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IntroductionMigration is a striking example of complex cellular behaviorthat is essential to development, wound healing and cellularimmunity. In the nervous system, cell migration is a commonstep in the final positioning of neuronal and glial cell bodiesprior to the assembly of neural circuitry. In the developingmammalian brain, neuronal precursors migrate along radialand tangential pathways to arrive at stereotyped destinationsin the developing telencephalon (reviewed by Marin andRubenstein, 2001). Schwann cell precursors migrate alongaxon tracts into the peripheral nervous system prior toensheathing axon fibers (Carpenter and Hollyday, 1992). Giventhe central role of migration in neural development, it is notsurprising that migration defects are common in humandiseases of cortical organization (Ross and Walsh, 2001).Nonetheless, the mechanisms by which migrating cells choosespecific pathways and locate appropriate destinations are notwell understood.

Drosophila melanogaster has been a favorable model systemin which to study cell migration. Genetic analysis has beenapplied to the mechanisms controlling movement of folliclecells around the developing oocyte (reviewed by Montell,1999) and gonadal precursors to the site of the developinggonad (Deshpande et al., 2001; Stein et al., 2002). In thenervous system, there are many instances of neuronal andglial migration from sites of cell birth to the locales ofdevelopmental and mature function, both in the CNS (Klambtet al., 1991; Klambt, 1993) and PNS (Giangrande, 1994; Choiand Benzer, 1994; Sepp et al., 2000).

It is evident that the migration of neural cells to proper

destinations can be a complex process, even in the relativelysimple nervous system of the fruit fly. The adult Drosophilavisual system contains glia of many distinct types andproperties that are localized to specific sites in the retina andoptic lobe (Tix et al., 1997; Saint Marie and Carlson, 1983;Trujillo-Cenoz, 1965). Some glia subtypes arise fromprogenitors located at sites well removed from their maturepositions. Retinal basal glia arise from progenitors located inthe optic stalk, the epithelial tube that connects the developingeye and brain. Basal glia precursors migrate to the basal surfaceof the retina (Choi and Benzer, 1994; Rangarajan et al., 1999).In the optic lobe, lamina epithelial and marginal glia migrateto their specific layers from progenitor zones located at theprospective dorsal and ventral margins of the ganglion (Perezand Steller, 1996; Huang and Kunes, 1998). This migrationdepends on local signaling from the photoreceptor axons, as itlargely fails to occur in mutants that do not producephotoreceptor neurons (Perez and Steller, 1996) or are deficientin the activity of the COP9 signalosome (Suh et al., 2002).Conversely, lamina glia are required for the guidance ofphotoreceptor axons, and provide an essential stop signal forthe R1-R6 subset of photoreceptor axons to terminate theiroutgrowth in the lamina (Poeck et al., 2001). These complexprocesses are controlled with precision in the three-dimensional milieu of the developing brain by mechanisms thatremain unclear.

An important aspect of glial cell migration is the targetingof particular subtypes to specific layers of the developingganglia. Given their common origin in dorsal and ventralmargin progenitor zones, selective mechanisms would be

In the developing Drosophila visual system, glia migrateinto stereotyped positions within the photoreceptor axontarget fields and provide positional information forphotoreceptor axon guidance. Glial migration converselydepends on photoreceptor axons, as glia precursors stall intheir progenitor zones when retinal innervation iseliminated. Our results support the view that thisrequirement for retinal innervation reflects a role ofphotoreceptor axons in the establishment of an axonalscaffold that guides glial cell migration. Optic lobe corticalaxons extend from dorsal and ventral positions towardsincoming photoreceptor axons and establish at least fourseparate pathways that direct glia to proper destinations in

the optic lobe neuropiles. Photoreceptor axons induce theoutgrowth of these scaffold axons. Most glia do not migratewhen the scaffold axons are missing. Moreover, glia followthe aberrant pathways of scaffold axons that projectaberrantly, as occurs in the mutant dachsous. The localabsence of glia is accompanied by extensive apoptosis ofoptic lobe cortical neurons. These observations reveal amechanism for coordinating photoreceptor axon arrival inthe brain with the distribution of glia to multiple targetdestinations, where they are required for axon guidanceand neuronal survival.

Key words: Glia, Drosophila, Axon, Migration

Summary

An axon scaffold induced by retinal axons directs glia todestinations in the Drosophila optic lobeRichard Dearborn, Jr and Sam Kunes*

Department of Molecular and Cellular Biology, Harvard University, Cambridge MA 02138, USA*Author for correspondence (e-mail: kunes@fas.harvard.edu)

Accepted 3 February 2004

Development 131, 2291-2303Published by The Company of Biologists 2004doi:10.1242/dev.01111

Research article

Development ePress online publication date 21 April 2004http://dev.biologists.org/lookup/doi/10.1242/dev.01111Access the most recent version at First posted online on 21 April 2004 as 10.1242/dev.01111

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needed to direct glia to different destinations. We show thatspecific highways for migration are provided by axons thatoriginate in the vicinity of glial progenitors. Photoreceptoraxons induce the outgrowth of these ‘scaffold axons’ upon theirarrival in the brain, and targeted migration follows. Theseobservations describe a cellular mechanism for the control ofoptic lobe glial migration by photoreceptor axons, an importantelement in the coordinated assembly of the precise neuralarchitecture of the optic lobe.

Materials and methodsFly stocksAll strains were grown on standard cornmeal medium (Ashburner,1989). The following strains and transgenic animals were used inthis study: wg-lacZ; so1; hh1; dpp-GAL4, UAS-CD8::GFP; omb-GAL4,UAS-CD8::GFP; ds1; ds33k; ds-lacZ; eyD; UAS-Ras1N17.

ImmunocytochemistryThird larval instar-staged animals were examinedimmunocytochemically essentially as described by Kunes et al. (Kuneset al., 1993). Primary antibodies were used at the following dilutions:mouse anti-Repo 1:10, goat FITC anti-HRP (Cappel) 1:200, rabbitanti-β-gal (Promega) 1:500, mouse anti-glial cells missing 1:100,monoclonal rabbit anti-human caspase 3 (BD PharMingen) 1:100 andrat anti-Elav 1:25. Secondary antibodies were used at the followingdilutions: Cy3 or Cy5goat anti-mouse (Jackson Immunochemical, Inc.)1:100, Cy3 or Cy5-goat anti-rabbit (Jackson) 1:500, HRP-conjugatedgoat anti-mouse IgG (Jackson) 1:100. Specimens were viewed on aZeiss LSM510 META confocal microscope.

Mosaic analysis and crossesMosaic analysis was carried out as described by Xu and Rubin (Xuand Rubin, 1993). Larvae were subjected to heat shock at 37°C for 5-7 minutes 24-36 hours after hatching to induce expression of an hsFLPtransgene. After growth at 20°C, larvae were dissected at late thirdinstar stage and processed for immunohistochemical analysis. Thefollowing crosses and strains were used in the experiments described:

(1) Identification of scaffold axons and temporal analysis of Wg+

neurons: wg-lacZ/y+CyO.(2) Marker analysis of wg-domains: (A) y,hsFLP122; wg-

lacZ/y+CyO X y,w; UAS-CD8::GFP, tubα1>y+,CD2>GAL4/y+CyO;(B) Omb-GAL4, UAS-CD8::GFP/FM7X wg-lacZ/y+CyO; (C) Omb-GAL4, UAS-CD8::GFP/FM7 X ds-lacZ/y+CyO; (D) Dpp-GAL4,UAS-CD8::GFP/TM6B, TbX wg-lacZ/y+CyO; (E) Dpp-GAL4, UAS-CD8::GFP/TM6B, TbX ds-lacZ/y+CyO; and (F) Repo-GAL4,UAS-CD8::GFP/TM3, Sb.

(3) Dependence of scaffold axon induction on retinal innervation:(A) so1::wg-lacZ/y+CyO X so1/y+CyO; and (B) eyD X wg-lacZ/y+CyO. Retina development was assessed in both so1 and eyD

animals by examining ommatida formation in the eye disc. For eachspecimen, regional brain development was assessed with respect tonormal, or a lack of, correlated eye development.

(4) Analysis of scaffold axon outgrowth: (A) ds1::wg-lacZ/y+CyOX ds1/y+CyO; (B) ds33k::wg-lacZ/y+CyO X ds33k/y+CyO; (C)y,hsFLP122; wg-lacZ/y+CyO X y,w, UAS-RasN17;tubα1>y+,CD2>GAL4; UAS-CD8::GFP.

(5) Analysis of optic lobe apoptosis: (A) y,hsFLP122; wg-lacZ/y+CyO X y,w, UAS-RasN17; tubα1>y+,CD2>GAL4; UAS-CD8::GFP; and (B) so1::wg-lacZ/y+CyO X so1/y+CyO; (C) wg-lacZ/y+CyO.

(‘>’ indicates the position of an FRT site in the respectiveconstruct.)

Quantitative analysis of apoptotic cellsQuantification of apoptotic cells in sine oculisand wild-type animals

relied on carefully matched confocal and developmental parameters.Optical slices (1.3 µm) were compared for age-matched specimens ata similar focal plane for all specimens analyzed. The number ofapoptotic cell profiles in 50 µm2 regions both proximal to the medullaneuropile and within the medulla cortex of sine oculisand wild-typeanimals were counted. Cortical neuron size (~3 µm) and overall brainsize did not differ between sine oculisand wild-type animals, so cellcounts were not biased by any intrinsic differences in cell sizesbetween these two genetic backgrounds. Differences in the number ofneuropile-proximal and cortical apoptotic cells in sine oculisanimalsrelative to wild-type animals were evaluated for significance usingStudent’s paired t-test.

ResultsAxons from dorsal and ventral optic lobe organizingcenters establish divergent pathways for glialmigrationThe Drosophila optic ganglia are derived from a pair ofneuroectodermal proliferative centers known as the inner andouter anlage, which are located on the ventrolateral surface ofeach larval brain hemisphere (Green et al., 1993; Hofbauer andCampos-Ortega, 1990; Nassif et al., 2003) (reviewed byMeinertzhagen and Hanson, 1993). We have previouslydescribed the onset of Wingless (Wg) expression in a few cellslocated at the presumptive dorsal and ventral margins of thedisk-shaped outer anlagen in young larvae (Fig. 2A)(Kaphingst and Kunes, 1994). These two ‘Wg domains’ arepositioned as adjacent wedges within the disk-shaped outeranlage (Figs 1, 2). In mid-metamorphosis, the disk opens at afissure located between the two Wg domains (yellow bar inFig. 1A); the disk unfurls linearly, such that the Wg domainsmove to the future dorsal and ventral poles of the ganglion.Interestingly, the Wg domains are roughly coincident with sitesrevealed by clonal analyses to be the origins of glia that migrateinto the lamina neuropile (Perez and Steller, 1996). We soughtto investigate this relationship in greater detail.

We used a marker of wingless expression, the lacZ reporterconstruct 17en40 (or wg-lacZ) (Kassis et al., 1992) to examinewg expression with respect to glial cell development andmovement. In cells expressing wg-lacZ, cytoplasmic β-galactosidase fills cellular processes and permits thevisualization of their detailed morphology. We observed thatthe paths of glial cell migration coincided with a stereotypedpattern of cytoplasmic extensions emanating from wg-lacZexpressing cells (Fig. 1C,D,F, arrowheads). The extensionsfollowed stereotyped routes and terminated at specificdestinations in the lamina, medulla and lobula. Glia formedchains along these extensions and accumulated in neuropiledestinations beyond the extension’s termini (Fig. 1D). Markeranalysis indicated that the glia undergo differentiation as theyprogress along these paths. Lamina glia express the early glialdifferentiation factor glial cells missing (gcm) (Hosoya et al.,1995; Akiyama et al., 1996; Egger et al., 2002) at their pointsof entry onto the extensions (Fig. 1E, white cells betweenarrowheads). Further along the path toward the neuropile, gliacommence expression of the homeodomain protein Repo(Xiong et al., 1994) a marker downstream of Gcm expression.Thus, in the case of lamina marginal glia shown in Fig. 1E(Gcm expression) and Fig. 1D (Repo expression),differentiation can be assessed relative to their progressionalong the migratory pathway.

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2293Glial migration in the Drosophila optic lobe

The wg-lacZ positive extensions are evidently axons. Theircell membranes were labeled by anti-horseradish peroxidaseantibody, a neuronal marker (Fig. 1C) (Jan and Jan, 1982).Their cell bodies were labeled by Elav, also a neuronal marker(Fig. 1F) (White et al., 1983). The axons extending toward the

same neuropile destination were bundled together in a fascicle,as indicated by labeling of individual axons in mosaic animals(described below; see Fig. 4). Four wg-lacZ labeled fascicleswere resolved emerging from each of the dorsal and ventralWg domains (Fig. 1A,B). One fascicle from each domain

Fig. 1.Axons from Wg-expressingneurons form pathways for glial migrationto different optic lobe target layers.(A) Schematic diagram of developingoptic lobe architecture and glial migrationviewed from a lateral perspective in a latethird instar larval stage brain. Dorsal isupwards and posterior rightwards. Innerchiasm glia (Xi, red), medulla neuropileglia (MNG, purple), marginal andepithelial lamina glia (Ma/Ep, yellow),and lobula neuropile glia (LoG, lightblue) exit the Wg domains (Wg, darkblue) via distinct scaffold axon fascicles.The eye disc (ed), photoreceptor axons(R-cell) and optic stalk (os) are indicated.The medulla (med, dark green), lamina(lam, light green) and lobula (lob, brown)neuropiles are indicated. (A′) An enlargedschematic view of the ventral Wg domain,showing the distinct scaffold axonpathways for different glial cell types.(B) Optic lobe architecture and glialmigration viewed from the horizontalperspective. Distal is upwards. Thescaffold axon fascicles are distributed onthe proximal distal axis. The pathway forlamina glia is the most distally positioned,while the tract for lobula neuropile glia ismost proximal. Labeling as in A. (B′) Anenlarged view of the ventral Wg domainin horizontal view, illustrating thelamination of scaffold axon fascicles andglial cell types. (C) Two scaffold axonfascicles (orange arrowheads) areobserved extending from each of thedorsal and ventral Wg domains (Wg;dorsoventral boundary indicated by theorange line in C-F), which are visualizedby expression of wg-lacZ(anti-β-gal;blue) in this late third instar stage brain.The two pairs of scaffold axon fasciclesvisible in this plane extend to the medullaneuropile (med n’pil, tract for MNG glia)and lobula neuropile (lob n’pil, tract forlobula neuropile glia). The neuropiles arevisualized by anti-HRP antibody staining(green). The location of medulla cortex(med cortex) is indicated. (C′) Image in C showing only wg-lacZexpression, to permit visualization of the scaffold axon fascicle pairs (orangearrows). (D) Glia (anti-Repo staining; red) seen during migration (white arrowheads) from the Wg domains (wg-lacZvisualized by anti-β-galstaining; blue). The layer of lamina marginal glia (Ma) is visible in this plane, as well as some inner chiasm glia (Xi) headed towards a moreproximal destination. (D′) Image shown in D with only wg-lacZexpression shown in order to detail the scaffold axon fascicles (orangearrowheads). (E) On their pathway towards the lamina (lam), glial differentiation is marked by the onset of Gcm expression (anti-Gcm, white,cells between arrowheads). The glia emerge from a region in which Optomotor Blind (Omb; anti-Omb, purple) is expressed under Wg control,and continue to express Omb as they migrate toward neuropile destinations. The area demarcated by white arrowheads, in which most cellsexpress Gcm, can be compared with the onset of Repo expression in the same region, indicated by white arrowheads in D. (F) Scaffold axonfascicles (orange arrowheads) originate from a subset of Wg expressing neurons (wg-lacZ; anti-β-gal staining; white), which co-express theneuronal marker Elav (anti-Elav; red). Neuronal populations and neuropiles are labeled as in C. Scale bar: 20 µm.

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extended to the border of the lamina field, terminating at aposition adjacent to layers of glia known as the laminaepithelial (Ep) and marginal (Ma) glia [establishednomenclature (http://www.flybrain.org, Accession NumberPP00003)] (Tix et al., 1997). These layers of glia lie,respectively, above and below the layer the axon termini of theR1-R6 photoreceptors. Glia could be observed migrating in achain along the fascicles (Fig. 1D, between arrowheads).Owing to the absence of specific markers, we could notdistinguish epithelial and marginal glia prior to their separationinto distinct layers. It seems likely, however, that both glialtypes migrate on the same pathway. One fascicle from each Wgdomain extended to the cortex, neuropile boundary of themedulla, and was associated with the chain-like migration ofmedulla neuropile (MNG) glia. One fascicle from each domainextended to the boundary between the medulla and lobula, and

corresponded to a pathway for migration of inner chiasm (Xi)glia, which demarcate the border between medulla and lobulaneuropiles (Tix et al., 1997). The final pair of fasciclesextended into the lobula neuropile (Fig. 1F) and formed apathway associated with lobula neuropile glia (LoG). We willrefer to these four sets of putative migratory guides as ‘scaffoldaxons’. Notably, the migration of these four glia types dependson retinal innervation of the optic lobe, a requirement that isexplored below.

Earlier stage specimens were examined in order to determinethe temporal relationship between glial migration and theoutgrowth of scaffold axons. During the first two larval stages,most neuroectodermal cells of the outer anlagen express thecell adhesion protein Fasciclin 2 (Fas2), with the exception ofthose in the two Wingless domains; Wingless-expressing cellsare Fas2 negative (Kaphingst and Kunes, 1994; Dumstrei et al.,

Development 131 (10) Research article

Fig. 2. Developmental profile of scaffold axon outgrowth, glial migration and retinal innervation. Wg expression commences in the mid-1stinstar larval stage in a pair of domains in the outer anlagen, preceding retinal innervation by several days. Scaffold axon fascicle elaborationwas visualized by wg-lacZexpression (blue in A-E or white in A′-E′). Glia were visualized by anti-Repo staining (red in all panels). Neuronalcell bodies and neuropiles were visualized by anti-HRP staining (green in A-E). Above each optic lobe image, which shows a different timepoint (youngest at left in A), is a schematic diagram indicating the state of ommatidial development in terms of the number of ommatidialcolumns. The developing optic lobe is outlined in white in A-E and yellow in A′-E′. (A,A′) An early 2nd instar stage specimen, lacking anyretinal innervation (0 columns), in which the two Wg domains contain a small number of cells. A small island of Repo-positive glia areobserved in the prospective photoreceptor axon target region (arrowhead). Scaffold axon fascicles are not detected. (B,B′) A mid-2nd instarspecimen, a time point just prior to ommatidial development. The anlage have grown in size, but scaffold axon tracts are not yet detectable.(C,C′) A late 2nd instar specimen in which one ommatidial column is detected in the retina, and a small amount of retinal innervation hasoccurred. The medulla neuropile has just begun formation. Scaffold axon fascicles are clearly visible emanating from the Wg domains andextending towards the neuropile (green arrowheads in C′). There is an increase in glia number within the neuropile region at this time point(yellow arrowhead). (D,D′) An early 3rd instar stage animal in which three columns of developing ommatidia were present in the retina.Scaffold axon fascicles begin to elaborate an architecture reminiscent of the late third instar (compare with Fig. 1C), in which tracts for medullaneuropile glia and lobula neuropile glia are distinguishable (green arrowheads in D′). Considerably more glia are visible in the neuropile region(yellow arrowhead in D′) in the focal plane shown. (E,E′) A mid-3rd instar stage specimen in which seven columns of ommatidia were detectedin the retina. Scaffold axon fascicles for MNG glial migration are indicated (green arrowheads in E′).

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2003). As development proceeds to the third instar stage, theneuroectodermal populations mostly convert to blast cells thatproduce the neurons and glia of the lamina and medulla. Thedifferentiation of wg-lacZ positive scaffold neurons and theextension of their axons follow this general temporal scheme(Fig. 2). As seen in Fig. 2A,B, a small population of gliaprecedes the arrival of the first photoreceptor axons in the targetfield of the retinal axon. Perez and Steller (Perez and Steller,1996) also described a small population of centrally locatedglia that preceded the arrival of photoreceptor axons in thelamina. At these early time points, when the Wg domainsconsist of fewer than a hundred cells; scaffold axon fascicleswere not detected. Photoreceptor axons subsequently arrive inthe temporal order that follows the posterior to anterior patternof eye development. The elaboration of wg-lacZ positivescaffold axons was first detected as the first photoreceptoraxons arrived in the target field, when only the first one or twocolumns of ommatidia had initiated differentiation in the eyedisc (Fig. 2C). As retinal innervation continued, the number ofglia in the neuropile regions increased steadily (Fig. 2D,E).Thus, Wingless-positive cells, though present long before thearrival of photoreceptor axons in the brain, appear to firstextend axons when retinal axons begin to arrive in the brain.

The wg-lacZ labeled-axons also were examined in pupalstage animals, where mature axon projections into the opticlobe neuropiles could be resolved (Fig. 3). wg-lacZ positiveaxons could still be detected extending from cell bodies locatedat dorsal and ventral cortical positions. The axons projected torespectively dorsal and ventral targets in the medulla and lobulaneuropiles (Fig. 3A,B). It is evident that the wg-lacZ-positiveneurons include intrinsic neurons of the proximal medulla (Pmneurons), which extend arbors tangentially within smallregions of the proximal medulla (Fischbach and Dittrich,1989). Projections into a specific tangential layer of the lobulawere also observed. Projections into the lamina neuropile werenot observed; at the third instar stage the extensions terminateat the border of the developing lamina neuropile. These axonsthus may not have become part of lamina circuitry, oralternatively have ceased wg-lacZ expression at the pupalstages examined.

Clonal analysis defines the origins of glia and theneurons that guide their migrationWe sought to determine the location of progenitors that giverise to the distinct types of migratory glia and the neurons thatformed their migratory pathways. The Wingless expressingcells of the dorsal and ventral domains are located in areas ofcomplex gene expression controlled by Wingless (Wg)signaling activity (Fig. 4A) (Kaphingst and Kunes, 1994; Songet al., 2000). Adjacent to the Wg domains are non-overlappingcell populations that express the TGF-β family memberDecapentaplegic (Dpp; Fig. 4C,E). Both the Wg- and Dpp-positive cell populations express the transcription factorOptomotor Blind (Omb; Fig. 4D) (Poeck et al., 1993; Huangand Kunes, 1998; Song et al., 2000). Dachsous (Ds), aCadherin family member (Clark et al., 1995), is expressed agraded fashion with respect to the Wg domains (Fig. 4D,E)(Song et al., 2000). These three genes, though expressed indifferent patterns, are under the control of Wg activity (Songet al., 2000).

By performing clonal analysis with tissue labeled to provide

positional landmarks, it was possible to localize glialprogenitors and scaffold neurons to distinct sites of origin (Fig.4B; Table 1). The FLP/FRT system (Golic and Lindquist, 1989;Xu and Rubin, 1993) was used to generate somatic clones thatwere positively marked by membrane-bound GFP (UAS-CD8::GFP) (Lee and Luo, 1999). Rare recombination eventswere induced such that most specimens harbored only one ora few labeled cell clones in the developing optic ganglia, whichwere examined in late third instar larvae. As reported by Perezand Steller (Perez and Steller, 1996), clones including laminamarginal and epithelial glia were found to label progenitors

Fig. 3.Fate of Wg-expressing neurons in the mature optic lobe.Specimens harboring the transgene wg-lacZwere examined duringthe late pupal stage [about 200 hours AEL (after egg laying)] for thelocation and axonal projection pattern of the Wg-positive neurons.(A) Most Wg-positive neurons are located at dorsal and ventralpositions in the medulla and lobula cortices. In this image, a dorsalcluster of neurons is visible in the upper right corner of the yellowbox (anti-β-gal staining; blue). MNG and Xi glia (anti-Repo staining;red) are indicated in their distinct positions relative to the medullaand lobula neuropiles (med n’pil, lob n’pil; anti-HRP staining;green). The medulla cortex (med ctx) is also indicated. (A′) Ahigher-magnification view of the yellow-boxed region in A. Clustersof wg-lacZ-positive neurons (anti-β-gal staining; grayscale) extendaxons (yellow arrowheads) towards the edge of the medullaneuropile and into the proximal medulla layer (green arrowhead).(B) A late pupal stage specimen with wg-lacZ-positive neuronsvisible (blue) along the dorsal edge of the cortices. In this image,glial layers of the lamina (lam) are visible. (B′) The image in B withanti-HRP staining omitted and wg-lacZexpression shown ingrayscale. wg-lacZ-positive neurons elaborate long axons that projecttangentially into the medulla and lobula neuropiles (yellow and greenarrowheads). Glia (red; yellow arrows) are associated with the wg-lacZ-positive axons In this specimen, the lamina marginal (Ma) andepithelial (Ep) glial layers are indicated, but wg-lacZ-positive axontracts are not found extending toward these layers at this stage. Scalebars: in A, 15 µm for A,B,B′; in A′, 10 µm.

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located at the dorsal and ventral margins of the outer anlagen(Fig. 4F,I). Clones that included lamina epithelial glia, laminamarginal glia, medulla neuropile glia, inner chiasm glia orlobula neuropile glia were all found within the domain of cellsthat express Wg, Omb and Ds (Domain I; Fig. 4A; Table 1).A majority of these clones contained both labeled scaffoldneurons and glia (16/26); clones with only neurons or glia wereless frequent. In the majority of specimens in which glia werelabeled, the clone extended into multiple domains and includedDomain I. With rare exception, these larger clones alsocontained neurons. Thus, at the time that somaticrecombination was induced (the mid-second instar, 75 hoursAEL (after egg laying)), most progenitor cells retained thepotential to produce both neurons and glia. When thespecimens were analyzed with respect to the glial types thatwere labeled, an interesting pattern emerged with regard to theposition of labeled progenitor cells in the Domain I region.Labeled progenitors for each of glial cell type appeared indistinct domains on the proximal, distal axis (Fig. 4B). Forexample, the lamina epithelial and marginal glial progenitorswere found in a more lateral position (Fig. 4F) than medullaneuropile glial progenitors (Fig. 4G). Inner chiasm glialprogenitors were observed in an even more proximal location(Fig. 4H). Hence, the progenitor domains for distinct glialtypes appear to be organized into a proximal distal stack withinDomain I, as illustrated in Fig. 4B.

Labeled scaffold neurons were most often included in theglial clones, and were found in close proximity to glialprogenitors that used the axons as migratory guides (Fig. 4I,J).In a minority of cases, small clones were recovered whichincluded only scaffold neurons (Fig. 4K). These clones werecontained within Domain I (Wg, Omb, Ds expression) in allbut two of nineteen cases. Our data do not resolve when theglial and neuronal lineages diverge. However, they do permitthe conclusion that glia and the neurons that appear to establishtheir migratory pathways are generated in close proximity andwith a lineage relationship.

Photoreceptor axons induce the outgrowth ofscaffold axonsThe migration of glia from the prospective dorsal and ventralmargins of the developing optic lobe depends on the arrival ofphotoreceptor axons in the target field (Perez and Steller, 1996;Huang and Kunes, 1998). When photoreceptor axons are

absent, as occurs in mutants that eliminate ommatidialdevelopment (sine oculis,eyes absentand eyeless) most gliaremain stalled in their progenitor domains (Perez and Steller,1996). When photoreceptor axons innervate only part of thelamina field, glia migrate to the region that receives retinalinnervation. It has thus been supposed that photoreceptor axonsattract glia into the lamina target field. We thus thought it mightbe informative to examine the glial migratory scaffold underconditions where glial migration did not occur.

To this end, the axon scaffold was examined in sine oculis1

(so) and EyelessD (ey) animals. These mutants display variablepenetrance, such that photoreceptor neurons can be completelyabsent, or develop in variably sized clusters in a particularregion of the developing retina. A lack of retinal innervationhas compound effects on optic lobe development. Laminaneurons fail to develop because of the absence of axon-bornesignals (Selleck and Steller, 1991; Huang and Kunes, 1996).The medulla is greatly reduced in cell number by extensiveapoptosis (Fischbach, 1983). Employing mosaic analysis,Fischbach and Technau (Fischbach and Technau, 1984)showed that so1 acts in the eye to bring about these effects onthe brain.

In our analysis, the scaffold axons were labeled by theexpression of wg-lacZ (Fig. 5). When photoreceptor axons wereabsent, the scaffold axons were likewise missing (Fig. 5B).However, wg-lacZ positive cells seemed to be present in normalnumber in the Wg domains (compare Fig. 5A′′ with 5B′′), andexpressed the neuronal HRP antigens (e.g. the ventral Wgdomain in Fig. 5C). In prior work, a number of markersexpressed in the vicinity of the Wg domains were expressednormally in the absence of retinal innervation (e.g. Dpp andOmb) (Huang and Kunes, 1996; Huang and Kunes, 1998).Therefore, we think it unlikely that retinal innervation is requiredfor the differentiation of these optic lobe neurons. When thescaffold axons were absent in so1 and eyD animals, gliaaccumulated at the edges of the Wg domains near the pointwhere they would have joined axon fascicles on paths towardneuropile destinations (arrowheads in Fig. 5B′,C′). Most so1 andeyD animals develop part of an eye, such that the correspondingoptic lobe receives partial innervation. In these cases,photoreceptor axons project to appropriate retinotopic locationsdespite the absence of the usual array of neighboring axons(Kunes et al., 1993). In such specimens, scaffold axons werefound only in parts of the brain that received retinal innervationand not in regions that completely lacked innervation (Fig. 5C).Thus, for example, in the specimen shown in Fig. 5C,photoreceptor axons innervate the dorsal half of the developingoptic lobe where scaffold axons have extended to the medullaneuropile, and medulla neuropile glia have migrated to normalpositions. The ventral half of the optic lobe by contrast lackedretinal innervation. Scaffold axons and medulla neuropile gliawere correspondingly absent; glia stalled at their exit point fromthe Wg domain (arrowhead in Fig. 5C). A correlation betweenretinal innervation, scaffold axon extension and glial migrationwas found in each of 32 so1 animals with partial eyedevelopment restricted to either dorsal or ventral regions. Theseobservations thus argue strongly that scaffold axon outgrowthand glial migration depend on retinal innervation.

The role of scaffold axons in glial migrationRetinal innervation might elicit scaffold axon outgrowth,

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Table 1. Glial and scaffold neuron clone distributionGlia and

Location Total Glia Neuron neurons

I (Wg/Omb/Ds) 26 7 3 16III (Dpp/Omb/Ds) 1 0 1 0IV (Omb) 1 0 1 0Wg and other 50 2 2 46

The clonal relationship and location of neurons and glia that originate fromthe Wg domains was examined by using the FLP/FRT method to generaterare, small mitotic clones marked by UAS-CD8::GFPtransgene expression.Glia were identified with anti-Repo antibody. For each specimen, clones werescored as labeling glia only (Glia), neurons only (neuron) or a mixedpopulation of glia and neurons (glia and neurons) and the location of eachclone was identified in accordance with the domain map shown in Fig. 4A. Intotal, 78 specimens were analyzed.

2297Glial migration in the Drosophila optic lobe

thereby establishing a necessary pathway for glial migration.Conversely, glial migration, elicited by retinal innervationdirectly, might establish a necessary pathway for scaffold axonoutgrowth. Two approaches were undertaken in an attempt toresolve this issue. In the first, scaffold axons were eliminatedby neuronal expression of activated Ras1 (Ras1N17) in order todetermine whether glial migration would occur in theirabsence. In the second, mutant animals with misdirectedscaffold axons were examined in order to determine whether

migratory glia follow the aberrant axon projections. Bothapproaches indicated that scaffold axons are necessary as glialmigratory guides.

In the first approach, we used the ectopic expression of adominant negative Ras protein (Ras1N17) (Lee et al., 1996),which was found to autonomously block the outgrowth ofscaffold axons (Fig. 6D,E). The UAS-Ras1N17 transgene wasexpressed in small somatic clones that were labeled by the co-expression of UAS-CD8::GFP. Occasional somatic clones of

Fig. 4.Wg domain regions that give rise to glia and theneurons that guide their migration. (A) The areasurrounding the Wg domains was divided into severalsubdomains on the basis of gene expression patterns. All ofthe genes categorized (omb, ds, dpp) are regulated by Wgactivity. The schematic diagram shows that the Wg domaincan be divided into two subdomains (I and III) on the basisof ds expression. wg-expressing cells all express omb, andin one area also express ds. The four subdomains shownare: I, wg/omb/ds; II, wg/omb; III, dpp/omb/ds; and IV, omb.The overlapping expression patterns of these genes areshown in color for the top domain, and in outline form inthe bottom domain. The location of the lamina (lam) andlobula (lob) are indicated. An arrow indicates the positionof the optic fissure, where the two Wg domains separateduring pupal metamorphosis. (B) A horizontal perspectiveof one Wg domain region. Distal is towards the top.Subdomain I is shown with wg-lacZpositive fasciclesextending toward glial migratory destinations. A map ofglial subtype origin is overlaid onto subdomain 1, showingthe origin of lamina Ma and Ep glia (#1, yellow), medullaneuropile glia (#2, purple), inner chiasm glia (#3, red) andlobula neuropile glia (#4, light blue). The position of theseprogenitor sites forms a stack on the proximal (#4) to distal(#1) axis. (C) Animal harboring wg-lacZ(anti-β-galstaining; blue) and a transgenic marker for dppexpression(dpp-GAL4, UAS-CD8::GFP; GFP expression is green). Inthis lateral view of a late third instar stage optic lobe, thenon-overlapping expression of wgand dppis evident. dppexpression is also found in the inner proliferation center ofthe lobula (lob). (D) A late third instar optic lobe, like thatshown in C, but harboring a ds-lacZreporter (anti-β-galstaining in grayscale) and a transgenic marker for ombexpression, omb-GAL4, UAS-CD8::GFP (GFP expressionin red). Ds expression intersects Omb expression,distinguishing subdomains II and IV from I and III. (E) Alate third instar stage optic lobe from an animal harboringthe ds-lacZreporter (anti-β-gal staining; grayscale), asshown in D, and stained with anti-Dpp antibody (green).The Dpp-positive cells form subdomain III. (F-K) Clonalanalysis was performed in order to determine the origins ofparticular glial subtypes, and the neurons that form theirmigratory pathways. Random clones were generated usingthe FLP/FRT system (see Materials and methods) and labeled by membrane-bound GFP (UAS-CD8::GFP, green in F-K). (F) A cloneoriginating within a distal layer of subdomain I (wg/omb/ds) labels lamina glia (Ep, epithelial glia). (G) A clone originating in subdomain I in aslightly more proximal focal plane than in F labels medulla neuropile glia (MNG). The medulla neuropile glia are also labeled by anti-Repoantibody (red). This clone is in the dorsal Wg domain (dorsal towards the top). (H) Clones originating in a more proximal layer of subdomain Ithan is shown in F or G label inner chiasm glia (Xi). The particular clone shown also labels the scaffold neurons and axons that project alongthe glial migratory tract (between arrowheads). In this specimen, expression of wg-lacZis shown in blue. Inset: Xi glia shown double labeledby anti-Repo (red) and repo-GAL4, UAS-CD8::GFP (green) in order to visualize their characteristic size and morphology, which permit theseglia to be easily distinguished from many other glial cell types. (I,J) Clones often labeled both scaffold neurons and glia (see Table 1). In thesetwo specimens, single clones labeled both lamina Ma glia (arrows) and a few neurons whose axons project along their migratory pathway(arrowheads). (K) A small (three-or four-cell) clone (green) within the Wg domain (subdomain I, blue) labels neurons (arrowhead) that extendscaffold axons. Scale bars: in C, 20 µm for C-I; in J, 20 µm for J,K. A white or yellow bar indicates the boundary between dorsal and ventralWg domains in all panels.

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Ras1N17-expressing cells within the Wg domain regionresulted in failure of scaffold axons to extend toward glialdestinations. By contrast, clones that labeled lamina epithelial,marginal or medulla neuropile glia did not affect theirrespective migration (data not shown). When the scaffoldaxons were absent, glia stalled prior to migrating into theneuropile regions, as they did in so1 animals (arrowheads inFig. 6D,E). That is, glia did not migrate when the scaffoldaxons were missing, even though retinal axons were present inthe target field. These observations indicate that the scaffoldaxon fascicles are required as migratory guides for glialmigration.

In the second approach, we examined mutants lacking thefunction of the Cadherin family member Dachsous (Ds) (Clarket al., 1995). As described above, Ds is expressed in a gradientthat decreases with distance from the Wg domains (Song et al.,2000) (Fig. 4D,E). Ds is highly expressed by neurons in theWg domains but not by migrating glia. Ds can mediatehomophilic binding, and so one might suppose that the Dsgradient could provide positional information for axonoutgrowth within the developing neuropiles.

Scaffold axons did indeed project aberrantly in the mutantsds1 and ds33k (Fig. 6B,C) and glia lined these aberrantpathways and accumulated in abnormal destinations. In the ds1

homozygous animal shown in Fig. 6B, scaffold axons whichshould project toward the medulla neuropile and form a pathfor medulla neuropile glia (as shown in Fig. 6A) were foundextending toward the lobula cortex. Glia formed a chain alongthe aberrant axons (Fig. 6B′) and accumulated abnormally inthe lobula, while the number that reached the medulla was

reduced. In the ds33k homozygous animal shown in Fig. 6C,the dorsal scaffold axons bifurcate and appear to direct gliaaway from paths toward neuropile destinations. Mutations incombgap, a negative regulator of optic lobe ds expression(Song et al., 2000) resulted in similar axon misrouting and gliadistribution abnormalities (data not shown). The correlationbetween patterns of scaffold axon misprojection and gliamigratory paths is a further indication that scaffold axons serveas migratory guides.

The migration of medulla neuropile glia is requiredfor cortical cell survivalPrior work revealed extensive apoptosis in the optic lobes of‘eyeless’ mutants of Drosophila(Fischbach and Technau,1984; Fischbach 1983). In the mutant sine oculis (so), mosaicanalysis has revealed that extensive cell death in the optic lobeis due to the lack of sofunction in the retina and not the brain.These and additional observations have led to the conclusionthat the optic lobe phenotype of sine oculis is due to lack ofretinal innervation. The ‘trophic’ function of photoreceptoraxons could be direct, via provision of a survival factor, orindirect, e.g. by eliciting the migration of glia that provide asurvival factor. We explore these distinct hypotheses below.

To address the issue, wild-type and so1 animals wereexamined for the onset of apoptosis by their expression ofactivated caspase, which can be monitored in Drosophilawithan antibody against the activated human caspase 3 protein(Baker and Yu, 2001). In third instar stage wild-type animals,few cortical cells are labeled by the anti-Caspase labeling (Fig.7A′′ ). By contrast, so1 third instar larval optic lobes displayed

Development 131 (10) Research article

Fig. 5. Retinal innervation is locally required forscaffold axon outgrowth. (A-A′′) A wild-type late thirdinstar stage optic lobe (lateral view) in which the Wgdomains (indicated by a pair of arrows) are marked bywg-lacZ(anti-β-gal staining; blue). Glia are marked byanti-Repo staining (red). MNG and Xi glia are visible inthis focal plane. These two glial types are more easilydiscerned when anti-HRP staining (green, A) is omittedin A′ and A′′. (A′′ ) An enlarged view of the area boxedin A′, where the alignment of MNG glia (yellowarrowheads) with a scaffold axon fascicle is evident.(A′′ ) wg-lacZstaining is shown in grayscale. (B-B′′) Anso1, wg-lacZ late third instar stage optic lobe in whichall photoreceptor axon innervation is absent. Themedulla neuropile (arrow in B) is somewhatdisorganized (med n’pil; anti-HRP, green). Glia (red) aremostly absent from the neuropile region, and appearaccumulated at the edge of the Wg domains (yellowarrowheads in B′,B′′). Scaffold axons are completelyabsent, as is clear in the higher magnification view (B′′ )of the boxed area in B′. (C-C′′) An so1, wg-lacZ latethird instar specimen in which photoreceptor axons haveinnervated only the dorsal half of the optic lobe. Thedorsal scaffold axon fascicles (anti-β-gal staining, blue)have extended (yellow arrow in C′, C′′) and glia (red)migrate into the neuropile. By contrast, ventral scaffoldaxons are missing and the glia stall (yellow arrowhead)as in B. Medulla neuropile organization is disrupted inthe ventral half of the optic lobe. White or yellow linesindicate the boundary between dorsal and ventral Wgdomains. Scale bars: 20 µm in all panels.

2299Glial migration in the Drosophila optic lobe

an increased number of caspase 3-positive cells throughout themedulla cortex (Fig. 7B). Putative apoptotic cells wereconcentrated in regions where glia were particularly few orabsent. Caspase 3-positive cells were also particularlyprevalent in cortical areas immediately adjacent to theneuropile (arrowheads in Fig. 7B′). As noted above, areasparticularly deficient in glia also displayed an irregularneuropile structure. These observations were subjected to aquantitative analysis (see Materials and methods). Areas of 50µm2 in 1.3 µm confocal optical sections of the medulla cortexwere counted for the number of caspase 3-positive cells. Inwild-type animals, an average of 0.6 (±1, n=10) activatedcaspase-positive cells were found per 50 µm2 area. No regionaldifferences in the density of apoptotic cells were observed inwild-type animals. so1 specimens displayed an average of 5.5(±2.9; n=37) apoptotic cells in 50 µm2 areas adjacent tomedulla neuropile regions that lacked medulla neuropile glia.Elevated apoptosis, an average of 6.5 (±5.2 n=37) activatedcaspase-positive cells, was also observed in distal cortical cellpopulations whose axons would normally innervate glia-starved regions of neuropile. By contrast, cell populationsadjacent to glia-rich regions of medulla in the same so1 animalsshowed only 2.4 (±2.5 n=37) apoptotic cells per 50 µm2, while

in the corresponding distal cortical cell populations thatinnervate these glia-rich regions, only 2.5 (±3 n=37) apoptoticcells per 50 µm2 were found. Thus, although so1 animalsdisplayed an overall increase in caspase-positive cells, thefrequency was significantly greater in cell populations thatinnervated glia-starved regions of neuropile. The results of thisanalysis are statistically significant: for medulla neuropileproximal regions within so1 animals a paired t-test analysisyielded a P-value of 1.3e–06(comparing glia-poor and glia-richregions), while the same statistical analysis comparing corticalregions yielded a P value of 4.8e–07. Our observations suggestthat both local and long-range trophic cues are provided by gliato cortical neurons.

To determine whether the increase in apoptotic cells was dueto the absence of retinal axons or the absence of glia, we usedRas1N17 expression to block the migration of medullaneuropile glia along the scaffold axons, as described above. Inthe specimen shown in Fig. 7C, a small clone of cellsexpressing the UAS-Ras1N17 and UAS-CD8::GFP transgenesis localized to the Wg domain region. Glia accumulated at theborder of this region instead of migrating into locationsadjacent to the medulla neuropile. Notably, photoreceptorinnervation was not markedly affected in such animals. The

Fig. 6.Evidence that scaffold axons are required for glia migration and direct glia along specific pathways. (A,A′) Wild-type scaffold axonfascicles (anti-β-gal; grayscale) in a wg-lacZlate third instar optic lobe project in a stereotyped fashion toward glial destinations. MNG and Xiglia (anti-Repo; red) are seen following along fascicles towards their appropriate destinations. A higher magnification view of the yellow boxedarea in A is shown in A′. (B,B′) In ds1 homozygous animals, scaffold axon fascicles project aberrantly, and the distribution of glia iscorrespondingly aberrant. A higher magnification image of the yellow-boxed region of B is shown in B′; glia (yellow arrowheads) on anabnormal trajectory are closely associated with the misprojecting scaffold axon fascicle (anti-β-gal; grayscale). (C,C′) Scaffold axon projectionsare likewise aberrant in ds33k homozygous animals. In this specimen, dorsal scaffold axon fascicles bifurcate onto aberrant trajectories, and gliaare likewise misdirected. The higher magnification image of the boxed region of C shown in C′ reveals mispositioned glia (anti-Repo; red) inassociation with the misrouted scaffold axon fascicles (anti-β-gal staining in grayscale). (D,D′) Random somatic clones were generatedexpressing both a GFP marker and an activated Ras protein (Ras1N17) that inhibits axon extension. wg-lacZ(anti-β-gal staining; blue) was usedto mark the migratory axon scaffold. In the late third instar optic lobe shown, a clone encompasses part of the dorsal Wg domain (white outlinein D and yellow outline in D′). Glia (anti-Repo, red), which do not express the UAS-Ras1N17 transgene, stalled at the edge of this Wg domain(yellow arrowheads in D,D′), much as they do in ‘eyeless’ mutant strains (see Fig. 5). Scaffold axon fascicles cannot be detected emanatingfrom the dorsal Wg domain, but are clearly visible extending from the ventral Wg domain (yellow arrows); ventral glia migrate normally.(E,E′) A specimen like that shown in D,D′ in which somatic clones (white outline in E, yellow outline in E′) express UAS-Ras1N17 (labeled byco-expression of GFP, green in E). Clones of particular interest encompass both the dorsal and ventral Wg domains. Scaffold axons are absentfrom both and glia stall at both the dorsal and ventral margins of the Wg domains (yellow arrowheads in E′). Scale bars: in A, 20 µm (forA-D,D′ ,E,E′); in A′, 8 µm (for A′,B′,C′).

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cortical area adjacent to this glia-deficient region displayed anincreased number of anti-caspase 3-positive cells (arrowheadsin Fig. 7C′′). Thus, the absence of glia, rather thanphotoreceptor axons, appears the more likely cause ofextensive apoptosis and neural cell loss observed in eyelessstrains of Drosophila.

DiscussionGlia and neurons are known to use one another’s extendedcellular processes to guide their migration over long distancesand through complex terrain. Well-known examples includethe migration of cortical precursors along radial cell processes(Rakic, 1988) and the movement of Schwann cell precursorsalong axon tracts into the PNS (Carpenter and Hollyday, 1992).The directed migration of glia along axons has also beendescribed in Drosophila (Choi and Benzer, 1994; Giangrande,1994; Sepp et al., 2000). This mode of cell dispersal has thusbeen conserved across significant evolutionary distances.

We show in this report that Drosophila optic lobe glia useaxon fascicles as migratory guides and that the extensionof these axon fascicles is induced by the ingrowth ofphotoreceptor axons from the developing retina (Fig. 5). Themigratory scaffold axons emerge from optic lobe regions thatare in close proximity to sites where glial cells originate; botharise in the dorsal and ventral domains where cells express themorphogen Wingless (Figs 1, 4) (Kaphingst and Kunes, 1994).When the scaffold axons were eliminated by the autonomousexpression of an activated Ras transgene, glia failed to migrate

and stalled at the borders of their progenitor sites (Fig. 6).Extensive cortical cell apoptosis ensued (Fig. 7). When thescaffold axons projected aberrantly (in animals mutant for thecadherin Dachsous; Fig. 6), glia followed the aberrant routesto incorrect destinations. The longstanding observation thatglial migration does not occur in eyeless mutant Drosophila(Perez and Steller, 1996; Huang and Kunes, 1998) might thusbe explained by an indirect mechanism in which innervationcontrols the establishment of an axon scaffold necessary todirect glial migration.

The migratory scaffold axons were identified by theircytoplasmic expression of β-galactosidase from lacZ under thecontrol of a wingless promoter (Kassis et al., 1992). Theneurons are thus residents of the Wg domains, a pointadditionally supported by labeling small numbers of neuronsthat projected their axons toward glial destinations (Fig. 4). Intotal, four different wg-lacZ delineated pathways wereidentified. These appear to account for all the pathways takenby optic lobe glia that have been identified as migratory byclonal studies. Separate pathways were identified for medullaneuropile glia, lobula neuropile and inner chiasm glia. A singlescaffold axon pathway was observed leading to the marginaland epithelial glial layers of the lamina, suggesting that bothof these glial types follow the same pathway. Perhaps these gliabecome separated only on the interposition of photoreceptorR1-R6 growth cones as they arrive in the lamina. Whether theepithelial and marginal glia arise from distinct precursors thatmigrate on the same pathway is unclear. In all cases, glia wereobserved to form migratory ‘chains’ along axonal extensions,

Development 131 (10) Research article

Fig. 7.A normal glial distribution is required for cellsurvival in the developing medulla. Cell death wascompared in wild-type and so1 animals in relation to thedistribution of glia. (A-A′′) A wild-type late third instaroptic lobe is stained to reveal a normal organization ofthe medulla neuropile (med. n’pil, anti-HRP staining,green) and distribution of MNG and Xi glia (anti-Repo,red). Very few apoptotic cells are evident by staining withanti-activated caspase 3 antibody (blue in A,A′, shown ingrayscale in A′′). (B-B′′) In so1 animals, an increasedfrequency of apoptosis was detected in the late thirdinstar optic lobe (see the text for details). In the specimenshown, retinal axons have innervated the dorsal (dorsal isupwards) optic lobe, resulting in a relatively normallyorganized medulla neuropile. The ventral optic lobe lacksinnervation and glia accumulate in the vicinity of the Wgdomain (white arrow in B′). Few glia are found in theventral neuropile region. An increased frequency of anti-activated caspase 3-positive cells (blue in B and B′,grayscale in B′′) is seen throughout the medulla cortex.More apoptotic cells are found in the ventral region(regions between white, yellow and blue arrowheads; seetext for quantification), where glia are rare. (C-C′′) Ananimal in which glial migration was blocked byexpression of UAS-Ras1N17 in somatic clones (markedby co-expression of UAS-CD8::GFP (green) (see thelegend to Fig. 6, and Materials and methods forexperimental details). A Ras1N17-expressing clone in thedorsal Wg domain region has blocked glial migrationinto the dorsal optic lobe in this late third instar stagespecimen. Glia (red) accumulate at the position of the yellow arrowhead shown in the boxed area in C. A locally increased frequency ofactivated Caspase 3-positive cells is visible (blue in C; grayscale in C′, shown alone in grayscale between arrowheads in C′′ ). Scale bar: 20 µm.

2301Glial migration in the Drosophila optic lobe

resembling a similar organization of migratory glia on retinalaxons en route from the optic stalk to the eye field (Choi andBenzer, 1994) and from midline progenitor sites to destinationsin the PNS (Sepp et al., 2000). In pupal stage animals, the wg-lacZ labeled neurons were observed in dorsal and ventralcortical locations, sending projections into neuropile targetsconsistent with the patterns of glial migration. However, noaxons from wg-lacZ positive neurons were observed extendinginto the lamina neuropile at this stage.

The optic lobe regions surrounding the Wg domains displaycomplex patterns of gene expression, mainly because of thesignaling activity of Wingless (Kaphingst and Kunes, 1994;Song et al., 2000). Our clonal analysis indicates that all fivemigratory glial cell types we examined arise from thesedomains (Fig. 4 and Table 1). Interestingly, the sites fromwhich particular glia arise are stacked on the proximal distalaxis (Fig. 4B) in a manner that correlates with targetdestinations in the developing ganglia. Thus, for example,somatic clones that label the medulla neuropile glia are locatedat a position that corresponds to the medial/distal position ofMNG glia relative to the lamina and lobula glia. Furthermore,on the basis of their expression of wg-lacZ, as well as clonalanalysis (Fig. 4), the neurons that extend scaffold axons arisein close proximity to the sites of glial origin. Indeed, as somaticclones induced in mid-second instar larval animals oftenlabeled both scaffold neurons and migratory glia, the glia andneurons must share common progenitors. It is curious that allof these distinct cell types express wingless. We have noevidence that axonally transported Wg functions in opticlobe development, as it does in the development of theneuromuscular junction (Packard et al., 2002). Partialelimination of wg+ activity (by the use of a conditional wgts

allele) did not result in a specific defect in glial migration inthe optic lobe (R.D. and S.K., unpublished) but other possiblefunctions of Wg were not addressed by this analysis.

Early studies on sine oculis mutants (Fischbach, 1983;Fischbach and Technau, 1984) demonstrated that lack of retinalinnervation results in excessive optic lobe cell death, especiallyin the medulla, which is reduced to less than half of its normalvolume without innervation (Power, 1943). Our analysissuggests that these earlier observations may be accounted forby a lack of glial migration. Though apoptosis was generallyelevated in the medulla of animals lacking retinal innervation,a higher level of cell death was found in regions particularlydevoid of medulla neuropile glia (Fig. 7). This effect could beproduced with retinal axons present when glial migration wasblocked by elimination of the scaffold axons (via Ras1N17

expression). Therefore, retinal innervation and its inductiveeffect on neuronal development in the lamina is not sufficientfor survival. Glial migration is required. A role for glia inneuronal survival has also been documented in the lamina,where neuronal cell death follows the elimination of glia inanimals carrying a visual system specific allele of repo (Xiongand Montell, 1995).

Our observations suggest a developmental mechanism forthe control of glial cell migration that depends on theestablishment of an axon scaffold for guidance of migratingglia (Fig. 8). In normal development, a small number of gliamigrate into the target field of the photoreceptor axon prior tothe arrival of the first photoreceptor axons (Fig. 2) (Perez andSteller, 1996). These glia, which migrate independently of

retinal innervation, may serve a necessary early role inphotoreceptor axon guidance. They may be targets for the firstretinal axons to arrive in the optic lobe, and provide the firstsignals that differentiate the outgrowth termination points ofthe R1-R6 and R7/8 axons (Poeck et al., 2001). We proposethat the first photoreceptor axons to arrive in the optic lobeelicit outgrowth of the scaffold axons from neurons at thedorsal and ventral margins. Subsequent migration of glia fromthe dorsal and ventral margin progenitors is then both permittedand directed along the specific pathways of the scaffold. Afterthe erection of the migratory scaffold, glial migration may beindependent of continued photoreceptor axon ingrowth. Onthis point, we note that glia migrate into the lamina inapproximately normal number in hh1 animals, in whichommatidial development ceases after 11-13 columns form atthe posterior of the developing retina (Huang and Kunes,1998). Therefore, glial migration may not depend on continuedarrival of new retinal axons in the lamina primordium.Nonetheless, this observation cannot rule out the alternativeinterpretation that retinal axons emit a continuous attractivesignal for glial migration that functions in adjunct with themigratory axon scaffold.

How do photoreceptor axons control the outgrowth of axonsfrom the Wg domains? This does not appear to be aconsequence of an affect of retinal innervation on neuronaldevelopment in the Wg domains, which appear normal in sizeand organization in eyeless mutant Drosophila strains (Song etal., 2000). Hedgehog, which is brought into the brain by retinalaxons, is not required for the expression of either Wg or Dpp

Fig. 8.A cellular model for thecontrol of glial migration byretinal innervation. A smallnumber of glia (red) migrate intothe target region forphotoreceptor axons prior toommatidial development, and areready to provide initial guidancecues to the first photoreceptoraxons (green; top two panels).These early photoreceptor axonstrigger the elaboration of theaxonal scaffold for glialmigration (blue; third panel fromtop), which extends instereotypical fashion to establishthe multiple pathways (notdepicted). Glia generated in theWg subdomain 1 (yellow) canthen migrate to targetdestinations, such as the lamina(shown, bottom panel).Subsequent migration may beindependent of continued retinalaxon ingrowth (see the text).Through this mechanism, thedistribution of glia throughout theoptic lobe is coordinated withinnervation by the photoreceptoraxons.

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at the dorsal and ventral margins of the optic lobe (Huang andKunes, 1998). It seems that the induction of scaffold axonoutgrowth by the photoreceptor axons may be direct, as theoutgrowth occurs in the hedgehog mutant, hh1, in which thefirst steps of lamina neuronal development fail to occur (Huangand Kunes, 1996). Thus, one might suppose that retinal axonsemit a chemoattractant for scaffold axon outgrowth, eithersynthesized in the retina or acquired from environmentalsources and redistributed by retinal axons (e.g. as for Netrin)(Hiramoto et al., 2000).

The system for glial migration guidance we describe permitsdiversified cell types to originate from a common site, and yettarget specific locations in complex neuropiles. One couldimagine that as more complex and diversified neuropilesevolved, relatively simple changes in the developmentalpathway of glial progenitors and the projections of scaffoldaxons would deliver glial support to new structures. Thissystem also has the feature of functioning as a developmentaltiming ‘checkpoint’ that fine-tunes the general hormonalcoordination of imaginal development. Thus, upon their initialarrival in the brain, retinal axons provide a fine level of localcellular control over the movement of glia, preparing the targetfield for the next steps of optic lobe development.

This work was supported by NIH-NEI grant EY10112 to S.K., andNIH-NEI grant EY07030 to R.D. We appreciate the receipt of strainsand antibody reagents from Michael Hoffman (Wisconsin, Madison),Seymour Benzer (Caltech), the Developmental Studies HybridomaBank (Iowa) and the Bloomington Drosophila Stock Center.

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