An Antigen Present in the Drosophila Central Nervous System Only during Embryonic and Metamorphic Stages Masahiro J. GO**+and Yoshiki Hotta

Department of Physics, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

SUMMARY W e report here about an antigen that is expressed in the central nervous system ( C N S ) of Drosophilu only during the embryonic and metamorphic stages. In Drosophilu, axonogenesis and synaptogenesis occur twice during the development: first in the embryonic and second in the metamorphic stages. We generated monoclonal antibodies (MAbs) in order to obtain molecular probes for analyzing axonogenesis or synaptogenesis in the CNS on the assumption that good candidates for molecules responsible for such phenomena must be present in the neuropil during those stages exclusively. As a result, we found MAb 66B2 whose intense immunoreactivity in the neuropi1 of the CNS was observed exclusively in the embryo and pupa, and not in the larva and adult. Immunoblot analyses showed that MAb 66B2 binds specifically to a protein with an apparent molecular weight of 350 K and

neutral PI in the prepupal CNS. A significant amount of the antigen was isolated in forms that were soluble without detergent. Results of immunohistochemistry with MAb 66B2 in a primary culture of embryos showed that some live cells in the ganglion-like cluster were stained, and that neuronal cell bodies and neurites emanating from there were negative. These results strongly suggest that the 66B2 antigen observed in the C N S is an extracellular matrix component secreted from nonneuronal cells. These developmental changes in the 66B2 immunoreactivity in the C N S presumably reflect dynamic changes of an extracellular matrix in the C N S that are accompanied by axonogenesis or synaptogenesis.

INTRODUCTION

sion correlated with the axon growth during development and also during regeneration from injury (Gordon-Weeks, 1989; Skene, 1989; Skene and Willard, 198 1a,b) . Drosophila offers a useful model for the study of early embryogenesis, such as cell differentiation and pattern formation (Campos-Ortega, 1988; Ingham, 1988; Mahowald and Hardy, 1985; Scott and O’Farrell, 1986), and also for the study of neural development, such as axonal guidance (Goodman et a]., 1984), because this organism is amenable to genetic and molecular investigations. In holometabolous insects, such as moths and flies, metamorphosis brings about substantial changes in body form and behavior. The nervous system of a holometabolous insect must also undergo an extensive reorganization during metamorphosis under hormonal control (Levine, 1989; Levine,

Neural development can be divided into several distinct but contiguous stages. In particular, such phenomena as axonogenesis and synaptogenesis are characteristic of neural development. The search for neuronal proteins specific to the growth state corresponding to axonogenesis and synaptogenesis has led to the discovery of axonal growthassociated proteins such as GAP-43, whose expresReceived April 27, 1992; accepted June 10, 1992 Journal of Neurobiology, Vol. 23, No. 7, pp. 890-904 (1992) 0 1992 John Wiley & Sons, Inc. CCC 0022-3034/92/070890-15 * To whom correspondence should be addressed. Present address: Howard Hughes Medical Institute, Yale University School of Medicine, Departments of Cell Biology and Biology, New Haven. CT 06536-08 12, U.S.A.

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0 1992 John Wiley & Sons, Inc.

Keywords: Drosophilu, central nervous system, metamor-

phosis, stage-specific antigen, extracellular matrix.

Stage-Specific Antigen in Drosophila CNS

Truman, Linn, and Bate, 1986; Technau and Heisenberg, 1982; Truman and Reiss, 1988; reviewed in Weeks and Levine, 1990). Thus, some larval neurons sprout new neuronal processes and form new synaptic connections as they are remodeled to serve new functions in the adult (Kent and Levine, 1988; Levine and Truman, 1982, 1985; Levine et al., 1986; Truman and Reiss, 1976; reviewed in Truman, 1990). On the other hand, new adult-specific neurons arise from stem cells (neuroblasts) that divide during larval life to generate postmitotic immature neurons, and at the onset of metamorphosis, some of these cells differentiate into mature functional neurons (Booker and Truman, 1987; Truman and Bate, 1988; reviewed in Truman, 1990). The reorganization of the nervous system during metamorphosis has many parallels with the events observed during embryonic development. Such phenomena as axonogenesis and synaptogenesis thus take place twice in holometabolous insects: once during the embryonic stage, and later during the metamorphic stage. We considered that good candidates for the molecules that play a critical and specific role in the process are those that are expressed only during the embryonic and metamorphic stages. In addition, they must be present in the neuropil, where neuronal and glial processes exist and synaptic contacts occur. In order to uncover such antigens, monoclonal antibodies ( MAbs) were generated using the central nervous system (CNS) of Drosophilu prepupae as an immunogen. As a result, we found MAb 66B2 whose immunoreactivity in the CNS showed marked changes with the developmental stages. Immunohistochemical and biochemical studies showed that the antigen is an extracellular matrix component. From the distribution in the embryo and its molecular weight, we concluded that this antigen is distinct from any of the extracellular matrix (ECM) molecules in Drosophila so far reported. The developmental changes in the 66B2 immunoreactivity are discussed in relation to the presumable changes of the extracellular matrix in the CNS that are accompanied by axonogenesis or synaptogenesis.

MATERIALS AND METHODS Drosophila Stocks

Embryos, larvae, and pupae were obtained from Cunton-S wild-type strain. Flies were fed with

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cornmeal-agar-yeast medium, and were raised at 25 "C under standard laboratory conditions. Generation and Screening of Monoclonal Antibodies

Homogenate of CNS dissected out of prepupae (812 h postpupariation ) of Drosophila was used as an immunogen for the production of MAb 66B2 studied in this work. The homogenate was lightly fixed in 10% formalin/ phosphate-buffered saline (PBS), pH 7.2, before homogenization in PBS. The homogenate was emulsified in Freund's complete adjuvant, and injected intrapentoneally into BALB/c mice (about 50 dissected, prepupal CNS per mouse). The mice were boosted 3 weeks later with an intraperitoneal injection of an equal amount of the homogenate emulsified in Freund's incomplete adjuvant. At 3 days before fusion, a final injection of the homogenate in PBS was administered intravenously. The hybridoma experiments were carried out according to Oi and Herzenberg ( 1980). Myeloma cells of X63-Ag8-653 strain were used. Hybridomas were cloned by limiting dilutions. Hybridoma supernatant was screened by immunofluorescent whole-mount staining of 12- to 15-h-old embryos, the CNS of the third instar larvae, and prepupae as described below. As a result, MAb 66B2 was obtained and determined to be an IgG2b by an Ouchterlony double-diffusion typing kit (Miles). lmmunohistochemistry

The samples of embryos for immunohistochemistry were prepared by standard procedures ( Mitchison and Sedat, 1983). For MAb staining, the embryos were incubated with MAb supernatant with shaking for 1-2 h, and then incubated overnight at 4°C. The embryos were washed three times for 10 min with PBS, and incubated for 2 h with a 1:100 dilution in PBS of fluorescein-conjuL ) (Cappel). gated goat anti-mouse IgG ( H After washing three times for 10 min with PBS, the embryos were mounted with 90% glycerol in PBS and viewed and photographed with a Zeiss epifluorescence microscope. For observation of the embryonic CNS, embryos after staining were dissected with a needle, and mounted and viewed as described above. For the observation of the CNS of larvae, prepupae, pupae, and adult flies, the CNS dissected in cold PBS was fixed by immersion in 10%formalin /

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PBS for 30 rnin to I h a t 4°C. Then, indirect immunofluorescent whole-mount staining was carried out as described above. For cryostat sections, the fixed specimens were frozen in OCT embedding medium (Miles). Cryostat sections of 10-pm thickness were cut at -25”C, taken up on gelatin-subbed slide glass. Indirect immunofluorescence staining was performed as described in the whole-mount staining, except that incubation time for the first and second antibodies was 30 min. For the staining of primary cultures derived from individual embryos, cells were fixed in 10% formalin/culture medium for 30 rnin in a culture dish. Preparation of the embryo cultures is described below. Indirect immunofluorescence staining was carried out as described above, except that incubation time for the first and second antibodies was 1 h. In the case of the double staining with fluorescein-conjugated goat anti-horseradish peroxidase (HRP) antibody (Cappel), rhodamineconjugated goat anti-mouse IgG ( H + L) (Cappel) was used as the second antibody. For the staining of cells without fixation, the cells were incubated with MAb supernatant for 30 min after the culture medium was removed and washed twice for 10 rnin with PBS or Schneider’s Drosophilu medium (GIBCO). Then the cells were fixed in 10% formalin/PBS or Schneider’s Drosophilu medium for 30 rnin at room temperature. Subsequent steps were the same as those described above. Electrophoresis and lmmunoblotting

For one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE ) , the samples were homogenized in a sample buffer containing 2.3%)SDS, 5% 2-mercaptoethanol, 63 m M Tris-HC1 and 10%glycerol ( p H 6.8), and heated in a boiling water bath for 5 min. Aliquots of the samples were subjected to electrophoresis in 5% polyacrylamide slab gels with 3% stacking gels using the buffer system of Laemmli ( 1970). Molecular weight standards (myosin, 200,000; /3-galactosidase, 116,250; phosphorylase b, 97,400; bovine serum albumin, 66,200; ovalbumin, 45,000; BioRad) were run on an adjacent lane of the same gel. The gels were stained with Coomassie brilliant blue R-250 (CBB). Two-dimensional gel electrophoresis was performed as described by O’Farrell ( 1975) using 5% polyacrylamide gels for the second dimension. The samples were homogenized in the lysis buffer (am-

pholines: four parts of pH 3.5-10 and one part of pH 4-6), and aliquots of the samples were subjected to electrophoresis for the first dimension. The gels were stained with silver following manufacturers’ instructions ( Daiichikagaku ) . The samples were separated by SDS-PAGE as described above, and transferred electrophoretically to nitrocellulose paper according to Burnette (1981) in a modified buffer containing 25 m M Tris, 0.192 M glycine, 20% methanol, and 0.05% SDS. After blocking with 50 m M Tris-HC1 buffer (pH 7.4) including 3% bovine serum albumin and 0.15 MNaCl (TBS), the blots were incubated for 2 h with the hybridoma supernatant, and washed in TBS (three times, 30 rnin each). The blots were then incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG ( H + L) (BioRad) diluted 1000 times in TBS, which contains 3% bovine serum albumin, and washed in TBS (three times, 30 rnin each). The blots were developed with 4-chloro- l-naphthol as the chromogen. Periodate Treatment

In order to determine whether the epitope of MAb 66B2 is a carbohydrate, the effect of periodate treatment on the MAb binding to the CNS was examined. This reagent oxidizes carbohydrates with adjacent hydroxyl groups (Woodward, Young, Jr., and Bloodgood, 1985). Fixed CNS of white prepupae were washed in 50 m M sodium acetate buffer (pH 4.5) containing 0.1 M NaCl, and incubated with 50 m M sodium meta-periodate in the acetate buffer for 2 h at 4°C in the dark. Control CNS were treated in an identical manner except that periodate was omitted from the acetate buffer. Both were washed thoroughly with PBS, and stained as described above. Trypsin Digestion

For trypsin digestion experiments, the CNS with discs of prepupae were homogenized in PBS (pH 7.2) containing 1% NP-40, 0.1% SDS, 1% 2-mercaptoethanol, and were boiled for 3 min. Digestion was initiated by the addition of 1 pg/ml trypsin to 50 p1 of the buffer containing about 12 CNS with discs; the reaction was allowed to proceed for 20 h at 37°C. Alternatively, in control mock digestion, the samples after the addition of the enzyme were boiled for 3 min, and incubated as above. Then, the reactions were terminated by the addition of an equal volume of 2 X SDS sample buffer ( 125 m M Tris-HCI, pH 6.8, 4% SDS, 10% 2-mercaptoeth-

Stage-SpeciJc Antigen in Drosophila CNS

anol and 20% glycerol) and boiling. Products of digestion were examined by immunoblotting with MAb 66B2. Preparation of Soluble and Particulate Fractions

For preparation of soluble and particulate fractions, 12- to 15-h-old dechorionated embryos were homogenized in a sample buffer (50 m M TrisHCI, pH 7.5, 250 m M sucrose, 5 m M MgCl,, 1 m M EGTA, 1 m M EDTA, 1 m M phenylmethylsulfonyl fluoride (PMSF), 5 pg/ml pepstatin A, 0.5 pg/ml antipain, and 0.5 pg/ml leupeptin). Unbroken tissues and nuclei were removed by centrifugation at 500 g for 5 min. The low-speed supernatant solution was subjected to centrifugation at 100,000 g for 60 min. This high-speed supernatant solution is referred to as a solubtefraction, and the pellet of this high-speed centrifugation as a particulate.fraction or membrane fraction. Each fraction was analyzed for the 66B2 antigen by immunobloting. Moreover, to investigate whether the 66B2 antigen remained in particulate fraction under the extraction conditions with a nonionic detergent or at high-salt concentrations, we added 1% Triton X-100 or 1 M NaCl to the the sample buffer for extraction. Soluble and particulate fractions were analyzed for the 66B2 antigen by immunoblotting as well. Preparation of Primary Cultures from Individual Embryos

Eggs were collected on agar plates, and allowed to age undisturbed on the plates for 2.5 h at 25°C. The eggs were dechorionated and sterilized with 2.5% sodium hypochlorite for 1.5-2 min. Subsequently, they were washed with autoclaved distilled water and kept in a culture medium. As the culture medium, M3( BF) or Schneider’s Drosophila medium was used. M3 (BF) was prepared according to Cross and Sang ( 1978). It contained 10% or 15% heat-inactivated fetal bovine serum (GIBCO). Insulin ( I0 mU/ ml) (Collaborative Research) was added to the medium just before use. Embryos at the early gastrula stage [stage 6 or 7 according to Campos-Ortega and Hartenstein ( 1985)l were selected (Seecof, Alleaume, Teplitz, and Gerson, 1971). The contents of an embryo were withdrawn with a glass capillary and plated directly onto a 35-mm culture dish (Falcon). The cells were further dissociated with a capillary by

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pipetting up and down once or twice under a stereomicroscope. After the cells were allowed to attach to the bottom of the dish, they were cultured in an incubator at 25°C in a humid atmosphere of 5% C02 and 95% air.

RESULTS Developmental Changes of Histochemical lmmunoreactivity to MAb 6682 in the CNS

In order to find antigens whose expression pattern correlates with axon growth and synapse formation in the CNS, we generated MAbs using homogenate of the CNS of prepupae as an immunogen. Screening was carried out by immunofluorescent whole-mount staining of both the middle stage ( 12- 15 h ) embryos, and the isolated CNS from the third instar larvae and prepupae (8- 10 h postpupariation). We screened about 500 hybridoma lines, and MAb 66B2 was obtained whose staining intensity in the neuropil of the CNS showed marked changes with the developmental stage. Whole mount of isolated CNS from various stages were studied by immunofluorescent histochemistry with MAb 66B2 (Figs. 1-3). In Drosophilu, the CNS is a bilaterally symmetrical structure, consisting of a pair of brain hemispheres and a ventral cord which includes a chain of relatively simple segmental ganglia. Neural cells within the ganglia have a typical invertebrate organization, with the cell bodies at the periphery forming the cortex, and the neurite elongations toward the center forming a neuropil where synapse formations occur. In the middle-stage ( 12- 15 h ) embryo, intense immunoreactivity to MAb 66B2 was observed in the entire region of the neuropil, whereas the cortex was almost negative [Fig. l (A), brain is not shown]. Axonogenesis-which begins between 8 and 9 h after fertilization-and synapse formation occur during this stage (Goodman et al., 1984; Johansen, Halpern, and Keshishian, 1989; Thomas, Bastiani, Bate, and Goodman, 1984). In contrast to the CNS, the 66B2 immunoreactivity was not detected in the peripheral nervous system (PNS). Thus, axons entering and leaving the CNS via the intersegmental and segmental nerves were not stained. Immunopositive thread-like structures in Figure 1 ( A ) (arrow) were tracheae, whose interpretation was confirmed by staining with a trachea-specific MAb (data not shown). A close inspection revealed that the 66B2 immunoreactivity was almost

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Figure 1 Immunofluorescence staining with MAb 66B2 of the CNS dissected out of the middle-stage (about 15 h ) embryo ( A ) and the first instar larva ( B ) of Drosophilu. lmmunofluorescent whole-mount staining was performed as described in Materials and Methods; anterior is toward the left. ( A ) Arrow indicates the tracheae which enters the CNS. ( B ) Scale bar = 50 Fm for ( A and B). Note that the intense immunoreactivity to MAb 66B2 in the neuropil of the embryo ( A ) disappears in the first instar larva ( B ) except for some regions of the brain.

negative in the midline region of each commissure, and that cells around these regions were faintly stained [Fig. I ( A ) ] . It is possible that these immunopositive cells are midline ectodermal cells or midline glia because of their positions (Fredieu and Mahowald, 1989; Jacob and Goodman, 1989; Jacob, Hiromi, Patel, and Goodman, 1989; Klambt, Jacobs, and Goodman, 199 1; Rothberg, Hartley, Walther, and Artavanis-Tsakonas, 1988). As development proceeded, the 66B2 immunoreactivity in the CNS decreased. Consequently, the CNS in the first instar larva was only weakly stained, although relatively intense 66B2 immunoreactivity remained in some regions of the brain [Fig. 1 (B)] . These regions roughly correspond to the neuropil area, which is close to the cortical area where cell proliferation is still active in the newly hatched larva (Truman and Bate, 1988). The weak

66B2 immunoreactivity in the CNS continued until the late third instar larva, so that the CNS in the second and early third instar larvae were only weakly stained [Fig. 2( A,B)] . The imaginal discs that are connected to the larval CNS, however, were stained intensely [Fig. 2( A,B)] . Throughout the developmental stages examined here, tracheae were always immunopositive (Figs. 1-3 ). In Drosophila, pupal moult is preceded by the formation of a white prepupa (at 0 h postpupariation). The prepupal period lasts about 12 h, and the pupal period lasts for 4 days. During the metamorphosis, the CNS undergoes dramatic changes in its shape and in its overall cellular organizations (Kankel, Ferrus, Garen, Harte, and Lewis, 1980). The 66B2 immunoreactivity in the CNS reappeared gradually during the late third instar larva, such that the entire region of neuropil and optic lobes in the CNS of white prepupa was stained again [Fig. 2( C)]. The intense 66B2 immunoreactivity in the CNS was maintained during metamorphosis, so that the CNS of prepupa ( 12 h postpupariation) and pupa (24 h postpupariation) were similarly stained [Figs. 2( D ) , 3 (A,D)] . This intense 66B2 immunoreactivity in the CNS decreased gradually again from 48-h postpupariation, such that the CNS of the newly hatched adult fly was only weakly stained [Figs. 3 ( B,E)], and that of mature (more than 1 week after eclosion), adult fly was stained only faintly [Figs. 3(C,F)]. Thus, the intense 66B2 immunoreactivity in the CNS was observed almost exclusively during the embryonic and pupal stages, and not during the larval and adult stages. Cross sections of the brains and imaginal discs of white prepupae were studied as well. In the cross section of a white prepupal brain, the 66B2 immunoreactivity was restricted to a neuropil region consistent with the whole-mount staining (Fig. 4). A closer inspection revealed that characteristic surface-associated staining of cell bodies was observable in the cortex (arrowhead in Fig. 4 ) . As for the disc, the inside corresponds to the outer (or apical) surface of a larval epidermis, and the outside corresponds to the inner (or basal) surface of the epidermis. Disc epidermal cells will eventually secrete pupal and adult cuticle from the apical surface, and constitute what is described as the disc epithelium proper. In the cross section of an imaginal disc (leg disc), the intense 66B2 immunoreactivity was detected only in the disc lumen, that is, most of the antigen was observed along the apical surface of the epithelium (Fig. 5 ) .

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Figure 2 lmmunofluorescence staining with MAb 66B2 of the CNS dissected out of the second (A) and early third ( B ) instar larvae, and 0 h (C) and 12 h ( D ) postpupariation prepupae. Immunofluorescent whole-mount staining was performed as described in Materials and Methods; anterior is toward the left. Scale bar in A = 70 pm for A, and scale bar in D = 100 pm for B-D. Note that the intense immunoreactivity to MAb 66B2 appears again in prepupae

( C , D) .

Distribution of the 66B2 Antigen in the Middle-Stage Embryo In middle-stage ( 12-15 h) embryos, MAb 66B2 intensely stained epidermis and trachea as well as neuropil of the CNS (data not shown). These tissues are all of ectodermal origin (Poulson, 1950). In addition, other ectodermal organs, such as foregut, hindgut, and Keilin’s organ, were also immunopositive. However, not all the ectoderm-derivatives were immunopositive. For example, salivary glands, Malpighian tubules, ring gland, and PNS were not stained. It is noted that immunopositive tissues except the CNS are those that secrete chitinous cuticle. It is unlikely, however, that MAb 66B2 bound to chitinous cuticle proper, because tracheal pits as well as the insides of stomodeum and proctodeum invaginations were already immunopositive in early-stage (6-7 h ) embryos (data not

shown), when chitinous cuticle was not yet secreted. Biochemical Identification of the 6682 Antigen

In order to identify the biochemical nature of the 66B2 antigen, one-dimensional electrophoresis and immunoblotting with MAb 66B2 were performed using homogenate of the CNS dissected out of prepupae (2-5 h postpupariation). On the blots of 5% separating gels with a 3% stack, one intense band at a position of molecular weight of 350 K was specifically detected [Fig. 6 (lane 3 ) ] . To obtain the biochemical basis of the observed histochemical changes, we examined homogenate of the brains dissected out of mature adult (more than 1 week after eclosion) for the presence of the antigen by immunoblotting [Fig. 6 (lane 4) J . Ap-

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Figure 3 Immunofluorescence staining with MAb 66B2 ofthe CNS dissected out of pupa (24 h postpupariation) ( A , D ) , newly hatched (B, E ) , and old (more than 1 week after eclosion) (C. F) adult flies. Immunofluorescent whole-mount staining was performed as described in Materials and Methods. The left and right columns show the posterior view of the brain and dorsal view of the ventral ganglion, respectively. Dorsal is toward the top in the left column, and anterior is toward the left in the right column. Unidentified specific cells corresponding to each ommatidium are immunopositive in the pupal retina ( A ) . Scale bar in F = 100 Fm for A-F. Note that the intense immunoreactivity to MAb 66B2 in the CNS disappears again in the old adult fly (C, F).

proximately equal amounts of protein judged by CBB staining were run as in the case of prepupa [Fig. 6 (lanes 1, 2 )]. Consistent with the results of immunohistochemistry, the 66B2 immunoreactivity was hardly detected in the mature adult brain [Fig. 6 (lane 4 ) ] . It was thus concluded that the histochemical changes ofthe 66B2 immunoreactivity in the CNS are due to the stage-specific appearance of the antigen with an apparent molecular weight of 350 K. Immunoblotting with MAb 66B2 was per-

formed on homogenate of the imaginal discs dissected out of the third instar larvae, and middlestage ( 12- I5 h ) embryos [ Fig. 6 (lanes 5, 6)]. Intense bands at the same position as that of the prepupal CNS (350 K ) were detected in both tissues [Fig. 6 (lanes 5, 6)]. Lower molecular weight bands detected in the embryos may be immunologically cross-reactive species or proteolytic fragments [Fig. 6 (lane 6 )]. The result in the case of the middle-stage embryos suggested that the various immunopositive tissues with MAb 66B2 in the em-

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-116 97 Figure 4 Immunofluorescence staining with MAb 66B2 of a cryostat section of a white prepupal(0 h postpupariation) brain. Immunofluorescence staining was performed as described in Materials and Methods. The optic lobes in the brain are not visible in this section. An arrowhead indicates characteristic surface-associated staining of cell bodies in the cortex. Scale bar = 50 pm.

bryo expressed the same antigen with an apparent molecular weight of 350 K. Only in the imaginal discs, however, another band at a position higher

Figure 5 Localization of the 66B2 antigen in a cryostat section of a white prepupal leg disc. (A and B) are the same section. (A) is a phase-contrast image, and (B) is immunostained with MAb 66B2. Scale bar in B = 50 pm for A, B. Note that the intense immunoreactivity to MAb 66B2 was detected at the inside lumen of the disc which corresponds to the apical surface of the adult epidermis (B).

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Figure 6 Immunoblot identification of the 66B2 antigens. Lanes 1 and 2 are one-dimensional gels and lanes 3 and 4 are the corresponding immunoblots of homogenate of both the prepupal(2-5 h postpupariation) CNS (lanes 1, 3 ) and the mature (more than 1 week after eclosion) adult fly brains (lanes 2, 4). Lanes 5 and 6 are immunoblots of homogenate of the imaginal discs of the third instar larvae (lane 5 ) and of the middle-stage ( 1215 h ) embryos (lane 6 ) . Electrophoresis and immunoblotting were performed as described in Materials and Methods. The gel was stained with Coomassie brilliant blue, and the blots were immunostained with MAb 66B2. Scale bars on the right of lane 6 indicate positions of molecular weight standards described in Materials and Methods. An arrow indicates the top of the gel. Approximately equal amounts of protein were loaded in lanes 3 and 4, as attested by protein staining with CBB (lanes 1,2). One intense band at a position of molecular weight of about 350 K is detected in the prepupal CNS (lane 3 ) , and not in the adult brain (lane 4 ) . Note that another band at a position of higher molecular weight than 350 K is detected only in the imaginal discs (lane 5 ) .

than 350 K was detected [Fig. 6 (lane 5 )]. Because this additional band entered the gel only for a short distance, it was not possible to estimate its molecular weight. It was considered that the band represented a molecule with a molecular weight of 400 K or more. The relation between these two bands (350 and 400 K antigens) is not understood clearly at present. To understand the biochemical nature of the 66B2 antigen further, we performed two-dimensional electrophoresis and immunoblotting with MAb 66B2 using homogenate of the CNS dissected out of prepupae ( 2-5 h postpupanation). The immunoPositive signals had an approximate neutral PI, though they were somewhat broad in pH range (data not shown). Although intense immunoposi-

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Figure 7 Effect of periodate treatment on the immunoreactivity to MAb 66B2. The CNS dissected out of white prepupa (0 h postpupariation) had been treated with periodate (B, D) and with buffer alone (A, C ) as described in Materials and Methods. The specimens in A and B were stained with MAb 66B2, and the specimens in C and D were stained with MAb 82E10, which was used as a control antibody. lmrnunofluorescent whole-mount staining was performed in Materials and Methods. Scale bar in D = 100 Fm for A-D. Note that only the immunoreactivity to MAb 66B2 is almost lost after the treatment ( B ) .

tive signals which corresponded to the 350 and 400 K antigens were detected on the blot, corresponding spots could not be identified clearly on the gel even by silver staining (data not shown).

Effect of Periodate and Trypsin Treatments on the 6682 lmmunoreactivity It is possible that the epitope of MAb 66B2 is a carbohydrate, so the effect of periodate treatment on the MAb binding to the CNS of white prepupae was examined by immunofluorescent wholemount staining. The 66B2 immunoreactivity was almost lost from the CNS after the treatment, whereas weak immunoreactivity remained in the discs [Fig. 7 ( B ) ] . In this experiment, MAb 82ElO was used as a control antibody, as its specificity toward highly phosphorylated components of neurofilament proteins of the chicken has been established (Go, Tanaka, Obata, and Fujita, 1989). In contrast to MAb 66B2, the 82E10 immunoreactivity was essentially unaffected by the treatment

[Fig. 7( D)]. This result suggested that the epitope of MAb 66B2 was a carbohydrate residue. In order to confirm that the 66B2 antigen has a protein moiety, we incubated homogenate of the CNS with discs of prepupae (2-5 h postpupariation) with trypsin, and assayed it by immunoblot analysis with MAb 66B2. The 66B2 immunoreactivity was almost lost as compared with a control mock digestion experiment carried out in the presence of heat-inactivated enzymes (data not shown). This result showed that the 66B2 antigen has a protein moiety. Together with the result of periodate treatment experiment, it is concluded that the 66B2 antigen is most likely a glycoprotein.

The 6682 Antigen Is Not Tightly Associated with the Membrane Soluble and particulate fractions were prepared from middle-stage ( 12- 1 5 h ) embryos, and analyzed for the 66B2 antigen by immunoblotting. A large amount of the 66B2 antigen in the embryos

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whereas a small amount is in the forms closely associated with the membrane. Expression of the 6682 Antigen in Primary Cultures of Embryos

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Primary cultures were prepared from individual embryos as described in Materials and Methods to examine the expression of the 66B2 antigen in dissociated cells. It has been reported that several cell types including neurons and muscle cells differentiate within 1 day in a culture (Cross and Sang, 1978; Shields, Dubendorfer, and Sang, 1975; Shields and Sang, 1970). Neurons were the most prominent cells in the culture at 24 or 48 h. It has

Figure8 Hydrophilic property ofthe 66B2 antigen. Soluble and particulate fractions were prepared from the middle-stage ( 12- I 5 h) embryos under conditions in the absence of detergent (lanes 1, 2), in the presence of detergent ( 1% Triton) (lanes 3,4), and at a high concentration of salt ( 1 M NaCl) (lanes 5 , 6 ) as described in Materials and Methods. Lanes 1, 3, and 5 are soluble fractions and lanes 2, 4, and 6 are particulate fractions under respective conditions. Approximately equal amounts of protein were loaded in each lane. Lanes 1-6 are immunoblots stained with MAb 66B2. Scale bars and an arrow are valued as per Figure 7. Note that the 66B2 antigen is extracted in the absence of detergent (lane 1 ).

were found to be extracted in the absence of detergent [Fig. 8 (lane l ) ] , whereas some remained in the particulate fraction [Fig. 8 (lane 2)]. The fact that the antigen was extractable without detergent showed that the 66B2 antigen is not an intrinsic membrane protein. To investigate whether the 66B2 antigen remained in the particulate fraction in the presence of nonionic detergent or high concentration of salt, we carried out extraction under respective conditions and analyzed for the antigen by immunoblotting. In the presence of 1% Triton, almost all the antigens in the embryos were extracted, so that the immunoreactivity was not detected in the particulate fraction at all [Fig. 8 (lanes 3,4)]. In the presence of 1 M NaC1, a large amount of the antigen was extracted, but a small fraction remained in the particulate fraction [Fig. 8 (lanes 5, 6)]. Approximately equal amounts of protein were run in each lane in Figure 8, as judged by CBB staining (data not shown). These results indicated that a large amount of the 66B2 antigen exists in the extracellular space in the forms soluble without detergent,

Figure 9 Expression of the 66B2 antigen in a primary culture of embryos. Primary cultures were prepared from individual embryos as described in Materials and Methods. The culture was fixed at 48 h, and immunostained with MAb 66B2 as described in Materials and Methods. ( A and B) are the same field. ( A ) is a phasecontrast image and ( B ) is a fluorescent image. Differentiated nerve and muscle (arrow) cells are identified in ( A ) . Scale bar in ( B ) = 50 Fm for (A, B). An arrowhead in ( B ) indicates immunopositive chitin-secreting cells. Note that a small number of immunopositive cells are observed in the ganglion-like clusters, whereas the neurite bundles connecting the clusters are not stained ( B ) .

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been reported that these begin to appear between 6 and 12 h, and continue to mature over the next several days (Cross and Sang, 1978; Shields et al., 1975; Shields and Sang, 1970). In the cultures at 24 or 48 h, most nerve cells were observed in ganglion-like clusters, and their neurite bundles connect the cell clusters [ Fig. 9 ( A ) ] . Dissociated cells in the cultures at 48 h were studied by immunofluorescent histochemistry with MAb 66B2. A small number of immunopositive cells were observed in the ganglion-like clusters, but the neurite bundles connecting the clusters and neurites emanating from the cluster were not stained [Fig. 9 ( B)] . Cells that appeared to secrete chitin (Cross and Sang, 1978; Shields et al., 1975) were also immunopositive [arrowhead in Fig. 9 ( B)] . Essentially, the same results were obtained in the cultures for several days (data not shown). To examine whether the immunopositive cells were neurons, we doubly stained the cells in the cultures with MAb 66B2 and antibodies against horseradish peroxidase (anti-HRP) which were specific to the surfaces of all the neurons in Drumphila (Jan and Jan, 1982; Snow, Patel, Harrelson, and Goodman, 1987). The results demonstrated that distributions of the 66B2 and anti-HRP immunopositive cells in the ganglion-like clusters were almost complementary (data not shown). Thus, in almost all the cases studied here, the 66B2-immunopositive cells were not stained with anti-HRP antibodies. Although the 66B2-immunopositive cells appeared to be anti-HRP positive in some cases, it might be due to two cells overlapping each other in the cluster. These results showed that neurons do not express the 66B2 antigen, and that the observed 66B2 immunoreactivity in the neuropil of the CNS is not due to the neurons themselves. Dissociated live cells were studied by immunofluorescent histochemistry with MAb 66B2 to confirm that the 66B2 antigen is expressed on the cell surface. Weakly immunopositive cells were observed in the ganglion-like cluster in essentially the same manner as that in the fixed specimens, and that characteristic surface-associated staining of the cells was observed (data not shown). In this experiment MAb 8C5 was used as a control antibody, as it specifically stained cell nuclei (Fujita, Zipursky, Benzer, Ferrus, and Shotwell, 1982). In contrast to MAb 66B2, no immunoreactivity was observed with MAb 8C5 (data not shown), demonstrating that the antibodies did not enter the cells under the experimental condition. It was thus concluded that the 66B2 antigen is expressed on the cell surface.

DISCUSSION The 6682 Antigen Appears in Parallel with the Axonogenesis and Synaptogenesis in the CNS

In Drosophila, the nervous system must undergo an extensive reorganization during metamorphosis; thus, some larval neurons show the growth of new neuronal processes and the formation of new synaptic connections (Kent and Levine, 1988; Levine and Truman, 1982, 1985; Levine et al., 1986; Truman and Reiss, 1976; reviewed in Truman, 1990). Such phenomena as axonogenesis and synaptogenesis, characteristic of the neural development, therefore, take place during the metamorphic stage as well as during the embryonic stage in Drosophila. Moreover, in the Drosophilu CNS, neurite extension and retraction or synaptic contacts occur in the center forming the neuropil. From this point of view, the 66B2 antigen is regarded as an axonal growth-associated antigen in the Drosophilu CNS, as the intense 66B2 immunoreactivity is observed in the neuropil only during the embryonic and metamorphic stages (Figs. 1-3 ). This antigen is thus appropriately distributed in time and space for axon growth and synapse formation in the CNS. Although the function of the 66B2 antigen is still unknown at present, it is likely that the antigen plays some important roles in axonogenesis or synaptogenesis. The 6682 Antigen as an Extracellular Matrix Component

Immunohistochemistry with MAb 66B2 of cells without fixation in a primary culture of embryos showed that the antigen was expressed on the cell surface (data not shown). It is hence concluded that the antigen is either an intrinsic membrane protein or a secreted protein in the extracellular space. MAb 66B2 staining in the cross section of a white prepupal brain also showed that characteristic, surface-associated staining of cell bodies was observed in the cortex (Fig. 4). In the cross section of an imaginal disc, the 66B2 antigen was observed inside the disc which corresponds to the apical surface of the epidermis, where the pupal and adult cuticle is secreted (Fig. 5 ) . It has been reported that components of extracellular matrix in imaginal discs are localized on the apical or basal surface of the epithelium ( Brower, Piovant, Salatino, Brailey, and Hendrix, 1987; Garzino, Berenger, and Pradel,

Stuge-Specific Antigen in Drosophila CNS

1989; Gratecos, Naidet, Astier, Thiery, and Semeriva, 1988). In the middle-stage embryo, the 66B2-immunoreactive tissues other than the CNS are those that secrete chitinous cuticle (data not shown). The extractability of the 66B2 antigen in the absence of detergent demonstrated that it is not an intrinsic membrane protein [Fig. 8 (lanes 1 , 2)], however, there seemed to be a fraction that is tightly associated with membranes, because a small amount of the antigen in the embryos could not be extracted in the presence of a high concentration of salt [Fig. 8 (lanes 5 , 6)]. Together with the results of immunohistochemistry, we conclude that the 66B2 antigen is an ECM component. Results of immunohistochemistry with both MAb 66B2 and anti-HRP antibodies in a primary culture of embryos [Fig. 9 (data not shown in the case of the double staining)] showed that the neurons themselves do not produce the 66B2 antigen, and that the 66B2 antigen in the neuropil is secreted from nonneuronal cells in the CNS. The results, however, do not exclude the possibility that dividing neuroblasts express the antigen, and the expression is transient as we have not established a thorough time course of embryonic expression in vitvo. Although it cannot be concluded at present which cells synthesize the 66B2 antigen in the CNS, we maintain that glial cells are the best candidate. Although functions of ECM during neural development have been little studied in insects, it is highly likely that axons navigate along, and presumably interact with basal laminae during the formation of the nervous system (Nardi, 1983). In Drosophila, some components of the ECM have already been identified and characterized ( Anderson, 1988; Semeriva, Naidet, Krejci, and Gratecos, 1989). These include laminin (Garzino et al., 1989; Fessler, Campbell, Duncan, and Fessler, 1987; Monte11 and Goodman, 1988, 1989), fibronectin (Gratecos et al., 1988), glutactin (Olson et al., 1990), and proteoglycan-like glycoprotein papilin (Campbell, Fessler, Salo, and Fessler, 1987). In addition, slit is an extracellular protein which is necessary for both the normal development of the midline of the CNS, and the formation of the commissural axon pathways ( Rothberg, Jacobs, Goodman, and Artavanis-Tsakonas, 1990). Clearly slit shares common characteristics with the 66B2 antigen, because the slit protein is secreted from the midline glial cells where it is synthesized and is eventually associated with the surfaces of the axons (Rothberg et al., 1990). The 66B2 antigen is, how-

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ever, quite distinct from those ECM proteins with regard to distribution in the embryo or molecular weight. It is unlikely that the 66B2 antigen is a typical proteoglycan-like molecule, because it has a nearly neutral PI (data not shown), whereas proteoglycan must have an acidic PI due to the presence of a large quantity of acidic glycosaminoglycan chains. The interesting aspect of this antigen is that the intense immunoreactivity is observed in the neuropil only during the growth state of the CNS. Moreover, this observation clearly demonstrates that a dramatic biochemical change occurs in the extracellular environment within the neuropil of the Drosophila CNS with the development. The developmental changes in 66B2 immunoreactivity presumably reflect dynamic changes of the ECM in the CNS that are accompanied by axonogenesis or sy naptogenesis. It is known that ECM serves as a substrate for the migration of neurons and the extension of axons of the PNS (Carbonetto, 1984; Lander, 1989; Sanes, 1989; Sanes and Covault, 1985; Thiery and Duband, 1986). On the other hand, until recently the possibility has been ignored that ECM molecules play a role in regulatory processes in the CNS development; it is now clear, however, that embryonic CNS contains extracellular spaces rich in substances such as proteoglycans and ECM glycoproteins (Sanes, 1989). It is likely that these ECM molecules serve as a substrate for the extension of CNS axons during development as well in the PNS. In view of this, the 66B2 antigen may serve as a substrate for the extension of the CNS neurons, or provide the CNS neurites with a flexible environment, because the intense immunoreactivity is observed only during the growth state of the CNS. The 6682 Antigen a s a Glycoprotein

Results of periodate treatment and trypsin digestion experiments showed that the 66B2 antigen is a glycoprotein and that the epitope that MAb 66B2 recognizes is a carbohydrate residue (Fig. 7 ) . It is, therefore, likely that specificity of MAb 66B2 is dependent on the carbohydrate structure attached to a protein backbone. It is not clear at present whether the observed developmental changes of the 66B2 immunoreactivity in the CNS are ascribed to either changes in the carbohydrate structure or those including protein backbone moiety. It has been suggested that the sugar moieties in glycoproteins play an important role in the mecha-

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nism by which cells recognize other cells or large molecules. In fact, in several vertebrate nonneural tissues there exists compelling evidence that interactions between carbohydrate and carbohydratebinding proteins are essential in cell adhesion and recognition (reviewed in Jessell, Hynes, and Dodd, 1990). In the nervous system there is no decisive evidence that carbohydrates play a role in similar adhesive or recognition functions. Suggestive evidence that carbohydrates play an essential role in neural adhesion and neurite outgrowth, however, has been derived from preliminary functional studies on HNK- 1 epitope, which is a common carbohydrate moiety shared by several neural adhesion molecules, and polysialic acid attached to a neural cell adhesion molecule (N-CAM) (Jessell et al., 1990). Recently, it has been reported that the level of nerve-associated polysialic acid plays an important role in regulating axon fasciculation and nerve branching patterns during initial innervation of chick muscle (Landmesser, Dahm, Tang, and Rutishauser, 1990). As for ECM glycoproteins, it has been reported that N-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein (Jones, Arumugham, and Tanzer, 1986). From this point of view, it is possible that carbohydrate moiety of the 66B2 antigen is involved in the functions of the antigen whether or not developmental changes of the immunoreactivity in the CNS involve changes in the protein backbone moiety. The authors are grateful for the helpful advice of both Dr. S. C. Fujita on fusion experiments, and Dr. K. Nakao on primary culture experiments, and would also like to express thanks to Ms. M. Akama, K. Hotta, and T. Naoi for their kind assistance.

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