Developmental Dynamics of Peripheral Glia in Drosophila melanogaster

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1 GLIA 30: (2000) Developmental Dynamics of Peripheral Glia in Drosophila melanogaster KATHARINE J. SEPP, JOOST SCHULTE, AND VANESSA J. AULD* Department of Zoology, University of British Columbia, Vancouver, Canada KEY WORDS gliogenesis; migration; sensory; motor neuron; development; bloodnerve-barrier; PNS ABSTRACT To study the roles of peripheral glia in nervous system development, a thorough characterization of wild type glial development must first be performed. We present a developmental profile of peripheral glia in Drosophila melanogaster that includes glial genesis, developmental morphology, the establishment of transient cellular contacts, migration patterns, and the extent of nerve wrapping in the embryonic and larval stages. In early embryonic development, immature peripheral glia that are born in the CNS seem to be intermediate targets for neurites that are migrating into the periphery. During migration to the PNS, peripheral glia follow the routes of pioneer neurons. The glia preferentially adhere to sensory axonal projections, extending cytoplasmic processes along them such that by the end of embryogenesis peripheral glial coverage of the sensory system is complete. In contrast, significant lengths of motor branch termini are unsheathed in the mature embryo. During larval stages however, peripheral glia further extend and elaborate their cytoplasmic processes until they often reach to the neuromuscular junction. Throughout the embryonic and larval developmental stages, we have also observed a number of similarities of peripheral glia to vertebrate Schwann cells and astrocytes. Peripheral glia seem to have dynamic and diverse roles and their similarities to vertebrate glia suggest that Drosophila may serve as a powerful tool for analysis of glial roles in PNS development in the future. GLIA 30: , Wiley-Liss, Inc. INTRODUCTION Glial cells have diverse roles important for nervous system development and the establishment and maintenance of mature function. During development, immature glia often pre-pattern neuronal migration pathways and provide molecular guidance cues for neurites (reviewed by Silver, 1993; Auld, 1999). Fully differentiated glia have ion channels, neurotransmitter transporters and receptors that are used to maintain the extracellular environment required for neuronal signaling as well as modulate synaptic activity (Treherne and Schofield, 1981; Pfreiger and Barres, 1997; Robitaille, 1998). Glia insulate axonal processes and provide trophic support for neurons (reviewed by Birling and Price, 1995). Glial wrapping also increases action potential efficacy and helps to maintain both axon fasciculation and the blood-nerve-barrier (Auld et al., 1995; Halter et al., 1995). The CNS of Drosophila has proven to be a powerful model system to understand glial roles in development. There are similarities between Drosophila and vertebrate nervous systems at both the cellular and molecular levels. Yet the simpler genetics of Drosophila allow for rapid isolation of genes essential for development. As in vertebrates, different subsets of glial cells are required to prepattern neuronal migration pathways (Jacobs and Goodman, 1989). Experiments where the glial subsets are genetically removed lead to the disruption of the axon pathways that normally traverse the glial scaffolds (Klämbt et al., 1991; Jacobs, 1993; Hidalgo et al., 1995). Also, many novel genes expressed by glia that code for Grant sponsor: Medical Research Council of Canada; Grant sponsor: Howard Hughes Medical Institute. *Correspondence to: V. Auld, Dept. Zoology, 6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada. auld@zoology.ubc.ca Received 17 November 1999; Accepted 25 November Wiley-Liss, Inc.

2 molecules important in axon guidance and gliogenesis have been cloned in Drosophila (e.g., Tear et al., 1996; Noordermeer et al., 1998; Hummel et al., 1999). Many of these genes have homologues in the vertebrate system (reviewed by Goodman, 1994). Compared to our knowledge of the roles of Drosophila CNS glia, our understanding of early developmental roles of glia in the PNS is limited. This has resulted largely from a lack of cellular markers specific to embryonic peripheral glia. Yet it is very likely that peripheral glia are also important for correct formation of PNS axonal tracts. Repo and gcm mutants that disrupt the differentiation of most glia cause PNS nerve tract defasciculation and axon pathfinding defects (Campbell et al., 1994; Xiong et al., 1994; Halter et al., 1995; Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996; Auld 1999). As these mutations do not selectively affect peripheral glia, it is yet to be demonstrated that the PNS glia directly mediate any aspect of axon guidance. To precisely understand what roles peripheral glia play in PNS development, we recently generated a series of GAL4 enhancer traps specific to PNS glia (Sepp and Auld, 1999). The GAL4 lines enable us to characterize the development of peripheral glial cytoplasmic processes by activating the expression of cytoplasmic markers fused to UAS promoters (Brand and Perrimon, 1993). Here we present a developmental profile of peripheral glial development that shows the changes in number, position, and morphology of glia from early gliogenesis to late larval stages. MATERIALS AND METHODS Fly Strains The enhancer trap lines rq286 and rl82 were generated in a standard enhancer trap screen (Klämbt and DYNAMICS OF PERIPHERAL GLIAL DEVELOPMENT Goodman, 1991). These lines express a -galactosidase protein that is fused with a nuclear localization signal. rl82 is a viable insertion in the gliotactin gene (Auld et al., 1995). The insert rq286 is a viable insertion into a novel peripheral glial gene that is currently being characterized. Enhancer trap lines rl82#29 and rq286#5 were obtained by converting the rl82 and rq286 lines to the GAL4 system via targeted transposition (Sepp and Auld, 1999). The UAS-tau-lacZ stock was provided by Andrea Brand. The UAS-gapGFP stock was obtained from the Bloomington Stock Center (donated by A. Chiba). Antibodies 123 Anti- -galactosidase (rabbit IgG) was purchased from Cappel (ICN Pharmaceuticals, Inc., Aurora, OH) and used at 1:1,000 or (mouse IgG) (Sigma BioSciences, St. Louis, MO) and used at a 1:250. The anti-repo (rabbit) antibody was used at 1:200. The monoclonal antibodies 1D4 (mouse anti-fasciclin II IgG) and 22C10 (mouse IgG) (Fujita et al., 1982) were used at a 1:2 dilution. The alkaline phosphatase conjugated goat anti-rabbit (1:1,500 dilution) and horseradish peroxidase (hrp) conjugated goat anti-mouse (1:300 dilution) were both purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). For larvae, rabbit anti-hrp polyclonal and hrp-conjugated goat anti-rabbit secondary (Jackson) were used for bright-field microscopy staining at 1:100 and 1:300 dilutions respectively. For larval confocal analysis, rabbit anti-gfp polyclonal IgG (Clontech, Palo Alto, CA) and goat antihrp (Jackson) primaries were both used at a 1:100 dilution. Fluorescent secondaries donkey anti-rabbit FITC and donkey anti-goat Texas Red (both purchased from Jackson) were used at 1:100. Fig. 1. (Overleaf.) Early stages of peripheral glial development. CNSderived peripheral glia may function as intermediate targets for axon outgrowth into the PNS. A: At Stage 12, glial cells appear on the lateral edge of the CNS with no associated neurons (arrowheads). Soon after its birth, the acc pioneer neuron extends a growth cone (arrows) toward the glial cells. B: acc makes growth cone contacts with peripheral glia before exiting the CNS (higher magnification at early Stage 13). Cytoplasmic labeling of peripheral glia is very similar to nuclear labeling at the same stage in (A) indicating compact cell morphology. C: To reach the PNS, motor neuron growth cones navigate between peripheral glia that have proliferated and arranged into a cone shaped matrix by Stage 13. Whole mount embryos were stained with anti- -galactosidase expression to label glia (blue). mab 22C10 was used to label neurons (brown). Peripheral glial nuclei were labeled using the rq286 nuclear enhancer trap (A,C), glial cytoplasm was labeled using rq286#5 (B). Anterior is at the top, CNS midline is in the center (A) and at the left (B,C). Fig. 2. (Overleaf.) Migration of peripheral glia in the PNS occurs along preformed neuronal pathways. Glial cells extend cytoplasmic processes while this migration occurs. Concave arrows show direction of glial migration. A: Stage 13. Peripheral glia (arrow) begin to migrate and extend cytoplasmic processes along the ISN motor root. B: Late Stage 13. Peripheral glia extend along the ISN, approaching the developing lateral chordotonal (lch) organ cell bodies (lower focal plane). The ISN motor axons meet with centrally migrating dorsal sensory cluster axons that contribute to the anterior fascicle (af). C: Stage 14. Ventral peripheral glia (vpg, arrowhead) migrate into the periphery on the posterior fascicle (pf) that is combined with the SN motor root in this region. D: Stage 15. PNS-derived dorsolateral peripheral glia (dlpg) extend along projections of the dorsal sensory cluster. Ventral peripheral glial cell body migration on the posterior fascicle is complete (arrowhead, lower focal plane). E: Early Stage 16. CNS-derived peripheral glial cytoplasmic processes (arrow) still reach only as far as the lateral region of the PNS. F: Stage 16. Glia migrating peripherally extend up to the lateral chordotonal organ (lch). The length of the unsheathed nerve between peripherally the centrally extending glia is decreased (compare to E). The vpg cell extends processes along the ventral (v) and ventral (v ) sensory cluster projections. G: Late Stage 16. Glial coverage of anterior fascicle/isn is contiguous as CNS and PNS derived glial processes meet in lch region. H: Stage 17. Glial wrapping seems more compact in the late embryo. The ventral peripheral glial cell (vpg, arrowhead) wraps all projections of ventral and ventral sensory clusters. Peripheral glia (blue) were labeled with anti- -galactosidase using enhancer traps rq286#5 (A C) and rl82#29 (D H). Sensory system neurons were labeled with mab 22C10 (brown). The CNS midline is to the left, anterior is at the top.morphology. C: To reach the PNS, motor neuron growth cones navigate between peripheral glia that have proliferated and arranged into a cone shaped matrix by Stage 13. Whole mount embryos were stained with anti- -galactosidase expression to label glia (blue). mab 22C10 was used to label neurons (brown). Peripheral glial nuclei were labeled using the rq286 nuclear enhancer trap (A,C), glial cytoplasm was labeled using rq286#5 (B). Anterior is at the top, CNS midline is in the center (A) and at the left (B,C).

3 Figure 1. Figue 2.

4 Immunohistochemistry DYNAMICS OF PERIPHERAL GLIAL DEVELOPMENT 125 For bright-field microscopy, embryos and larvae were fixed and stained as described previously (Klämbt et al., 1991; Ito et al., 1995). Embryos of the nuclear lacz enhancer trap lines were stained with the anti- -galactosidase antibody. The anti-repo antibody was used to label glial nuclei in wild type embryos. The GAL4 enhancer trap lines were crossed to the UAS-tau-lacZ line, embryos collected and stained using the anti- galactosidase antibody. All embryos were also double stained using either 1D4 or 22C10 monoclonal. Larvae were stained with anti-hrp to detect neurons and then were stained with Xgal (American Biorganics, Niagara Falls, NY) to detect glia. Embryos were cleared in a series of 50%, 70%, and 90% glycerol (in PBS) and dissected. Stained and dissected larvae were also cleared in a glycerol series. All dissections were mounted with 90% glycerol and viewed with a Zeiss Axioskop microscope with Nomarski optics. Thirty-five mm slides were made using Kodachrome ISO 64 film, digitized and assembled into figures using Adobe Photoshop 4.0. For confocal analysis, larvae were prepared as for bright-field, substituting PBS with TBS (25 mm Tris- Cl, ph 7.4; 137 mm NaCl; 5 mm KCl; 0.6 mm Na 2 HPO 4 ; 0.7 mm CaCl 2 ; 0.5 mm MgCl 2 ) and using antibodies for fluorescence as detailed above. Fluorescently labeled larvae were mounted with 90% glycerol, 2.5% DABCO (Sigma) in TBS and viewed under a Biorad MRC 600 confocal microscope. Digital images were processed using NIH Image 1.61 and Adobe Photoshop 4.0. RESULTS To understand the functional roles of PNS glia, we previously generated a series of GAL4 enhancer trap lines that are peripheral glial selective (Sepp and Auld, 1999). When the GAL4 lines are used to drive the expression of a UAS-tau-lacZ marker, an accurate profile of peripheral glial morphological development is feasible. By double labeling glia along with sensory or motor axons, the selectivity and extent of cell contacts that peripheral glia make over the course of development can be discerned. For simplicity, we focus on the abdominal segments (A2 A7) in our characterization. Peripheral glia of thoracic segments develop in a highly analogous manner, the main difference being that there are fewer thoracic peripheral glia compared to abdominal segmental numbers (data not shown). Fig. 3. Nuclear labeling of peripheral glia shows proliferation and movement of cell bodies. A; Stage 13. A glial cell body (arrow) begins to migrate into the PNS along a pre-established neuronal pathway. B: Stage 13. vpg cell body (arrowhead) begins to migrate into the PNS along the posterior fascicle/sn pathway. Concave arrow indicates direction of migration. C: Repo antibody staining labels almost all glial nuclei. Part of its pattern matches peripheral glial enhancer trap staining as peripheral glia are a smaller subset of all PNS glial cells (compare to panel B). D: Final location of peripheral glial cell bodies in the stage 17 embryo. Peripheral glia tightly associate with the anterior fascicle (af) and the posterior fascicle (pf). The position of the vpg nucleus (arrowhead) is only slightly variable, as are the other peripheral glial cell bodies. Whole mount embryos were stained with anti- -galactosidase using nuclear enhancer trap lines rq286 (A,B) and rl82 (D). Anti-Repo antibody was used to label nuclei of wild type embryos (C). Sensory system neurons are labeled with mab 22C10.

5 126 SEPP ET AL. Early Embryonic Peripheral Glial Development Because glial cells of the Drosophila CNS are known to have integral roles in axon guidance during early neurodevelopment, we wanted to examine whether peripheral glia may have analogous roles in the PNS. Previous lineage analysis has shown that almost all peripheral glia are born in the early embryonic CNS. The CNS-derived peripheral glia vary in number from 6 to 8 and arise from neuroblasts 1 3 and 2 5 (Schmidt et al., 1997). In the PNS, a single intersegmental nerve (ISN) associated peripheral glial cell is born and remains roughly in its initial position throughout embryonic development (Campbell et al., 1994; Halter et al., 1995). GCM and Repo, the earliest glial specific markers, are detected in all peripheral glial nuclei by stage 11 (Jones et al., 1995). Two of the enhancer trap lines used in this study label peripheral glia beginning at Stage 11, hence we are able to follow the development of peripheral glia from their origin. One such enhancer trap, rq286, is a P[lacZ, ry ] insertion that labels peripheral glial nuclei. The other enhancer trap, rq286#5, is a P[GAL4, w ] insertion to the same position and allows the staining of peripheral glial cytoplasmic processes. During Stage 12, peripheral glia are situated at their birthplace at the nerve exit/entry region of the CNS/ PNS border, with no associated motor or sensory neurons. Soon after its birth, the acc neuron, that pioneers the intersegmental nerve (ISN) tract, projects a growth cone directly toward the peripheral glia and makes contact with them. After this contact acc subsequently migrates further into the PNS (Fig. 1, 2A,B). This approach, touch and pass of the nerve tract pioneer is stereotypical for all hemi-segments and suggests that the peripheral glia act as intermediate targets for pioneering growth cones. The nature of the molecular signals controlling this interaction has yet to be established. As Stage 12 progresses, peripheral glia proliferate until Stage 13 at which point 8 to 10 glial cells per abdominal hemi-segment can be counted. The peripheral glial enhancer traps also label the subperineurial glia (Ito et al., 1995), that would account for the evidence that only up to 8 CNS-derived peripheral glial nuclei are ever seen by lineage analysis (Schmidt et al., 1997). At all stages, the location of peripheral glial cell bodies (as detected by the nuclear enhancer traps) correspond to Repo staining patterns, confirming that the enhancer traps are indeed glial (example shown in Fig. 3B,C). During the proliferation stage, the glia arrange into a cone shaped array at the CNS/PNS border. During early neurodevelopment, the peripheral glia have a rounded, compact morphology the appearance of nuclear and cytoplasmic labeling preparations are almost identical (Fig. 1A,B). In instances where glia function as guidepost cells, the rounded immature morphology is often observed in both vertebrates and invertebrates (e.g., Silver et al., 1982; Jacobs and Goodman, 1989). It is during Stage 13 that the peripheral glial cytoplasm and membrane processes begin to expand (compare Fig. 1C and 2A). Axons migrating across the CNS/PNS border make extensive growth cone contacts with the glia as they arrive at the cone array and often their migration paths seem to accommodate the glial matrix (Fig. 1C). The compact glial conduit through which neurites project bears striking similarity to the vertebrate CNS/PNS ventral transition zone (TZ) and the dorsal root entry zone (DREZ) (Fraher, 1997; Golding et al., 1997; O Brien et al., 1998). Glial Migration Into the PNS Another major question we sought to answer in our study was to examine how peripheral glia migrate into the PNS. Do glial processes precede migrating growth cone fronts? What are their preferred migrational substrates? Cytoplasmic labeling of peripheral glia has provided great insight into glial migration patterns. We observe that toward the end of Stage 13, the peripheral glial cone shaped formation at the CNS/PNS border loosens as peripheral glia start their migration into the PNS. The migration begins after the first motor neuron pioneers have extended past the glia into the periphery. The glial cell bodies move peripherally whereas their cytoplasmic processes elongate in advance of the cell bodies (compare Fig. 2 and 3). Peripheral glial processes are never observed to extend beyond the leading tip of a pioneer growth cone. PNS glia are also observed to trail behind (and not precede) pioneer growth cones projecting peripherally in vertebrates (Carpenter and Hollyday, 1992). It is therefore likely that glia require axons as substrates to migrate into the PNS. By examining peripheral glial migration in relation to motor or sensory axon development, we are able to gain insight into the preferred migrational substrates and possible trophic dependencies of glia. Peripheral glia associate with mab 22C10-positive axonal tracts throughout all migration in embryogenesis (Fig. 2). The 22C10 antigen is expressed on all sensory neurons as well as some motor neurons and its staining pattern in the embryo has been characterized in detail previously (Ghysen et al., 1986). Briefly, sensory cell clusters in dorsal (d) and lateral chordotonal (lch) regions project axons centrally in a common anterior fascicle (af) in the mature embryo (Fig. 3D). In addition, ventral and ventral (v and v ) sensory cell clusters project axons centrally along a common posterior fascicle (pf) (Fig. 3D). Fas II motor axon staining overlaps with sensory axon labeling in the ventral PNS region (Krueger et al., 1996; Davis et al., 1997) but is very divergent for all motor branches toward their termini. We find that this overlap and divergence is of significance in embryonic glial development. Hence we use the terms anterior fascicle/isn and posterior fascicle/sn to refer to combined sensory/motor tracts, anterior fascicle and posterior fascicle to refer to pure sen-

6 sory tracts, as well as ISN, ISNb, ISNd, SNa and SNc to refer to pure motor branches. Ensheathment of the posterior fascicle/sn Migration of all centrally derived peripheral glia during Stages 13 and 14 occurs along combined sensory and motor axon projections in the ventral region of the embryo. The glia initially associate with the anterior fascicle/isn and posterior fascicle/sn (Fig. 2B,C, 4A,B). One cell, that we designate ventral peripheral glial cell (vpg), migrates on the SNa/posterior fascicle whereas all other peripheral glia migrate along the ISN/anterior fascicle (Fig. 2C H, 3B,D). By Stage 15, the vpg soma arrives at its final embryonic destination (Fig. 2D, 3D) that is usually at the junction of the ventral and ventral (v and v ) sensory cluster projections. The vpg extends processes along the v and v cluster projections and seems to have fully wrapped them by stage 17 (Fig. 5C). This cell only reaches part way along the SNa nerve by the end of embryogenesis (Fig. 4D, 5B). The vpg does not associate with the ISNb, ISNd, or SNc motor tracts to any significant extent in the embryo (Fig. 5A,B). Ensheathment of the anterior fascicle/isn To establish ensheathment of the anterior fascicle/ ISN, peripheral glial migration and wrapping occurs in an analogous manner to the vpg. After migration along the combined sensory/motor tracts (Fig. 2A C, 4A,B) during Stages 13 15, CNS-derived peripheral glia seem to preferentially extend their processes along sensory tracts in regions where the sensory and motor bundles diverge (Fig. 2E H, 4C,D, 5). This is apparent where peripheral glia extend along projections of the lateral chordotonal organ as well as the dorsal sensory cluster (Fig. 5D,E). Peripheral glia extend further dorsally until late Stage 16 when their processes meet the ventrally extending process of the only PNS-derived DYNAMICS OF PERIPHERAL GLIAL DEVELOPMENT 127 glial cell, that we designate the dorsolateral peripheral glial cell (dlpg). The dlpg soma moves very slightly ventrally during embryonic development. Its cytoplasmic processes extend from Stage 15 onward both dorsally along the anterior fascicle and ventrally along the combined anterior fascicle/isn (Fig. 2D, 4C,D). The ventral extension reaches at least as far as the dorsally migrating CNS derived glial processes (Fig. 2D H). The dlpg is most likely not involved in embryonic motor axon guidance. Motor axons often run parallel to the dlpg and its associated sensory nerve instead of along with them (Fig. 5E,F). This suggests a preferential affinity of peripheral glia for sensory axon substrates in the embryo. By the end of embryogenesis at Stage 17, anterior fascicle coverage by peripheral glial cells is complete (Fig. 2H). At this stage, a significant distal portion of the ISN is not covered by these glial cells. It is possible, however, that non Repo-positive cells may provide coverage of the dorsal motor nerve region. For example, with Nomarski optics we are able to detect a persistent twist cell associating with the ISN just dorsal to the dlpg (data not shown). In contrast, for axon branch termini of SNa, SNc, ISNb and ISNd that are not sheathed by peripheral glia we can not detect any other wrapping cells with Nomarski in the mature embryo. Because motor projections have formed functional synapses by the end of embryogenesis (Davis et al., 1997), our observations suggest that peripheral glia are not directly required for initial generation and consolidation of the neuromuscular junction. Finally, in the mature Stage 17 embryo, a portion of peripheral glial cytoplasmic processes remain spanning the CNS/PNS border. This is shown by comparing the gliotactin nuclear enhancer trap with the corresponding gliotactin GAL4 enhancer trap staining patterns (Fig. 6). Although peripheral glial cell bodies are located in the PNS, their processes extend into the CNS. The glial processes wrap the ISN/af and SN/pf nerve roots as they cross the CNS/PNS boundary. Hence peripheral glia, that bear morphological similarity to the vertebrate DREZ and TZ during sensory and Fig. 4. (Overleaf.) Migration of peripheral glia into the PNS follows well behind the growing tips of pioneer motor neurons. A: Stage 14. After its initial contacts with peripheral glia in the CNS at Stage 12, the ISN pioneer migrates into the PNS well ahead of the glial cell bodies. B: Cytoplasmic extension of glial processes (arrow) lags behind the ISN pioneer growth cones (Stage 14). The vpg cell (arrowhead) migrates along SNa nerve root that combines with the posterior sensory fascicle in this region. C: Early Stage 16. The dlpg cell begins to extend cytoplasmic processes along the ISN that is combined with the anterior fascicle sensory neurons in more lateral regions of the hemi-segment. The vpg cell further extends processes along the SNa motor branch. D: Stage 17. Glial coverage of motor tracts is contiguous but not complete as significant lengths of branch termini are not sheathed. The focal plane shows SNc and SNa motor branch termini not wrapped by peripheral glia. Glia are stained with anti- -galactosidase (blue) using nuclear enhancer traps rq286 (A) and cytoplasmic enhancer traps rq286#5 (B) and rl82#29 (C,D). Motor neurons are labeled using mab 1D4 (brown). CNS midline is to the left, anterior is at the top. Concave arrows indicate directions of glial migration. Fig. 5. (Overleaf.) Peripheral glia preferentially extend processes along sensory neuronal tracts in regions where sensory and motor fascicles diverge during embryogenesis. A: Late Stage 16. The ISNb motor branch has no associated glia in its target muscle region (indicated by asterisk). B: Stage 17. The motor branches SNa, SNc, and ISNd are largely unensheathed by peripheral glia. C: Late Stage 16. The vpg cell wraps all projections of the ventral and ventral sensory organs. D: Stage 16. Peripheral glia extend processes along lateral chordotonal organ sensory projections (arrow). E: The dlpg tightly associates with the projections emanating from the dorsal sensory organ cluster (D). F: The dlpg has little affinity for motor tracts in the dorsal region. This particular cell has extended its centrally directed process parallel to the ISN motor branch, along the sensory neurons (not labeled). Glia are labeled with anti- -galactosidase using cytoplasmic enhancer trap rl82#29. Sensory and motor systems are labeled with mabs 22C10 (C E) and 1D4 (A,B,E) respectively.

7 Figure 4. Figure 5.

8 Figuue 6. Figure 8. Fig. 6. The CNS/PNS boundary is penetrated by peripheral glia. A: Stage 17. Peripheral glial cell bodies (arrowheads) are situated in the PNS and associate with combined ISN and SN roots. B: Stage 17. The cytoplasmic processes of peripheral glia penetrate into the CNS along the peripheral nerve roots. Glia are labeled with anti- galactosidase (blue) using nuclear enhancer trap rl82 (A) and cytoplasmic enhancer trap rl82#29 (B). Motor neurons are labeled with mab 1D4. Fig. 7. Glial coverage of peripheral nerves during larval development. Peripheral glia elaborate and extend their cytoplasmic processes along motor branch termini during early larval development. In second instar, glia continue their extension along motor branches that is completed by third instar. A: First instar larval staining of peripheral glia (blue). The ISNb branch is now covered by peripheral glia (arrowhead). Peripheral glia begin to extend along the ISN terminus (arrow) past the region where the ISN diverges from the anterior sensory fascicle. B: A lower focal plane of the same PNS segment as in (A). Peripheral glia now begin to ensheathe SNc motor branches (arrowhead) and SNa motor branches (asterisk). C: Second instar larva. Glial coverage of the ISN motor terminus is now far more extensive (arrow), as peripheral glia have grown well past the ISN/anterior fascicle branch point. The anterior fascicle is slightly anterior to the ISN terminus in this region. Similarly, the SNa root also has more extensive peripheral glial coverage (asterisk). SNc and ISNd are covered by peripheral glia at these stages but cannot be seen in this focal plane. D: Third instar larva. Peripheral glia cover all motor branches of the PNS and have maintained their sensory projection coverage established in embryogenesis. Glial coverage of ISNd is visible (arrowhead) as well as coverage of the forked projections of the SNa terminus (asterisk). Peripheral glia are also observed reaching to the synapses of the ISN distal tip. Larvae of the rl82#29 enhancer trap expressing tau-lacz were stained with Xgal to label glia and anti-hrp to label neurons (brown). Figure 7. Fig. 8. Confocal imaging of third instar larval neuromuscular junctions. Peripheral glia extend as far as the larval neuromuscular junction in the third instar. A: Peripheral glia are indicated in green, neurons in red and overlapping regions are in yellow. Peripheral glia extend to both type I (Ib and Is) and II synapses and small processes are often observed covering the first bouton of a synapse (arrows). B: A second projection as in (A). Peripheral glial projections to neuromuscular junctions are indicated (arrows). Larvae carrying the rl82#29 enhancer trap driving gapgfp expression in glia were labeled with anti-gfp. Neurons were labeled with anti-hrp.

9 130 SEPP ET AL. motor neuronal migration across the CNS/PNS border, remain linking the CNS and PNS in the mature embryo. Larval Development of Peripheral Glia As peripheral glia do not entirely wrap peripheral motor tracts by the end of embryogenesis, we wished to determine whether there is continued glial growth in following larval stages such that pure motor tracts are eventually covered. We labeled larval glia with Xgal that is cleaved into a blue product by the -galactosidase expressed in the glia. Larvae were counterstained with an anti-hrp neuronal marker (Bodmer and Jan, 1987). The hrp epitope is expressed on all neuronal cells. Therefore, as larval neuronal circuitry is analogous to late embryonic, the larval hrp staining pattern essentially represents the 22C10 and Fas II patterns combined. The most significant difference between larval and embryonic neuronal patterning is size. The larvae grow many times their initial body size as they mature over their three instar molts. In first instar larvae, peripheral glia further elongate and elaborate their processes such that distal motor neuron branches that were unsheathed in the embryo begin to acquire peripheral glial wrapping. The basic pattern of glial sensory tract wrapping in the embryo is maintained, yet new cytoplasmic projections of glia that reach to neuromuscular junctions continue to appear. Typically, wrapping extends past the af/isn divergence site, as well as on the ISNb, ISNd, SNa and SNc motor branches (Fig. 7A,B). For the shorter branches ISNb and ISNd, peripheral glia already seem to extend to the synapse at this stage. The remaining motor branches are typically not entirely covered in the first instar although coverage is more extensive than in the embryo. There seems to be no further proliferation of peripheral glia in the larval stages of development as the number of oval shaped peripheral glial nuclei seen with the gliotactin enhancer trap staining does not increase (data not shown). Throughout larval development, however, peripheral glia elongate their cytoplasmic processes to accommodate the growth of peripheral nerves. The peripheral glia of the second instar larva also show more extensive coverage of the distal motor branches compared to the first instar. We observe glial wrapping of the entire lengths of SNa, SNc, ISNb, and ISNd at this stage (Fig. 7C). The ISN distal tip also has greater glial coverage than in first instars, but still is not entirely ensheathed at this time. The peripheral glial staining pattern of the third instar larva is very similar to the second instar but on a larger scale in accordance with overall growth of the larva. By third instar, all motor axon branches are entirely wrapped by peripheral glia, including the distal tip of the ISN (Fig. 7D). With bright-field microscopy, we detected glial processes extending very close to larval neuromuscular junctions. To observe these structures at greater detail, we labeled glia and neurons for confocal imaging. Larvae whose peripheral glia express gap-gfp that associates with cell membranes, were double labeled with anti-gfp and anti-hrp. We observe fine structures of glia associating with both type I and type II synapses (Gorczyca et al., 1993). The glial processes often extend up to and cover the first bouton of many NMJs (Fig. 8) similar to glial caps observed at the blowfly larval NMJ (Osborne, 1967). Labeling of peripheral glia is not always penetrant enough to determine what percentage of synapses have glial caps, however, we note that glia almost always cover the first boutons of synapses at ventral muscles 6,7,12, and 13. It is not known whether the presence of peripheral glia at the larval NMJ is of functional significance. DISCUSSION Peripheral glia, sensory neurons, and motor neurons form the major cellular constituents of the PNS. Unfortunately, peripheral glia have been omitted from most phenotypic analyses of mutants for PNS development because strong cytoplasmic markers have not been available. Thus the advantages of Drosophila as a model organism to study peripheral glial roles in development have been underutilized. To overcome this, we have generated GAL4 enhancer traps selective to peripheral glia on both the second and third chromosomes that can be used in variety of functional analyses. The glial GAL4 marker lines can be crossed into mutant backgrounds for assessment of glial phenotypes, and they can also drive the ectopic expression of genes specifically in the glia (Brand and Perrimon, 1993). In this paper, we present the wild type development of peripheral glia and report the contacts that the glia make with sensory and motor axons. This profile examines glia from their origin in the embryo through to the third instar larva where glia extend to the neuromuscular junction, thus expanding on previous investigations of PNS glia (Fredieu and Mahowald, 1989; Klämbt and Goodman, 1991; Nelson and Laughon, 1993). Our results suggest that there are numerous functional phases for peripheral glia during embryonic nervous system development. We show that PNS glia appear very early in neurogenesis and are contacted by pioneer motor neuron growth cones before exiting the CNS. Therefore, peripheral glia have the potential to provide contact mediated guidance cues and act as an intermediate target for motor axon pioneers. Glial cells have been observed to be critical intermediate targets in axon pathfinding in many instances, most notably the midline glia (Klämbt et al., 1991) and the mesodermally derived transverse nerve exit glia of Drosophila (Gorczyca et al., 1994), the segment boundary cell in grasshoppers (Bastiani and Goodman, 1986) and the great cerebral commissures in mice (Silver et al., 1982). Interestingly during this phase, peripheral glia are

10 DYNAMICS OF PERIPHERAL GLIAL DEVELOPMENT 131 Fig. 9. Schematic diagram of the PNS showing glial coverage of sensory and motor tracts at the end of embryogenesis. A hemi-segment with no glia is shown at the top, indicating all major motor projections: ISN, ISNb (b), ISNd (d), SN, SNa (a), and SNc (c). Sensory cell clusters are also indicated: dorsal (d), lateral chrodotonal (lch), ventral (v ) and ventral (v). The d and lch neurons contribute to the anterior nerve fascicle and the v and v neurons contribute to the posterior fascicle. In the lower hemi-segment, peripheral glia (shaded) are included as well as typical locations of glial nuclei (open circles). The extent of peripheral glial penetration into the CNS (top right) is shown (stippled shading). The distinctive glia, the dorsolateral peripheral glial cell (dlpg) and the ventral peripheral glial cell (dpg) are indicated. Peripheral glia ensheathe all sensory projections by Stage 17. Regions where motor fascicles do not associate with sensory axons are not covered by glia at this stage. In larvae, neuronal patterning is essentially the same, but glial processes ensheathe the motor branches. clustered together in a cone-shaped array at the CNS/ PNS border that bears structural similarity to the vertebrate TZ and DREZ regions that are suggested to be important in axon sorting. The TZ is a dense, funnel shaped collar composed of astrocytes, extending deeply into the spinal cord through which motor axon bundles migrate (Fraher, 1997; O Brien et al., 1998). The DREZ is a similar glial interface of Schwann cells and astrocytes through which sensory axons project into the spinal cord (Golding et al., 1997). In Drosophila, the repo and gcm mutants affect differentiation of most glia (including the peripheral glia) and have abnormal axon tracts at the CNS/PNS border region (Campbell et al., 1994; Xiong et al., 1994; Halter et al., 1995; Hosoya et al., 1995; Jones et al., 1995; Vincent et al., 1996; Auld, 1999) suggesting that peripheral glia also may be involved in axon sorting. After nerve tract pioneers have crossed the CNS/ PNS border, the array of peripheral glia begins to disperse. The peripheral glia begin to migrate into the PNS along axonal tracts while extending cytoplasmic processes begin to wrap the nerve bundles. Glial processes never extend further than the migrating front of the peripheral nerve growth cones, presumably allowing neuronal growth cone steering to occur without interference. This phenomenon is similar to observations that Schwann cell precursors migrate into the PNS along axonal tracts (Carpenter and Hollyday; 1992). The peripheral glial morphological changes at this time are analogous to the changes seen in subsets of CNS glia that initially prefigure pathways as undifferentiated compact glia and then mature after nerve tract generation into glial cells that ensheathe axons (Jacobs and Goodman, 1989). Initially, peripheral glial migration into the PNS occurs along motor axon bundles in the ventral region of each hemi-segment. The ventral motor axon roots soon meet centrally projecting sensory neurons creating combined sensory/motor tracts along which the glia continue to migrate. In regions where the motor axon tracts diverge from sensory tracts, peripheral glia preferentially extend their processes along sensory tracts. Motor and sensory axons express different axonal markers that could confer such different glial cell affinities. Perhaps sensory neurons provide trophic support for peripheral glia that embryonic motor neurons lack. Peripheral glial cells are in constant contact with sensory neurons as they migrate as a chain into the PNS, and never leap ahead of other glia. Also, peripheral glial cell bodies are always well spaced from each other along the PNS nerve tracts by the end of glial migration. These observations suggest trophic interactions occur between peripheral glia and neurons in the embryo to limit the numbers of glial cells as has been shown in the CNS (Sonnenfeld and Jacobs, 1995). In the mature Stage 17 embryo, glia extend and wrap all sensory axonal tracts entirely but leave many motor axon branches untouched (Fig. 9). Perhaps glia do not reach up to the tips of motor axons during this time as they could interfere with muscle target recognition and synapse consolidation. During first instar larval development, however, peripheral glia continue to extend

11 132 SEPP ET AL. their cytoplasmic processes so that they reach toward the distal reaches of motor axons. It will be interesting to determine whether there is a change in glial trophic dependence such that glia are highly attracted to motor neurons after embryo hatching. One surprising observation was that glia seem to not proliferate during the rapid growth phase of the larva, but rather extend their processes along the elongating peripheral nerves to match their growth. The trophic response of glia to neurons might be of the same mechanism as that responsible for extension of glia along the motor axon termini. In the larva, many glia extend processes to the neuromuscular junction and cover the first synaptic bouton. These coverings are possibly similar to the glial cap of the blowfly larval NMJ (Osborne, 1967). To understand the significance of peripheral glial presence at the NMJ, more detailed analyses of glial structure must be performed. Staining of glia does not always penetrate the distal glial processes and detergents required for membrane permeabilization damage such fine membranous structures. With gapgfp labeling of live glia in larvae, we observe significantly greater detail of glia in these regions compared to fixed fluorescent preparations (unpublished data). In vertebrates, Schwann cells cover the neuromuscular junction and these glia can modulate NMJ function (Robitaille, 1998). The Drosophila model system could be very useful to understand glial-synapse interactions as single bouton electrophysiological recordings can be made from the larval NMJ. In the course of characterization of peripheral glial development, we have found that all peripheral glia seem to develop according to a similar program with the exception of two glia, the ventral and lateral peripheral glia. The dlpg cell is distinctive in that it is the only peripheral glial cell born in the PNS. The vpg cell is unique in that it is the only peripheral glial cell born in the CNS to associate with a nerve tract other than the ISN/anterior fascicle. We feel it is unnecessary to number the peripheral glia, as their numbers are slightly variable from segment to segment and because there are no distinguishing qualities amongst the peripheral glial cells. We also agree with Halter et al. (1995) that exit glia are simply a transient configuration of peripheral glia born in the CNS and suggest a simpler nomenclature that may reflect more the differences in differentiation or determination of these cells. Hence, we call all peripheral glia by the same name except for the more distinctive glia, vpg and dlpg, that have previously been called PG1 and PG3 respectively. In summary, we have characterized the changing morphologies, cell numbers, and cell cell associations of peripheral glia during embryonic and larval development. Our characterization indicates that glia may act as intermediate targets during axonal pathfinding early in nervous system development. We conclude that glia migrate into the PNS via preexisting nerve tracts, and preferentially associate with sensory neuron fascicles. Glia extend their cytoplasmic processes during their migrations and by the end of embryogenesis, glial wrapping of sensory axon tracts is complete, however many motor neuron branches are bare. During larval stages, the extension of peripheral glial cytoplasmic processes continues until glia are seen extending to the neuromuscular junction. The study shows that the peripheral glia have many qualities similar to vertebrate Schwann cells and suggests that Drosophila may be a good model organism to gain insight into how glial cells help to form the PNS. Lastly, we have provided a basis from which models of axon guidance, trophic support, and glial maturation can be better understood in mechanistic analyses of mutants. ACKNOWLEDGMENTS The authors wish to thank the Bloomington Stock Center and Andrea Brand for fly stocks; Corey Goodman for the 1D4 and 22C10 monoclonal antibodies and Sarb Ner for the repo antibody. As well, thanks to May Dang and Linda Matsuuchi for the production of the monoclonal antibodies and to Jennifer Bonner and Jane Roskams for helpful comments on the manuscript. This work was supported by the Medical Research Council of Canada and the Howard Hughes Medical Institute. REFERENCES Auld VJ, Fetter RD, Broadie K, Goodman CS Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. Cell 81: Auld VJ Glia as mediators of growth cone guidance: studies from insect nervous systems. Cell Mol Life Sci 55: Bastiani MJ, Goodman CS Guidance of neuronal growth cones in the grasshopper embryo. III. Recognition of specific glial pathways. J Neurosci 6: Birling MC, Price J Influence of growth factors on neuronal differentiation. 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