INTRODUCTION

Early studies indicate that insect glial cells represent a hetero-geneous population of cells displaying different features andmechanisms of development depending on their position (Wig-glesworth, 1959; see Klämbt and Goodman, 1991 for areview). For example, in the case of the embryonic centralnervous system (CNS), glial and neuronal cells originate at thesame time within the nervous system. During embryogenesis,glial cells display a metameric and stereotyped pattern ofmigration (longitudinal glia: Jacobs et al., 1989; midline glia:Klämbt et al., 1991). In contrast, in the enteric nervous system,glial cells originate in multiple waves from regions that havepreviously given rise to neurons, and migrate for long andvariable distances following neuronal paths (Copenhaver,1993).

As for the adult peripheral nervous system (PNS), littleinformation is available on the origin of glial cells. Althoughit has already been shown that cells of the appendages are ableto give rise to epithelium and sensory organs, it is not clearwhether they also have the potential to become glial cells.Tissue culture studies showed that at least some glial cells orprecursors are present in the wing imaginal disc by the end oflarval life but it could not be established whether glial precur-sors were present in the disc at earlier stages (Giangrande etal., 1993). Moreover, those results could not exclude the pos-sibility that some glial cells differentiate locally whereas someothers migrate from outside the wing. Another aspect that hasnot yet been investigated is the ability of glial cells in the PNSto migrate. Glial migration is a feature common to vertebratesand invertebrates (see Discussion), therefore it would berelevant to see whether peripheral glia also display thisbehaviour and, if they do migrate, to investigate the modalitiesof the process.

To assess unambiguously whether glial cells in the wing areclonally related to epithelial cells and whether they migrateduring development, I have marked cells using the recombi-nase

flp system (Golic and Lindquist, 1989; Struhl and Basler,1993). I show that glial cells present on the wing anteriormarginal nerve originate within the disc from regions that alsocontain the precursors of the sensory organs, and migrate forrelatively short distances along the nerve. Migration starts at astage at which axonogenesis is actively taking place and occursin the same direction as that taken by axons. Finally migrationof glial cells is dramatically affected by two mutations alteringaxonal navigation Notch (N) and fused (fu) (Palka et al., 1990;M. Schubiger unpublished observations and the present study),which indicates that glial migration and axonal growth aretightly linked processes.

MATERIALS AND METHODS

StocksThe wild-type stocks were Oregon R and Sevelen. For clonal analysis,I used a line carrying two transposons, one with the flp gene fused tothe heat-shock promoter, the other with the FRT sites between theactin promoter and the

β-galactosidase (β-gal) gene, as described inStruhl and Basler (1993). The β-gal product accumulates in thenucleus, due to the presence of a nuclear localization signal. The fliesfrom this line were crossed to the Oregon R stock, and eggs werecollected for approx. 4 hours at 25°C. Flies were heat shocked (37-38°C) for 30 minutes early during the first larval instar. This leads toa functional β-gal gene in the FRT-carrying chromosome, and givesrise to a clone of β-gal-expressing cells. The mutations AxP8 and fuP1

were identified by M. Schubiger as being viable Abruptex and fualleles, respectively. Both mutations originated from an EMS muta-genesis in W. Pak’s laboratory. The glial-specific enhancer trap linerA87 was provided by V. Auld and C. Goodman. For a description of

523Development 120, 523-534 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

The

Drosophila major wing nerve collects axons from theanterior margin sensory organs. Using the flp recombinaseto make clones, I show that all glia present on this nerveare clonally related to wing epithelial cells. Glial cells ariseonly from regions that also give rise to sensory organs andmigrate along the nerve following the direction taken byaxons. As in vertebrates, wing glial cells start migrating at

a stage at which axons are growing. The migration of wingglial cells is affected by two mutations altering axonogene-sis, fused and Notch, which suggests that the two processesare tightly associated.

Key words: gliogenesis, cell migration, mitotic clones, fly,Drosophila, wing, nerve, fused, Notch

SUMMARY

Glia in the fly wing are clonally related to epithelial cells and use the nerve as

a pathway for migration

Angela Giangrande

Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Unité 184 de Biologie Moléculaire et de Génie Génétique del’INSERM, Institut de Chimie Biologique, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cedex, France

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the wing rA87 profile of staining, see Giangrande et al. (1993). Theen1 stock was kindly provided by P. Johnston.

ImmunohistochemistryAfter heat shock, larvae were kept at 25°C. White prepupae werecollected and kept at 25°C until the desired stage; therefore, the stageis expressed as hours after pupariation (AP). Dissection, fixation andantibody incubation were performed following the protocol describedin Giangrande et al. (1993) with the following modifications: 0.01%sodium azide was added during incubations with primary andsecondary antibodies; anti-β-gal (Promega) and anti-HRP (US Bio-chemical) were used at 1:4000; secondary antibodies were, respec-tively, Cy3-conjugated-donkey-anti-mouse IgG (Jackson) at 1:600and FITC-conjugated goat anti-rabbit IgG (Jackson) at 1:400. Vec-tashield (Vector) mounting medium was used to prevent bleaching.

To analyze glia in the AxP8 mutant, AxP8/AxP8 females were crossedto rA87/rA87 males. Female and male white pupae were separatelycollected and kept at 25°C until the desired stage and were stained asabove. AxP8 heterozygous females showed weaker and less frequentdefects than hemizygous males.

The glial-specific RK2 rat antibody was used at 1:1000 andrevealed with a Cy3-conjugated goat anti-rat antibody (Jackson) at1:600. For double labelling with mouse anti-β-gal and rat RK2,secondary antibodies for multiple labelling were used.

RESULTS

Organization of glial cells in the wingOn the wing blade, two nerves (L1 and L3) travel along theveins L1 and L3, and merge proximally (Fig. 1A) (for adetailed description of wing sensory organ development, seeMurray et al., 1984; Hartenstein and Posakony, 1989). Glial-specific enhancer trap lines have shown that glial nuclei arepresent on these nerves; however, each line showed differentand variable number of glial cells (Giangrande et al., 1993).To analyze the number and variability of glial cells with anindependent marker, I have now used a glial-specific antibody

A. Giangrande

Fig. 1. Neuronal and glial organization in a developing wing. (A) 37 hour AP wing labelled with anti-HRP, an antibody that recognizes neurons(open arrows). L1, L3, c and tg indicate the L1, L3, costal and tegula nerves, respectively. The L1 nerve comprises two regions. The first region(anterior margin) extends from the distal tip to the twin sensillum on the margin (TSM) and contains the ventral and dorsal rows of sensoryneurons (dendrites indicated by arrowheads). The second (TSM) region, devoid of sensory neurons, extends from the TSM to the junctionbetween the L1 and L3 nerves (L1-L3) and carries the proximally growing axons from the margin neurons. Proximal to the L1-L3 junction, themerged nerve travels within the radius, parallel to the costal nerve, and collects the axons from more proximal sensory neurons. GSR indicatesthe neuron of the giant sensillum on the radius. (B) The same wing labelled with RK2, an antibody that recognizes glial nuclei. Although theRK2 staining is clearly specific for glial nuclei, high background due to non-specific staining was often present on the wing blade. Closeobservation of wings stained with RK2 showed that the non-specific staining was not nuclear. Note the presence of glial nuclei (arrows) alongall the wing nerves. Bar, 50 µm.

525Origin and migration of peripheral glia

(A. Tomlinson, personal communication) (Fig. 1B). Table 1shows the average number and variability of glial cells indifferent regions of the wing.

Glial cells on L1 originate within the wing andmigrate along the nerveIf glial cells originate within the disc, they have to be clonallyrelated to the wing epithelium. Therefore, I have inducedmitotic recombination early during the first larval instar, whendisc cells start proliferating, and determined whether themarked clones contained both epidermis and glial cells. A highfrequency of those clones was obtained by using the flp recom-binase system (Golic and Lindquist, 1989). Struhl and Basler(1993) have constructed a line carrying an engineered Pelement in which the β-gal-coding sequences are separatedfrom an actin promoter by FRTs, the target sites for the flprecombinase. Ubiquitous expression of the β-gal only takesplace in clones of cells in which the flp has induced recombi-nation between the FRT sites. β-gal-expressing clones wereanalyzed between 17 and 42 hours AP, a stage at which thewing has acquired adult morphology. The conditions of heatshock were chosen so that 0 to 7-8 large clones were presentin each wing. The size and the frequency of the clones weresomewhat variable because no attempt was done to stage thelarvae precisely. Two lineage compartment boundaries exist inthe wing blade: the anteroposterior boundary, just anterior tothe fourth longitudinal vein, and the dorsoventral boundary,between the dorsal and ventral wing blade epithelia (see Blair,1993 and references therein). β-gal-expressing clonesrespected the boundary between the anterior and posteriorcompartments, which is established very early, but didsometimes cross the dorsoventral boundary, which is estab-lished at later stages of disc development. In this report, I willmostly concentrate on clones present along the L1 nerve,

Table 1. Average number of nuclei stained on the wingusing the glial-specific antibody RK2

Sevelen No. of stained nuclei

L1 (8)* 83 (69-94)TSM (8)* 27 (22-30)L3 (10)* 22 (13-30)

L1, TSM, L3 indicate the regions where nuclei were scored. L1: glialnuclei of the anterior margin from the distal tip of the vein to the L1-L3junction; TSM: glial nuclei between the twin sensillum on the margin and theL1-L3 junction; L3: glial nuclei between the last neuron on the L3 nerve andthe L1-L3 junction.

*The number of wings analyzed (Sevelen control flies).For each region, the average number is indicated on the left part of the

column, the range (maximum and minimum values) on the right.

Fig. 2. Glial cells originate from epithelial cells. Clone located at thedistal tip (B) or in the middle region (D) of the anterior margin,detected with anti-β-gal. Anti-HRP labelled neurons are shown in(A,C). The filter used in this and in the following experiments todetected the FITC signal is not a band pass filter, therefore there waspartial bleed through of the Cy3 signal, so that in a single exposurewith the FITC filter both signals can be detected. Symbols as in Fig.1. Wings at 34 hours AP (A,B) or 17 hours AP (C,D). Note the shapeand position of glial nuclei, elongated and wrapping the nerve, andcompare with those of epithelial, blood and tracheal cell in thefollowing pictures. Bar, 25 µm.

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where glial nuclei expressing β-gal can be easily distinguishedfrom the nuclei of other stained cells by their shape andposition along the nerve.

Clones induced by the flp recombinase were present atdifferent positions within the wing blade. In cases where theyincluded cells of the L1 vein, marked glial cells were detectablewithin the clone, indicating that wing cells have the potentialto become glia (Fig. 2). Since it was possible that some glialcells arise from wing cells and other from the CNS, wings con-taining no clones were examined, in order to see whetherlabelled glia had migrated from a clone located outside thewing. Whenever clones were not found in the wing epithelium(at least ten such wings were scored) or were present in regionsoutside the L1 vein, no labelled glial cells could be observed(Fig. 3). In contrast, labelled blood cells and trachea comingfrom outside were usually found in those wings (Fig. 3 anddata not shown). These results demonstrate that all wing glialcells differentiate locally and that only some wing regionsdrive glial differentiation. In addition to glial cells found withinclones, some labelled glial cells were observed along the nerve,adjacent to the clones (Figs 2, 4). This implies that glial cellsor precursors are able to migrate for a certain distance alongthe nerve. Both results were confirmed in experiments in whichwings were double labelled with anti-β-gal (the clone marker),and the RK2 antibody, to identify glial cells independently(data not shown).

Since glial cells outside a clone could be seen in a varietyof clones located at different positions along the L1 nerve (Fig.4), it seems that cell migration is a general feature of glial cells.The extent of migration seems variable, but rather limited: inno case did a very distal clone give rise to marked glial cellsin the proximal part of the wing (Fig. 4). Two observations

suggest that some glial cells may not migrate at all: first, β-gal-positive glial cells adjacent to a clone make up only a subsetof the RK2-positive cells present at that location; second, β-gal-positive glial cells were found within a clone, even in caseswhere the clones were very small (data not shown).

Glial cell and sensory organ developmentNot all the regions carrying the L1 vein are gliogenic. Labelledglial cells in L1 were observed when the clones were along theanterior margin and included sensory organs (Figs 2, 4), butnot when clones were in the ‘TSM region’, which has nosensory organs (Fig. 5).

Notably, as the three rows of innervated bristles of theanterior margin (Palka et al., 1979; Hartenstein and Posakony,1989), L1 glial cells originate from both dorsal and ventralepithelium. This was shown by analyzing clones that did notstraddle the boundary between the dorsal and the ventral com-partments present along the anterior margin (Fig. 4A,D anddata not shown). Finally, glial cell development is induced atpositions that are not normally gliogenic in mutants that carryectopic nerves. In wild-type wings, the rows of innervatedbristles extend until the point at which the L3 vein reaches themargin; however, in flies mutant for fused (fu) (Nusslein-Volhard and Wieschaus, 1980), innervated bristles are foundposterior to this position. The sensory axons of these addi-tional bristles form a nerve that travels along the margin,reaches the distal tip of the L1 nerve and starts following it(Fig. 6). Glial cells are present along this ectopic axon bundle,at the same density as that observed on the L1 nerve. Similarresults were obtained with the engrailed (en) mutation, whichinduces partial duplication of anterior structures in theposterior of the wing (Garcia-Bellido and Santamaria, 1972;

A. Giangrande

Fig. 3. Glial cells originate from specific regions of the wing. 24-34 hour AP wings. No labelled glial cells can be detected in wings that haveclones (indicated by curved arrows) in the hinge region (A,B) or in the posterior compartment (C,D). (A,C) Labelled with anti-HRP; (B,D)same wings, but labelled with anti-β-gal. L1 and L3 indicate the L1 and L3 nerves, respectively. Marked blood cells (open arrowheads) wereobserved along all veins as well as marked trachea along L3 (not shown), even in cases where clones were completely absent (data not shown),indicating that these cells originate outside the wing and enter it during development. Bar, 100 µm.

527Origin and migration of peripheral glia

Lawrence and Morata, 1976). As can be seen in Fig. 7, theectopic L4 and PM nerves induced by en both carry glial cells.These three findings show that gliogenesis is associated withneurogenesis.

Glial cells on the anterior margin only migrate in onedirectionGlial migration could be random or monodirectional. Todetermine the direction of glial migration, I looked at wings inwhich only one clone was present along the gliogenic region,the anterior margin. In some cases, clones did not include thedistal part of the margin. Notably, whereas marked glial cellswere found proximal to the clone, they were never observeddistal to it (Figs 8, 9). Thus, glial cells only travel from distalto proximal, the same direction as that taken by axons. This

type of migration is specific to glial cells, since blood cells andtrachea can enter the wing proximally and therefore migrate inthe opposite direction (Fig. 3). It is worth noting that, in somemutants (Fig. 7 and data not shown), nerve fibres can growalong the wing blade from proximal to distal, even though thegeneral polarity of the epithelium has not changed. In thesecases, glial cells are present all along these nerves showingopposite polarity. Finally, proximodistal migration of glialcells was observed along the nerve in the leg, which containsaxons directed distally and originating from motor neurons inthe CNS (data not shown).

While on the anterior margin glial cells always migratedtowards the base of the wing, in more proximal regions, it ispossible that they also migrated in the opposite direction.These regions were more difficult to analyze because they wereoften damaged by the dissection. However, in a few cases ofclones along the proximal radius, labelled glial cells seemed tobe located distal to a clone (Fig. 8C,D). This proximal-to-distalmigration involved few glial cells and only occurred for a shortdistance since no stained nuclei were found on the anteriormargin.

Glial cell migration and axonogenesisTo determine at which stage glial cells start moving, Iexamined clones at different stages of wing development.Labelled glial cells outside a clone were clearly detectable at21-22 hours AP (Fig. 9), although some migration was occa-sionally found at 17-18 hours AP (data not shown). Comparedto older wings, both the number of migrating glial cells andtheir distance from the clone were smaller (compare Figs 9,4D). Although at 17 hours AP most anterior margin axons arestill actively growing, the final axonal pathway has alreadybeen established (Murray et al., 1984).

The fact that glial cells migrate along the nerve suggeststhat axonal growth and glial migration are interconnected

Fig. 4. Glial cells migrate along the nerve. 24-39 hour AP wings.(A-C) Two examples of glia leaving distal clones. (A,B) A doubleexposure in order to see the position of marked epithelial and glialcells (orange) relative to that of neurons and axons, labelled withanti-HRP (green). (C) Same wing as in B, anti-β-gal staining.(D) Example of a clone in the proximal anterior margin. Glial nucleiare indicated by arrows, neurons by open arrows. Note in D thepresence of the trachea (t) running adjacent to the L3 nerve (L3).Marked blood cells are indicated by open arrowheads. All clonesinclude cells that form sensory organs (so), see also thecolocalization of anti-HRP and anti-β-gal staining in neurons inA,B,D. Note that staining in B and C includes cells from the anteriorand the posterior compartments (the boundary runs few cells anteriorto the fourth vein which is indicated by (IV)), this is due to thepresence of several juxtaposed clones in the wing blade. The clonesin A and D contain only cells from the dorsal epithelium. In allexperiments, the dorsoventral position of a given clone wasestablished by comparing it to the position of other wing structures:the neuron of the L3-v sensillum is located ventrally, whereas theother neurons of L3 sensilla and those of the TSM at the base of theAM are located dorsally. Along the AM, the dorsal epitheliumcarries only a row of chemosensory organs, each innervated by acluster of five neurons; on the other side, the ventral epitheliumcarries both chemosensory and mechanosensory organs, which arerespectively, polyinnervated and monoinnervated (Murray et al.,1984). Bar, 25 µm (A,D), or 50 µm (B,C).

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processes. To test this hypothesis, I have analyzed the effectsof mutations altering axonogenesis on glial cell migration. Inwings mutant for Abruptex (Ax), a N allele affecting veinformation, axons along the anterior margin often form athickened nerve (neuroma) (Palka et al., 1990). In general, afew fibres continue to travel proximally. In some cases,however, the nerve stops growing beyond the neuroma. Asshown in Fig. 10, a high number of glial nuclei accumulate onthe neuroma, indicating that glial migration is altered whenaxonal growth is abnormal. Since N seems to affect the totalnumber of glial cells (embryonic PNS: Hartenstein et al.,1992; adult PNS: Giangrande et al., 1993; Giangrande, unpub-lished results), it is possible that its effects on glial migrationare due to altered glial development. For this reason I alsoanalyzed flies mutants for fuP1, which display aberrant growthof wing axons (M. Schubiger and A. Giangrande, unpublishedobservations). As it can be seen in Table 2 and in Fig. 6, glialand neuronal cells along the anterior margin are present innormal cell numbers in fuP1. The fact that there is no obviouscell fate alteration in the nervous system makes fu a usefultool to investigate glial behaviour when axonogenesis isaffected.

In a number of fuP1 males, the L1 nerve thickens in theTSM region, giving rise to different mutant phenotypes. Inone case, L1 fibres show abnormal growth and form aneuroma; some fibres continue to grow beyond the neuroma,meet with the L3 nerve and connect with more proximalfibres, as in the wild-type wing (Fig. 11A,B). Despite thepresence of a nerve leaving the neuroma, only one or two glialnuclei are present in this proximal part of the nerve, most glialcells accumulating on the neuroma, as if they had beentrapped there. In other cases, the L1 nerve makes a 90° turn

in the TSM region (Fig. 11C-G), meets L3 in a more distalposition than in the wild type, and starts growing along it butin the opposite direction, that is, towards the distal tip of thewing. The abnormally growing L1 fibre bundle thickens theL3 nerve and either forms a neuroma on L3, or progressivelystops. Although most L1 fibres turn, a few of them growtowards the base of the wing but stop before reaching the

A. Giangrande

Fig. 5. Only regions carrying sensory organsare gliogenic. (A) Example of a clone in theTSM region (TSM), same wing as in Fig.4A; (B) magnification of the TSM region.Double exposure to detect β-gal-expressingcells (red) and neurons (green)simultaneously: only epithelial cells can bedetected within the clone in the TSM region(TSM). Asterisks and open arrowheadsindicate marked tracheal nuclei and bloodcells, respectively. Glial cells are indicatedby arrows. A tracheal branch (t) leaves theL1-L3 junction (L1-L3) and travel in the L3vein.

Table 2. Average number of RK2-positive nuclei in wild-type and fuP1 males

No. of stained Genotype nuclei on L1

Sevelen/Y 80 (*6)fuP1/Y without neuroma 94 (*22)fuP1/Y with neuroma 105 (*12)

Wings were scored between 30 and 30

G hours AP. The slight difference (80versus 83 nuclei) between wild-type values in this table and Table 1 might bedue to the fact that here only males were scored, whereas in Table 1 noattempt to sex animals was done. Males are slightly smaller than females, andit has already been shown that the smaller, male wings carry fewer sensoryorgans (Palka et al., 1979) than female wings. Note that the number of nucleiin fu wings is higher than in wild type (rows 2 and 3). There are two reasonsfor this difference. In all fu wings, the innervated parts of the margin reachmore posterior positions compared to the wild type, due to the presence ofadditional sensory organs at the distal tip of the margin (see also Fig. 6). Thisectopic portion of the nerve is also wrapped by glial cells, which wereincluded in the L1 counting. In addition, in fu wings containing a neuroma(row 3), numbers were even higher than in wings with no neuroma. The mostlikely explanation for this finding is that glial cells that would normallymigrate beyond the L1-L3 junction and therefore would not be scored, wereincluded in the L1 counting. As shown in Fig. 11, glial cells accumulate distalto and within the neuroma present on the margin or in the TSM region, andtherefore remain within the L1 region instead of migrating to more proximalpositions.

529Origin and migration of peripheral glia

sensory neurons on the proximal radius,leading to the formation of a truncated L1nerve. As in the first case, glial migration isdisrupted: glial nuclei increase in the TSMregion and cluster at the site just preceding thebend of the bundle. The number of glial nucleion the L3 nerve does not seem to increasecompared to wild type, suggesting that L1glial cells do not follow the abnormallygrowing fibres.

DISCUSSION

Wing glial cells differentiate fromimaginal disc cellsAlthough the existence of peripheral glial cellshas been known for a long time, the analysisof their development has started only recentlyand little is known about their origin (Auldand Goodman, 1992; Fredieu and Mahowald,1989; Hartenstein et al., 1992; Giangrande etal., 1993; Giniger et al., 1993; Nelson andLaughon, 1993). It was not known whetherglial cells or precursors develop locally, as theneurons of the sensory organs, or, since glialcells in general migrate during development(see below), whether they migrate to theperiphery from the CNS. A third possibilitywas that the wing is populated by two types ofglial cells, one differentiating locally, theother migrating from the CNS. In the adult flyPNS, migration could occur either duringpupal development, through the nerves con-necting the appendages to the CNS, or duringlarval development, becoming associated atearly stages with imaginal disc cells. Therecently developed flp recombinase systemmakes it possible to obtain internally markedclones at high frequency (Golic and Lindquist,1989; Struhl and Basler 1993). Using thisapproach, I here show that all wing glial cellsare clonally related to the surrounding epithe-lium. It was already known that cells on thewing blade are not monopotent, since theygive rise to epithelial and sensory organ cells.The present results clearly indicate that thesecells have at least another choice and can takeon a third, glial, fate. Although clonal analyseshave not yet been performed, it seems thatembryonic peripheral glial cells also differen-tiate locally, through delamination from theposterior-lateral ectoderm (Hartenstein et al.,1992).

Whether local differentiation represents thegeneral rule for adult peripheral glial cells stillremains an open question. The mechanisms ofglial development may be different in the legdiscs, where motor and larval fibres (Bolwig,1946; Zipursky et al., 1984; Jan et al., 1985;Tix et al., 1989), absent in the wing (Jan et al.,

Fig. 6. fu induces the formation of glial cells on ectopic nerves. fuP1 36 hours APwing. (A,D) Anti-HRP and (B,C) RK2 staining, respectively. (A,B) Bar, 50 µm.(C,D) Detail of the wing shown in A and B, distal tip, Bar, 25 µm. L1 and L3 indicatethe L1 and L3 nerves, respectively. Neurons and glial nuclei are shown by open andfilled arrows, respectively. The nerve on the margin extends to more posteriorpositions than in the wild type (compare with Fig. 1). The overall number andorganization of neurons, however, is normal. The bracket indicates the region carryingthe ectopic nerve, between the points at which L3 and L4 veins reach the margin. Notethat glial nuclei are present on this ectopic portion of the nerve.

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1985; Tix et al., 1989), may constitute a pathfor glial migration from the CNS. Interest-ingly, at least one type of peripheral glia donot differentiate locally: bromodeoxyuridineincorporation studies indicate that subretinalglial cells migrate into the eye disc through theoptic stalk (Choi and Benzer, personal com-munication). The fact that different develop-mental strategies can be adopted suggests that,as in the CNS, different types of glial cellsexist in the PNS.

Glial cells migrate during adultdevelopmentThe clonal analysis shows for the first timethat glial cells in the PNS migrate during adultdevelopment. The ability to migrate seems tobe a feature shared by different types of glialcells, in both invertebrates and vertebrates:insect central and enteric glial cells (Jacobs etal., 1989; Klämbt et al., 1991; Copenhaver,1993); Schwann cells (Le Douarin, 1982; LeDouarin et al., 1991; Bronner-Fraser, 1993);oligodendrocytes (Zhou et al., 1990; Gans-muller et al., 1991 and references therein).Some migration was already observed ataround 17 hours AP. Since the number of glialnuclei detected by several markers is stillincreasing at that stage (Giangrande, unpub-lished results), it is likely that some cells startto migrate before the entire population of pre-cursors has stopped dividing, as has alreadyproposed for oligodendrocytes (Gansmuller etal., 1991 and references therein).

Glial migration in the wing may accountfor the variable organization found withdifferent types of glial markers (Giangrandeet al., 1993 and the present study). Variabil-ity, which has not been observed in othertypes of glial cells such as embryonic midlineand longitudinal glia, may be typical of glialcells associated with non-metameric struc-tures and reflect some plasticity during devel-opment.

Glial migration and axonogenesis areintimately connected processesGlial cells on the anterior margin migratefollowing growing axons. Since it has beenshown that CNS and PNS glia from vertebratealso use growing axons as substrata formigration (Zhou et al., 1990; Carpenter andHollyday, 1992), it is likely that similarcellular and molecular mechanisms areinvolved, making it possible to use glialmigration in the fly wing as a model to inves-tigate the genetic bases of vertebrate glial cellmigration. Glial migration and axonal naviga-tion are both affected by the N and fumutations: whenever neuromas form or axons

A. Giangrande

Fig. 7

531Origin and migration of peripheral glia

Fig. 7. en induces the formation of glial cells on ectopic nerves. en1

24-32h AP wing. (A,C) Anti-HRP staining, (B,D) RK2 staining;(C,D) a detail of the wing in A,B. Two nerves indicated by L1 andL3 display the normal polarity and are present at normal positions. Inaddition, there are sensory organs and nerve fibres, indicated by L4and PM, in the L4 vein and on the posterior margin, respectively, dueto the transformation of posterior into anterior structures. Thetransformation is not complete: the organization of sensory organs isnot normal, with some parts of the PM carrying more sensory organs

Fig. 8. Glial cells migrate following the axon direction.(A,B) Example of monodirectional glial migration:double exposures of a 37 hour AP wing. The clone onthe anterior margin (curved arrow) starts in the middle

region. (B) Marked glia (arrows) are present proximal to the clone but not distal to it (see bracketed region). Note that no glia were foundassociated to the clone at the distal tip (open arrow), which ends posterior to the sensory organs. Blood cells are indicated by open arrowheads.(C,D) Example of proximodistal glial migration from a clone (curved arrow) in the hinge region of a 21-22 hour AP wing. r and tg indicate thenerves on the proximal radius and on the tegula, respectively. (C) Labelled with anti-β-gal; (D) double exposure. Bar, 25 µm (A,C,D), or 50 µm(B).

than others; the axons form abnormal nerves that never connect tothe CNS. (A,C) The neuron on the L4 vein sends two fibres, onegoing proximally, the other distally, neither of which connects withother nerves (see asterisks at the end of the two fibres). Thedirections of the L4 and L3 fibres are compared and indicated bylarge arrows. Glial cells, indicated by small arrows, are present onthe ectopic fibres; all but one of the glial cells on L4 are located onthe distally growing fibre.

Fig. 9. Glial migration during development.21-22h AP wing: (A) Anti-β-gal;(B) double exposure. Although most glialnuclei (arrows) are contained within theclone present on the anterior margin (L1),some have migrated proximally. Thepresence of a blood cell on the blade isindicated by an open arrowhead. The TSM(TSM) cannot be detected because it is outof focus. Note that no glial cells are presentdistal to the clone (region in bracket).

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take wrong directions, glial cells stop migrating and accumu-late at the point at which axons behave abnormally. Althoughit is possible that, in the case of N, altered gliogenesis accountsin part for defects in migration (Hartenstein et al., 1992; Gian-grande et al., 1993; Giangrande, unpublished results), thiscannot be the case for fu, in which no cell fate change wasobserved in the nervous system (present study).

The present results allow us to formulate some hypotheseson the cellular mechanisms involved in glial migration. Glialmigration starts later that axonal navigation: anterior marginaxons start growing by 13 hours AP (Murray et al., 1984),whereas migrating glial cells were only occasionally observedat 17 hours AP. Thus, glial cells could migrate using molecularcues present on the axons. Alternatively, since N and fumutations also affect the vein pattern (Lindsley and Zimm,1992), both axons and glial cells could respond to cues presentin the extracellular matrix or on the wing epithelium sur-rounding the nerve. At any rate, it seems clear that glialmigration is not due to the presence of a general proximodis-tal gradient of molecular cues, since, in en, a mutant that doesnot affect epithelium polarity, glial cells were observed alonga single nerve fibre growing distally.

Gliogenesis is strictly associated with sensoryorgan developmentThe clonal analysis shows that differentregions of the wing behave differentlywith respect to their abilities to give riseto glial cells. Moreover, it shows that thegliogenic region corresponds to the onethat gives rise to sensory organs, theanterior margin. Like the sensory organs,glial cells can differentiate from bothventral and dorsal epithelia. Finally, infuP1 and en mutants, where sensoryorgans form at ectopic positions, glialcells were found on the ectopic nerves.The consequences of these results aretwofold. First, the potential to becomeglial cells is not evenly distributed in thewing blade; second, gliogenesis and neu-rogenesis are linked. Further experi-ments will be necessary to unravel thedevelopmental mechanisms underlyinggliogenesis: induction of glial cells bysensory organs, presence of a precursorcommon to glia and sensory organs, orexistence, on the anterior margin, ofglioblasts and sensory organs precursorcells.

Bodmer et al. (1989) have shown thatone precursor cell divides twice to giverise to the cells of the sensory organ, oneof which has often been called glial-likecell because it wraps the dendrite of thesensory neuron. While the lineage rela-tionship existing between glial cells andsensory organs has not yet beenexplored, it is clear that peripheral glialcells along the nerve bundle and theglial-like cells of the sensory organ are

different populations of cells (Giangrande et al., 1993). As forthe lineage of CNS glial cells, two situations have been found:glial cells in the first optic ganglion, longitudinal glia andmidline glia originate from a separate lineage from that givingrise to neurons (Winberg et al., 1992; Jacobs et al., 1989;Klämbt et al., 1991), whereas glial cells and neurons originatefrom the same precursor, previously called neuroblast 1-1 (NB1-1) (Udolph et al., 1993).

The anterior margin, which is both neurogenic andgliogenic, is a region of high expression of the genes of theachaete-scute complex (ASC) (Skeath and Carroll, 1991;Cubas et al., 1991), a cluster of genes required for neurogen-esis in both CNS and PNS. Since loss-of-function mutationsin the ASC result in lack of neural precursors (Garcia-Bellidoand Santamaria, 1978; Ghysen and O’Kane, 1989; Cabrera etal., 1990; Martin-Bermudo et al., 1991; Skeath and Carroll,1992), it is tempting to speculate that the genes of the ASCaffect gliogenesis in the same way they induce neurogenesis,namely by promoting the differentiation of the precursor cell.Although the analysis of glial development in proneuralmutants will not clarify the lineage relationship existingbetween glial cells and sensory organs, it will make it possibleto identify genes required for gliogenesis and to establishwhether neurons and glial cells share the same geneticpathway.

A. Giangrande

Fig. 10. Effects of the Ax mutation on axonogenesis and glial migration. 33 hour AP wingfrom a AxP8/+; rA87/+ female. The rA87 enhancer trap line labels glial nuclei (arrows)present on the wing nerves (see Giangrande et al., 1993). Wing stained with (A) anti-HRP,(B) anti-β-gal, and (C) double exposure. The L1 axons grow along the anterior margin (L1),but form a neuroma (n) once they get to the middle region. Note the accumulation of glialnuclei along the neuroma.

533Origin and migration of peripheral glia

The rat RK2 antibody was a gift of A. Tomlinson. The fuP1, en1,the hsflp; FRT β-gal and the rA87 stocks were kindly provided by W.Pak, P. Johnston, K. Basler and V. Auld, respectively. I am gratefulto S. Blair, E. Borrelli, P. Lawrence, J. Palka, M. Schubiger and P.Simpson for helpful discussions and to P. Lawrence, M. Murray, M.Schubiger and P. Simpson for many thoughtful comments on the man-uscript. I thank C. Carteret for excellent technical assistance and ColorSystem, B. Boulay, J. M. Lafontaine and S. Metz for help with thefigures. This work was supported by CNRS and INSERM.

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(Accepted 2 December 1993)

Note added in proofThe previously unpublished results of Choi and Benzer arenow in press: Choi, K.-W. and Benzer, S. (1994). Migration ofglia along photoreceptor axons in the developing Drosophilaeye. Neuron (in press).

A. Giangrande