Glia in the fly wing are clonally related to epithelial cells and use the nerve as a pathway for migration

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1 Development 120, (1994) Printed in Great Britain The Company of Biologists Limited 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 de l INSERM, Institut de Chimie Biologique, Faculté de Médecine, 11 rue Humann, Strasbourg Cedex, France SUMMARY The Drosophila major wing nerve collects axons from the anterior margin sensory organs. Using the flp recombinase to make clones, I show that all glia present on this nerve are clonally related to wing epithelial cells. Glial cells arise only from regions that also give rise to sensory organs and migrate along the nerve following the direction taken by axons. As in vertebrates, wing glial cells start migrating at a stage at which axons are growing. The migration of wing glial cells is affected by two mutations altering axonogenesis, fused and Notch, which suggests that the two processes are tightly associated. Key words: gliogenesis, cell migration, mitotic clones, fly, Drosophila, wing, nerve, fused, Notch INTRODUCTION Early studies indicate that insect glial cells represent a heterogeneous population of cells displaying different features and mechanisms of development depending on their position (Wigglesworth, 1959; see Klämbt and Goodman, 1991 for a review). For example, in the case of the embryonic central nervous system (CNS), glial and neuronal cells originate at the same time within the nervous system. During embryogenesis, glial cells display a metameric and stereotyped pattern of migration (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 have previously given rise to neurons, and migrate for long and variable distances following neuronal paths (Copenhaver, 1993). As for the adult peripheral nervous system (PNS), little information is available on the origin of glial cells. Although it has already been shown that cells of the appendages are able to give rise to epithelium and sensory organs, it is not clear whether they also have the potential to become glial cells. Tissue culture studies showed that at least some glial cells or precursors are present in the wing imaginal disc by the end of larval life but it could not be established whether glial precursors were present in the disc at earlier stages (Giangrande et al., 1993). Moreover, those results could not exclude the possibility that some glial cells differentiate locally whereas some others migrate from outside the wing. Another aspect that has not yet been investigated is the ability of glial cells in the PNS to migrate. Glial migration is a feature common to vertebrates and invertebrates (see Discussion), therefore it would be relevant to see whether peripheral glia also display this behaviour and, if they do migrate, to investigate the modalities of the process. To assess unambiguously whether glial cells in the wing are clonally related to epithelial cells and whether they migrate during development, I have marked cells using the recombinase flp system (Golic and Lindquist, 1989; Struhl and Basler, 1993). I show that glial cells present on the wing anterior marginal nerve originate within the disc from regions that also contain the precursors of the sensory organs, and migrate for relatively short distances along the nerve. Migration starts at a stage at which axonogenesis is actively taking place and occurs in the same direction as that taken by axons. Finally migration of glial cells is dramatically affected by two mutations altering axonal 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 are tightly linked processes. MATERIALS AND METHODS Stocks The wild-type stocks were Oregon R and Sevelen. For clonal analysis, I used a line carrying two transposons, one with the flp gene fused to the heat-shock promoter, the other with the FRT sites between the actin promoter and the β-galactosidase (β-gal) gene, as described in Struhl and Basler (1993). The β-gal product accumulates in the nucleus, due to the presence of a nuclear localization signal. The flies from this line were crossed to the Oregon R stock, and eggs were collected 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 to a functional β-gal gene in the FRT-carrying chromosome, and gives rise to a clone of β-gal-expressing cells. The mutations Ax P8 and fu P1 were identified by M. Schubiger as being viable Abruptex and fu alleles, respectively. Both mutations originated from an EMS mutagenesis in W. Pak s laboratory. The glial-specific enhancer trap line ra87 was provided by V. Auld and C. Goodman. For a description of

2 524 A. Giangrande the wing ra87 profile of staining, see Giangrande et al. (1993). The en 1 stock was kindly provided by P. Johnston. Immunohistochemistry After heat shock, larvae were kept at 25 C. White prepupae were collected and kept at 25 C until the desired stage; therefore, the stage is expressed as hours after pupariation (AP). Dissection, fixation and antibody incubation were performed following the protocol described in Giangrande et al. (1993) with the following modifications: 0.01% sodium azide was added during incubations with primary and secondary antibodies; anti-β-gal (Promega) and anti-hrp (US Biochemical) were used at 1:4000; secondary antibodies were, respectively, Cy3-conjugated-donkey-anti-mouse IgG (Jackson) at 1:600 and FITC-conjugated goat anti-rabbit IgG (Jackson) at 1:400. Vectashield (Vector) mounting medium was used to prevent bleaching. To analyze glia in the Ax P8 mutant, Ax P8/ Ax P8 females were crossed to ra87/ra87 males. Female and male white pupae were separately collected and kept at 25 C until the desired stage and were stained as above. Ax P8 heterozygous females showed weaker and less frequent defects than hemizygous males. The glial-specific RK2 rat antibody was used at 1:1000 and revealed with a Cy3-conjugated goat anti-rat antibody (Jackson) at 1:600. 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 wing On the wing blade, two nerves (L1 and L3) travel along the veins L1 and L3, and merge proximally (Fig. 1A) (for a detailed description of wing sensory organ development, see Murray et al., 1984; Hartenstein and Posakony, 1989). Glialspecific enhancer trap lines have shown that glial nuclei are present on these nerves; however, each line showed different and variable number of glial cells (Giangrande et al., 1993). To analyze the number and variability of glial cells with an independent marker, I have now used a glial-specific antibody 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 sensory neurons (dendrites indicated by arrowheads). The second (TSM) region, devoid of sensory neurons, extends from the TSM to the junction between the L1 and L3 nerves (L1-L3) and carries the proximally growing axons from the margin neurons. Proximal to the L1-L3 junction, the merged nerve travels within the radius, parallel to the costal nerve, and collects the axons from more proximal sensory neurons. GSR indicates the neuron of the giant sensillum on the radius. (B) The same wing labelled with RK2, an antibody that recognizes glial nuclei. Although the RK2 staining is clearly specific for glial nuclei, high background due to non-specific staining was often present on the wing blade. Close observation of wings stained with RK2 showed that the non-specific staining was not nuclear. Note the presence of glial nuclei (arrows) along all the wing nerves. Bar, 50 µm.

3 Origin and migration of peripheral glia 525 Table 1. Average number of nuclei stained on the wing using 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: glial nuclei of the anterior margin from the distal tip of the vein to the L1-L3 junction; TSM: glial nuclei between the twin sensillum on the margin and the L1-L3 junction; L3: glial nuclei between the last neuron on the L3 nerve and the 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. (A. Tomlinson, personal communication) (Fig. 1B). Table 1 shows the average number and variability of glial cells in different regions of the wing. Glial cells on L1 originate within the wing and migrate along the nerve If glial cells originate within the disc, they have to be clonally related to the wing epithelium. Therefore, I have induced mitotic recombination early during the first larval instar, when disc cells start proliferating, and determined whether the marked clones contained both epidermis and glial cells. A high frequency of those clones was obtained by using the flp recombinase system (Golic and Lindquist, 1989). Struhl and Basler (1993) have constructed a line carrying an engineered P element in which the β-gal-coding sequences are separated from an actin promoter by FRTs, the target sites for the flp recombinase. Ubiquitous expression of the β-gal only takes place in clones of cells in which the flp has induced recombination between the FRT sites. β-gal-expressing clones were analyzed between 17 and 42 hours AP, a stage at which the wing has acquired adult morphology. The conditions of heat shock were chosen so that 0 to 7-8 large clones were present in each wing. The size and the frequency of the clones were somewhat variable because no attempt was done to stage the larvae precisely. Two lineage compartment boundaries exist in the wing blade: the anteroposterior boundary, just anterior to the fourth longitudinal vein, and the dorsoventral boundary, between the dorsal and ventral wing blade epithelia (see Blair, 1993 and references therein). β-gal-expressing clones respected the boundary between the anterior and posterior compartments, which is established very early, but did sometimes cross the dorsoventral boundary, which is established at later stages of disc development. In this report, I will mostly concentrate on clones present along the L1 nerve, Fig. 2. Glial cells originate from epithelial cells. Clone located at the distal 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 to detected the FITC signal is not a band pass filter, therefore there was partial bleed through of the Cy3 signal, so that in a single exposure with 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 shape and position of glial nuclei, elongated and wrapping the nerve, and compare with those of epithelial, blood and tracheal cell in the following pictures. Bar, 25 µm.

4 526 A. Giangrande Fig. 3. Glial cells originate from specific regions of the wing hour AP wings. No labelled glial cells can be detected in wings that have clones (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) were observed 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. where glial nuclei expressing β-gal can be easily distinguished from the nuclei of other stained cells by their shape and position along the nerve. Clones induced by the flp recombinase were present at different positions within the wing blade. In cases where they included cells of the L1 vein, marked glial cells were detectable within the clone, indicating that wing cells have the potential to become glia (Fig. 2). Since it was possible that some glial cells arise from wing cells and other from the CNS, wings containing no clones were examined, in order to see whether labelled glia had migrated from a clone located outside the wing. Whenever clones were not found in the wing epithelium (at least ten such wings were scored) or were present in regions outside the L1 vein, no labelled glial cells could be observed (Fig. 3). In contrast, labelled blood cells and trachea coming from outside were usually found in those wings (Fig. 3 and data not shown). These results demonstrate that all wing glial cells differentiate locally and that only some wing regions drive glial differentiation. In addition to glial cells found within clones, some labelled glial cells were observed along the nerve, adjacent to the clones (Figs 2, 4). This implies that glial cells or precursors are able to migrate for a certain distance along the nerve. Both results were confirmed in experiments in which wings 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 variety of 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: in no case did a very distal clone give rise to marked glial cells in the proximal part of the wing (Fig. 4). Two observations suggest that some glial cells may not migrate at all: first, β-galpositive glial cells adjacent to a clone make up only a subset of the RK2-positive cells present at that location; second, β- gal-positive glial cells were found within a clone, even in cases where the clones were very small (data not shown). Glial cell and sensory organ development Not all the regions carrying the L1 vein are gliogenic. Labelled glial cells in L1 were observed when the clones were along the anterior margin and included sensory organs (Figs 2, 4), but not when clones were in the TSM region, which has no sensory organs (Fig. 5). Notably, as the three rows of innervated bristles of the anterior margin (Palka et al., 1979; Hartenstein and Posakony, 1989), L1 glial cells originate from both dorsal and ventral epithelium. This was shown by analyzing clones that did not straddle the boundary between the dorsal and the ventral compartments present along the anterior margin (Fig. 4A,D and data not shown). Finally, glial cell development is induced at positions that are not normally gliogenic in mutants that carry ectopic nerves. In wild-type wings, the rows of innervated bristles extend until the point at which the L3 vein reaches the margin; however, in flies mutant for fused (fu) (Nusslein- Volhard and Wieschaus, 1980), innervated bristles are found posterior to this position. The sensory axons of these additional 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. Similar results were obtained with the engrailed (en) mutation, which induces partial duplication of anterior structures in the posterior of the wing (Garcia-Bellido and Santamaria, 1972;

5 Origin and migration of peripheral glia 527 Fig. 4. Glial cells migrate along the nerve hour AP wings. (A-C) Two examples of glia leaving distal clones. (A,B) A double exposure in order to see the position of marked epithelial and glial cells (orange) relative to that of neurons and axons, labelled with anti-hrp (green). (C) Same wing as in B, anti-β-gal staining. (D) Example of a clone in the proximal anterior margin. Glial nuclei are indicated by arrows, neurons by open arrows. Note in D the presence of the trachea (t) running adjacent to the L3 nerve (L3). Marked blood cells are indicated by open arrowheads. All clones include cells that form sensory organs (so), see also the colocalization of anti-hrp and anti-β-gal staining in neurons in A,B,D. Note that staining in B and C includes cells from the anterior and the posterior compartments (the boundary runs few cells anterior to the fourth vein which is indicated by (IV)), this is due to the presence of several juxtaposed clones in the wing blade. The clones in A and D contain only cells from the dorsal epithelium. In all experiments, the dorsoventral position of a given clone was established by comparing it to the position of other wing structures: the neuron of the L3-v sensillum is located ventrally, whereas the other neurons of L3 sensilla and those of the TSM at the base of the AM are located dorsally. Along the AM, the dorsal epithelium carries only a row of chemosensory organs, each innervated by a cluster of five neurons; on the other side, the ventral epithelium carries both chemosensory and mechanosensory organs, which are respectively, polyinnervated and monoinnervated (Murray et al., 1984). Bar, 25 µm (A,D), or 50 µm (B,C). Lawrence and Morata, 1976). As can be seen in Fig. 7, the ectopic L4 and PM nerves induced by en both carry glial cells. These three findings show that gliogenesis is associated with neurogenesis. Glial cells on the anterior margin only migrate in one direction Glial migration could be random or monodirectional. To determine the direction of glial migration, I looked at wings in which only one clone was present along the gliogenic region, the anterior margin. In some cases, clones did not include the distal part of the margin. Notably, whereas marked glial cells were found proximal to the clone, they were never observed distal to it (Figs 8, 9). Thus, glial cells only travel from distal to proximal, the same direction as that taken by axons. This type of migration is specific to glial cells, since blood cells and trachea can enter the wing proximally and therefore migrate in the opposite direction (Fig. 3). It is worth noting that, in some mutants (Fig. 7 and data not shown), nerve fibres can grow along the wing blade from proximal to distal, even though the general polarity of the epithelium has not changed. In these cases, glial cells are present all along these nerves showing opposite polarity. Finally, proximodistal migration of glial cells was observed along the nerve in the leg, which contains axons directed distally and originating from motor neurons in the CNS (data not shown). While on the anterior margin glial cells always migrated towards the base of the wing, in more proximal regions, it is possible that they also migrated in the opposite direction. These regions were more difficult to analyze because they were often damaged by the dissection. However, in a few cases of clones along the proximal radius, labelled glial cells seemed to be located distal to a clone (Fig. 8C,D). This proximal-to-distal migration involved few glial cells and only occurred for a short distance since no stained nuclei were found on the anterior margin. Glial cell migration and axonogenesis To determine at which stage glial cells start moving, I examined clones at different stages of wing development. Labelled glial cells outside a clone were clearly detectable at hours AP (Fig. 9), although some migration was occasionally found at hours AP (data not shown). Compared to older wings, both the number of migrating glial cells and their distance from the clone were smaller (compare Figs 9, 4D). Although at 17 hours AP most anterior margin axons are still actively growing, the final axonal pathway has already been established (Murray et al., 1984). The fact that glial cells migrate along the nerve suggests that axonal growth and glial migration are interconnected

6 528 A. Giangrande Fig. 5. Only regions carrying sensory organs are gliogenic. (A) Example of a clone in the TSM region (TSM), same wing as in Fig. 4A; (B) magnification of the TSM region. Double exposure to detect β-gal-expressing cells (red) and neurons (green) simultaneously: only epithelial cells can be detected within the clone in the TSM region (TSM). Asterisks and open arrowheads indicate marked tracheal nuclei and blood cells, respectively. Glial cells are indicated by arrows. A tracheal branch (t) leaves the L1-L3 junction (L1-L3) and travel in the L3 vein. processes. To test this hypothesis, I have analyzed the effects of mutations altering axonogenesis on glial cell migration. In wings mutant for Abruptex (Ax), a N allele affecting vein formation, axons along the anterior margin often form a thickened nerve (neuroma) (Palka et al., 1990). In general, a few fibres continue to travel proximally. In some cases, however, the nerve stops growing beyond the neuroma. As shown in Fig. 10, a high number of glial nuclei accumulate on the neuroma, indicating that glial migration is altered when axonal growth is abnormal. Since N seems to affect the total number of glial cells (embryonic PNS: Hartenstein et al., 1992; adult PNS: Giangrande et al., 1993; Giangrande, unpublished results), it is possible that its effects on glial migration are due to altered glial development. For this reason I also analyzed flies mutants for fu P1, which display aberrant growth of wing axons (M. Schubiger and A. Giangrande, unpublished observations). As it can be seen in Table 2 and in Fig. 6, glial and neuronal cells along the anterior margin are present in normal cell numbers in fu P1. The fact that there is no obvious cell fate alteration in the nervous system makes fu a useful tool to investigate glial behaviour when axonogenesis is affected. In a number of fu P1 males, the L1 nerve thickens in the TSM region, giving rise to different mutant phenotypes. In one case, L1 fibres show abnormal growth and form a neuroma; some fibres continue to grow beyond the neuroma, meet with the L3 nerve and connect with more proximal fibres, as in the wild-type wing (Fig. 11A,B). Despite the presence of a nerve leaving the neuroma, only one or two glial nuclei are present in this proximal part of the nerve, most glial cells accumulating on the neuroma, as if they had been trapped there. In other cases, the L1 nerve makes a 90 turn Table 2. Average number of RK2-positive nuclei in wildtype and fu P1 males No. of stained Genotype nuclei on L1 Sevelen/Y 80 (*6) fu P1 /Y without neuroma 94 (*22) fu P1 /Y with neuroma 105 (*12) Wings were scored between 30 and 30G hours AP. The slight difference (80 versus 83 nuclei) between wild-type values in this table and Table 1 might be due to the fact that here only males were scored, whereas in Table 1 no attempt to sex animals was done. Males are slightly smaller than females, and it has already been shown that the smaller, male wings carry fewer sensory organs (Palka et al., 1979) than female wings. Note that the number of nuclei in fu wings is higher than in wild type (rows 2 and 3). There are two reasons for this difference. In all fu wings, the innervated parts of the margin reach more posterior positions compared to the wild type, due to the presence of additional sensory organs at the distal tip of the margin (see also Fig. 6). This ectopic portion of the nerve is also wrapped by glial cells, which were included 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 most likely explanation for this finding is that glial cells that would normally migrate beyond the L1-L3 junction and therefore would not be scored, were included in the L1 counting. As shown in Fig. 11, glial cells accumulate distal to and within the neuroma present on the margin or in the TSM region, and therefore remain within the L1 region instead of migrating to more proximal positions. in the TSM region (Fig. 11C-G), meets L3 in a more distal position than in the wild type, and starts growing along it but in the opposite direction, that is, towards the distal tip of the wing. The abnormally growing L1 fibre bundle thickens the L3 nerve and either forms a neuroma on L3, or progressively stops. Although most L1 fibres turn, a few of them grow towards the base of the wing but stop before reaching the

7 Origin and migration of peripheral glia 529 sensory neurons on the proximal radius, leading to the formation of a truncated L1 nerve. As in the first case, glial migration is disrupted: glial nuclei increase in the TSM region and cluster at the site just preceding the bend of the bundle. The number of glial nuclei on the L3 nerve does not seem to increase compared to wild type, suggesting that L1 glial cells do not follow the abnormally growing fibres. DISCUSSION Wing glial cells differentiate from imaginal disc cells Although the existence of peripheral glial cells has been known for a long time, the analysis of their development has started only recently and little is known about their origin (Auld and Goodman, 1992; Fredieu and Mahowald, 1989; Hartenstein et al., 1992; Giangrande et al., 1993; Giniger et al., 1993; Nelson and Laughon, 1993). It was not known whether glial cells or precursors develop locally, as the neurons of the sensory organs, or, since glial cells in general migrate during development (see below), whether they migrate to the periphery from the CNS. A third possibility was that the wing is populated by two types of glial cells, one differentiating locally, the other migrating from the CNS. In the adult fly PNS, migration could occur either during pupal development, through the nerves connecting the appendages to the CNS, or during larval development, becoming associated at early stages with imaginal disc cells. The recently developed flp recombinase system makes it possible to obtain internally marked clones at high frequency (Golic and Lindquist, 1989; Struhl and Basler 1993). Using this approach, I here show that all wing glial cells are clonally related to the surrounding epithelium. It was already known that cells on the wing blade are not monopotent, since they give rise to epithelial and sensory organ cells. The present results clearly indicate that these cells have at least another choice and can take on a third, glial, fate. Although clonal analyses have not yet been performed, it seems that embryonic peripheral glial cells also differentiate locally, through delamination from the posterior-lateral ectoderm (Hartenstein et al., 1992). Whether local differentiation represents the general rule for adult peripheral glial cells still remains an open question. The mechanisms of glial development may be different in the leg discs, 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. fu P1 36 hours AP wing. (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 indicate the L1 and L3 nerves, respectively. Neurons and glial nuclei are shown by open and filled arrows, respectively. The nerve on the margin extends to more posterior positions than in the wild type (compare with Fig. 1). The overall number and organization of neurons, however, is normal. The bracket indicates the region carrying the ectopic nerve, between the points at which L3 and L4 veins reach the margin. Note that glial nuclei are present on this ectopic portion of the nerve.

8 530 A. Giangrande 1985; Tix et al., 1989), may constitute a path for glial migration from the CNS. Interestingly, at least one type of peripheral glia do not differentiate locally: bromodeoxyuridine incorporation studies indicate that subretinal glial cells migrate into the eye disc through the optic stalk (Choi and Benzer, personal communication). The fact that different developmental strategies can be adopted suggests that, as in the CNS, different types of glial cells exist in the PNS. Glial cells migrate during adult development The clonal analysis shows for the first time that glial cells in the PNS migrate during adult development. The ability to migrate seems to be a feature shared by different types of glial cells, in both invertebrates and vertebrates: insect central and enteric glial cells (Jacobs et al., 1989; Klämbt et al., 1991; Copenhaver, 1993); Schwann cells (Le Douarin, 1982; Le Douarin et al., 1991; Bronner-Fraser, 1993); oligodendrocytes (Zhou et al., 1990; Gansmuller et al., 1991 and references therein). Some migration was already observed at around 17 hours AP. Since the number of glial nuclei detected by several markers is still increasing at that stage (Giangrande, unpublished results), it is likely that some cells start to migrate before the entire population of precursors has stopped dividing, as has already proposed for oligodendrocytes (Gansmuller et al., 1991 and references therein). Glial migration in the wing may account for the variable organization found with different types of glial markers (Giangrande et al., 1993 and the present study). Variability, which has not been observed in other types of glial cells such as embryonic midline and longitudinal glia, may be typical of glial cells associated with non-metameric structures and reflect some plasticity during development. Glial migration and axonogenesis are intimately connected processes Glial cells on the anterior margin migrate following growing axons. Since it has been shown that CNS and PNS glia from vertebrate also use growing axons as substrata for migration (Zhou et al., 1990; Carpenter and Hollyday, 1992), it is likely that similar cellular and molecular mechanisms are involved, making it possible to use glial migration in the fly wing as a model to investigate the genetic bases of vertebrate glial cell migration. Glial migration and axonal navigation are both affected by the N and fu mutations: whenever neuromas form or axons Fig. 7

9 Origin and migration of peripheral glia 531 Fig. 7. en induces the formation of glial cells on ectopic nerves. en h 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 and L3 display the normal polarity and are present at normal positions. In addition, there are sensory organs and nerve fibres, indicated by L4 and PM, in the L4 vein and on the posterior margin, respectively, due to the transformation of posterior into anterior structures. The transformation is not complete: the organization of sensory organs is not normal, with some parts of the PM carrying more sensory organs than others; the axons form abnormal nerves that never connect to the CNS. (A,C) The neuron on the L4 vein sends two fibres, one going proximally, the other distally, neither of which connects with other nerves (see asterisks at the end of the two fibres). The directions of the L4 and L3 fibres are compared and indicated by large arrows. Glial cells, indicated by small arrows, are present on the ectopic fibres; all but one of the glial cells on L4 are located on the distally growing fibre. 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 on the 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 found associated 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 hour AP wing. r and tg indicate the nerves 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). Fig. 9. Glial migration during development h AP wing: (A) Anti-β-gal; (B) double exposure. Although most glial nuclei (arrows) are contained within the clone present on the anterior margin (L1), some have migrated proximally. The presence of a blood cell on the blade is indicated by an open arrowhead. The TSM (TSM) cannot be detected because it is out of focus. Note that no glial cells are present distal to the clone (region in bracket).

10 532 A. Giangrande take wrong directions, glial cells stop migrating and accumulate at the point at which axons behave abnormally. Although it is possible that, in the case of N, altered gliogenesis accounts in part for defects in migration (Hartenstein et al., 1992; Giangrande et al., 1993; Giangrande, unpublished results), this cannot be the case for fu, in which no cell fate change was observed in the nervous system (present study). The present results allow us to formulate some hypotheses on the cellular mechanisms involved in glial migration. Glial migration starts later that axonal navigation: anterior margin axons start growing by 13 hours AP (Murray et al., 1984), whereas migrating glial cells were only occasionally observed at 17 hours AP. Thus, glial cells could migrate using molecular cues present on the axons. Alternatively, since N and fu mutations also affect the vein pattern (Lindsley and Zimm, 1992), both axons and glial cells could respond to cues present in the extracellular matrix or on the wing epithelium surrounding the nerve. At any rate, it seems clear that glial migration is not due to the presence of a general proximodistal gradient of molecular cues, since, in en, a mutant that does not affect epithelium polarity, glial cells were observed along a single nerve fibre growing distally. Gliogenesis is strictly associated with sensory organ development The clonal analysis shows that different regions of the wing behave differently with respect to their abilities to give rise to glial cells. Moreover, it shows that the gliogenic region corresponds to the one that gives rise to sensory organs, the anterior margin. Like the sensory organs, glial cells can differentiate from both ventral and dorsal epithelia. Finally, in fu P1 and en mutants, where sensory organs form at ectopic positions, glial cells were found on the ectopic nerves. The consequences of these results are twofold. First, the potential to become glial cells is not evenly distributed in the wing blade; second, gliogenesis and neurogenesis are linked. Further experiments will be necessary to unravel the developmental mechanisms underlying gliogenesis: induction of glial cells by sensory organs, presence of a precursor common to glia and sensory organs, or existence, on the anterior margin, of glioblasts and sensory organs precursor cells. Bodmer et al. (1989) have shown that one precursor cell divides twice to give rise to the cells of the sensory organ, one of which has often been called glial-like cell because it wraps the dendrite of the sensory neuron. While the lineage relationship existing between glial cells and sensory organs has not yet been explored, it is clear that peripheral glial cells along the nerve bundle and the glial-like cells of the sensory organ are different populations of cells (Giangrande et al., 1993). As for the lineage of CNS glial cells, two situations have been found: glial cells in the first optic ganglion, longitudinal glia and midline glia originate from a separate lineage from that giving rise to neurons (Winberg et al., 1992; Jacobs et al., 1989; Klämbt et al., 1991), whereas glial cells and neurons originate from the same precursor, previously called neuroblast 1-1 (NB 1-1) (Udolph et al., 1993). The anterior margin, which is both neurogenic and gliogenic, is a region of high expression of the genes of the achaete-scute complex (ASC) (Skeath and Carroll, 1991; Cubas et al., 1991), a cluster of genes required for neurogenesis in both CNS and PNS. Since loss-of-function mutations in the ASC result in lack of neural precursors (Garcia-Bellido and Santamaria, 1978; Ghysen and O Kane, 1989; Cabrera et al., 1990; Martin-Bermudo et al., 1991; Skeath and Carroll, 1992), it is tempting to speculate that the genes of the ASC affect gliogenesis in the same way they induce neurogenesis, namely by promoting the differentiation of the precursor cell. Although the analysis of glial development in proneural mutants will not clarify the lineage relationship existing between glial cells and sensory organs, it will make it possible to identify genes required for gliogenesis and to establish whether neurons and glial cells share the same genetic pathway. Fig. 10. Effects of the Ax mutation on axonogenesis and glial migration. 33 hour AP wing from a Ax P8 /+; 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 glial nuclei along the neuroma.

11 Origin and migration of peripheral glia 533 Fig. 11. Effects of the fu mutation on axonogenesis and glial migration. Three examples of mutant fu P1 /Y wings at hours AP. Labelled with (B,D,F) the glial-specific RK2 antibody, (G) anti- HRP and (A,C,E) double exposures. (A,B) Wing showing a neuroma (n) in the TSM region (TSM). The GSR neuron is indicated by (GSR), the costal nerve by (c) and the L1-L3 junction by (L1-L3). Most glial nuclei (arrows) concentrate on the neuroma, although one nucleus is also present proximal to it. (C,D) Wing with L1 nerve (L1) turning and following the L3 nerve. Glial cells accumulate at the point at which fibres start bending. Some fibres do not bend, continue to grow proximally, but stop once they get to the GSR. No glial nuclei were observed along these disconnected fibres. (E-G) Wing with L1 fibres bending and forming a neuroma on L3. As in the previous case, some fibres grow proximally until the GSR and then stop. Glial cells accumulate at the turning point, although a few stained nuclei were observed proximal to it. On L3, no glial nuclei were found distal to those clustered on the neuroma. Bar, 25 µm (A-G). The rat RK2 antibody was a gift of A. Tomlinson. The fu P1, en 1, 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 grateful to 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 manuscript. I thank C. Carteret for excellent technical assistance and Color System, B. Boulay, J. M. Lafontaine and S. Metz for help with the figures. This work was supported by CNRS and INSERM. REFERENCES Auld, V. J. and Goodman, C. S. (1992). Molecular characterization of embryonic glia in Drosophila. Society for Neuroscience, 22nd Annual Meeting, Anaheim, USA. Blair, S. S. (1993). Mechanisms of compartment formation: evidence that nonproliferating cells do not play a critical role in defining the D/V lineage restriction in the developing wing of Drosophila. Development 119, Bodmer, R., Carretto, R. and Jan, J. N. (1989). Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages. Neuron 3, Bolwig, N. (1946). Senses and sense organs of the anterior end of the house fly larvae. Vidensk. fra Medd. Dansk Naturh, Foren. 109, Bronner-Fraser, M. (1993). Mechanisms of neural crest cell migration. BioEssays 15, Cabrera, C. V., Martinez-Arias, A. and Bate. M. (1990). The expression of three members of the achaete-scute gene complex correlates with neuroblast segregation in Drosophila. Cell 50, Carpenter, E. M. and Hollyday, M. (1992). The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb. Dev. Biol. 150,

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