic assemblies incorporat ing a number of proteins including talin, vinculin, and a- actinin [Burridge and Fath, 1989; Pavalko and Otey, 1994). Whether integrins play a similar role in retinal stress fibers is not known, Drosophila a-actinin has been identified as the protein product of the fliA gene [Fyrberg et al, 1990) and immunolocalization using an anti a-actinin antibody (Saide et al, 1987) shows that the protein is distributed evenly along the stress fibers and at their terminals (R. Long- ley, unpublished results) Stress fibers and a-actinin stain ing appears normal in the retinas of fi” mutants, hypo: morphs of nonmuscle a-actinin (R. Longley, unpublished results). Drosophila homologs of both talin and vinculin have yet to be identified. In addition to their role in cell attachment, integrins have been implicated in the regulation of eell growth, differentia- tion, and gene expression (Hynes, 1992, Juliano and Haskill, 1996, Sastry and Horwitz, 1993), From this perspective, it is notable chat mys” animals raised at the restrictive eem- perature for the entirety of the pupal staze and clonal patches homozygous for the null allele mys‘ (Zusman et al, 1990} do not show defects in cell division, cell reenuit- ‘ment, cell death, or pattern formation. Retinal phenotypes for mys mutants appear to stem solely from a failure of cell attachment to the ECM, Such a role is consistent with analyses of integrin function during Drosophila embryogen esis and wing morphogenesis. ACKNOWLEDGMENTS ‘We ae grateful to Pr, Danny Brower fr fly stocks and technical advice. We thank Dan Kichart for anti-specerin and antimyasia “antibodies, Drs, John and Lisa Fessler for ant-laminin antibody, and Dr, J Saide for anti-a-actinin antibody. This work was supported by the National Bye Insitate. REFERENCES Albelda, $, and Buck, C. (1990), Integrins and other cell adhesion molecules. FASEB |. 4, 2868-2880, “Anikawa, K,, Hicks, |, and Wiliams, D. (1990). Mdentification of ‘actin laments inthe ehabdomeral mictavill of Drosophila pho loreceprors. J. Gel! Biol. 110, 1993-1998, Baumann, O,, and Walz, B {1989}. Topography of Ca” sequestering ‘endoplasmic reticulum in photoreceptors and pigmented glial cells in the compound eye of the honeybee drone, Cell Tissue Fes. 255, 511-522, Baumann, O., Lautenschliger, B, and Takeyasu, K. (1994), Iman rolcalization of Ne, K-ATPase in blowily photoreceptor cells. Call Tssue Res, 275, 225-236, Bogaert, T, Brown, N, and Wileox, M. (1987. 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Zool. 43,77 8. ‘Yee, G., and Hynes, B |1993}.A novel, sssue-specific integrin sub: unit, , expressed in the midgut of Drosophila melanogaster. Developraent 118, 845-858. Zosman, 5, PatelKing, R, Hreaeh-Constant, C,, and Hynes, R [1990], Requirements for integrins during Drosophila develop- ‘ment. Development 108, 91-102 Zusmas, S, Yee, C., Caveats, F, and Hynes, R. (1993). Analyses (of PS integrin funesions during Drasophila development. Devel ‘opment 118, 737-750. Received for publication March 28, 1995 ‘Accepted June 14, 1995 Conright © 1985 Wy Adon Peak Is A ht epcatan a any arm eee,