Specific cell surface labels in the visual centers of Xenopus laevis tadpole identified using monoclonal antibodies

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122,90-100

(1987)

Specific Cell Surface Labels in the Visual Centers of Xenopus laevis Tadpole Identified Using Monoclonal Antibodies SHIN

TAKAGI,’

TOSHIAKI

TSUJI,*

TAKASHI

AMAGAI,~

TETSURO

Departments of Anatomy, *Ophthalmology, tMicrobiology, University of Medicine, Kawaramachi-Hirokoji, Received

September

22, 1986; accepted

TAKAMATSU,*

and *Pathology, Kamikyo-ku, Kyoto

in revised

fern

AND

HAJIME

FUJISAWA

Kyoto Prefectural 602, Japan

February 20, 1987

Monoclonal antibodies (MAbs) against the optic tectum of Xenoms tadpoles were generated and screened by the immunofluorescent staining of frozen sections of tadpole brains. MAb-A5 stains the 8th and 9th plexiform layers of the optic tectum, whereas MAb-BZ stains all but the eighth and ninth plexiform layers of the optic tectum. MAb-A5 antigen is also detectable in the nucleus of Belonci, the corpus geniculatum thalamicum, the pretectal area, and the basal optic nucleus, all targets of the optic nerve, but is not detectable in the optic nerve or the optic tract. On the other hand, MAb-B2 does not stain any of these visual centers, though many fibers surrounding them are stained. Eyeenucleation experiments showed that MAb-A5 antigen is expressed in the optic tectum even when it is not innervated by optic nerves. Staining of viable brains with these MAbs indicates that these antigens are cell surface molecules. Immunoadsorption followed by SDS-PAGE suggests that proteins are constituents of these antigens. The MAb-A5 antigen in the diencephalon and the mesencephalon is not detectable at stage 35/36, but is detectable at stage 39 when the optic nerves begin to innervate the optic tectum. The spatial as well as the temporal patterns of the expression of the MAb-A5 antigen suggest that this molecule may be involved in the target recognition of optic nerve fibers. 8 isa? Academic

Press, Inc.

INTRODUCTION

Neurons interconnect in a highly specific and stereotyped fashion. Since Sperry demonstrated the reestablishment of a topographically ordered connection between regenerating optic nerve fibers and optic tectal neurons (1943, 1944, 1945), the retinotectal projection systems of lower vertebrates have been targets of study by developmental neurobiologists attempting to elucidate the development of specific neuronal connections. Through surgical manipulations of the retina and/or optic tectum (reviews: Gaze, 1978; Edds et al., 1979; Fraser and Hunt, 1980; Levine, 1984: Sharma and Romeskie, 1984) and anatomical mappings of growing optic nerve fibers (Fujisawa, 1981, 1984,1986; Fujisawa et ah, 1981,1982; Reh et ab, 1983; Constantine-Paton et ah, 1983; Reh and Constantine-Paton, 1984), it became evident that developing or regenerating optic nerve fibers possessthe ability to recognize appropriate target neurons. Recent studies on developing invertebrate nervous systems also showed that interneuronal recognition phenomena play critical roles at various phases of specification of neuronal connections (Bastiani et ab, 1985). The chemoaffinity hypothesis, proposed by Sperry (1963), attributing specific neuronal recognition to specific cell surface labels is a prevailing idea. However, molecular mechanisms underlying specific neuronal i To whom Anatomy).

all correspondence

0012-1606/87

$3.00

Copyright All rights

should

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

be addressed

(Department

recognition remain obscure because conventional biochemical techniques used to test this hypothesis are not adequate. A recently developed hybridoma technique provides a direct means to examine molecular diversity in the nervous system, to detect adhesion molecules in the nervous tissues (Edelman et ab, 1983; Rutishauser, 1984; Schachner et al., 1983; Kruse et al., 1985; Hatta and Takeichi, 1986), and to test the possible existence of marker molecules for specific neuronal connections (Kotrla and Goodman, 1984). Several attempts to detect identification tags on the neural retina cells with monoclonal antibodies (MAbs) were made (Trisler et ah, 1981; Henke-Fahle and Bonhoeffer, 1983), but an analysis of the targets of the optic nerve fibers has not been reported. Thus, molecules involved in specific recognition between the optic nerve fibers and visual center neurons have remained unknown. We attempted to generate MAbs against the optic tectum of Xenops tadpoles. Using these MAbs, we showed that the visual centers of the Xenops tadpoles have specific molecular labels. MATERIALS

AND

METHODS

Xenopus laevis were purchased from Hamamatsu Seibutsu Kyozai (Shizuoka, Japan).

Immunization

and Production

of Hybridomas

Xenopus tadpoles (stages 51-53, following the criteria of Nieuwkoop and Farber, 1956) were anesthetized with

of

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ET AL.

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0.015% ethyl m-aminobenzoate methanesulfonate (Nakarai), and the brains were dissected out and placed in Holtfreter’s solution. About 100 optic tecta without meningeal membranes were suspended in 0.5 ml Hanks’ saline, triturated by several passages through a 25-gauge needle, and then given intraperitoneally to BALB/c mice. Mice were immunized two to four times, at 3-week intervals. Three days after the final booster immunization, spleen cells and myeloma cells (P3X63Ag8Ul) were fused, according to Oi and Herzenberg (1981). The supernatants of hybridoma cultures were screened immunohistochemically as described below. For cloning, a single cell was picked up using a glass capillary with a fine tip and then transferred to a well of a 96-well culture plate (Sumitomo) containing 5 X lo5 mouse spleen feeder cells.

brains were dipped into twice-diluted supernatants of hybridoma cultures for 1 hr at room temperature; washed with five changes of Holtfreter’s solution, each for 30 min; and then fixed with 4% paraformaldehyde for 2 hr. Then they were processed for either the wholemounted specimens or sections. The former were subsequently reacted with ABC staining reagents diluted in Holtfreter’s solution. After coloration with DAB, the brains were postfixed with 1% glutaraldehyde and 1% paraformaldehyde in PBS for 30 min and mounted with 90% glycerine. As for immunofluorescent staining of the sections, the frozen sections were prepared as described and reacted with FITC-conjugated rabbit Ig against mouse IgG.

Immunohistochemical

To improve the contrast and resolution of the immunofluorescence, the fluorescence image was processed using a color image analyzer (Olympus CIA, Japan; Takamatsu et al., 1986). With fluorescence microscopy and an epiillumination apparatus (Zeiss Standerd 18FL, West Germany), fluorescence images were taken by a silicon intensifier target camera (Hamamatsu Photonics ClOOO-12, Japan) and g-bit input images were integrated from 16 to 128 times by successive addition to a 16-bit resolution (see Results and Fig. 3~). An adequate g-bit image was sliced out from the integrated l&bit image. Finally the specific object image was extracted by the thresholding technique (see Results and Figs. 3d and 3e).

Procedures

Frozen sections were prepared as follows. Tadpole brains were fixed with 4% paraformaldehyde in halfstrength of phosphate-buffered saline (PBS) for 3 hr at 4”C, immersed overnight in 0.1 Mphosphate buffer (pH 7.4) containing 20% sucrose at 4”C, embedded in OCT compound (Tissue Tek II, Miles), and then frozen immediately. Frozen sections 20 pm thick were prepared by cryostat (Histo-stat, American Optical), thawmounted on cover glasses coated with gelatin/chrome alum, dried under air for 1 hr, and immersed in PBS to dissolve the OCT compound. To screen the hybridoma lines, the sections were dipped into supernatants of hybridoma culture for 15 min, washed with PBS for 15 min, reacted with fluorescein isothiocyanate(FITC) conjugated rabbit Ig against mouse IgG (Miles, 70 times diluted with PBS) for 15 min. All steps were carried out at room temperature. After being mounted with 90% glycerine in PBS, sections were examined with an epifluorescent microscope (Olympus, BHT-RFK). For whole-mount staining of fixed brains, the brain roof was cut along the dorsal median line, fixed with 4% paraformaldehyde for 2 hr, washed in Holtfreter’s solution for 2 hr, reacted with MAbs for 2 hr, washed in Holtfreter’s solution, and then stained by streptoavidinbiotin complex (ABC) methods following the instruction manuals of the manufacturer (Amersham), with diaminobenzidine (DAB) as a chromogen for horseradish peroxidase (HRP). Staining of viable brains with MAbs was performed as follows. The brains were dissected out into Holtfreter’s solution, and meningeal membranes were removed, with care not to injure the brain tissue. To facilitate penetration of the staining solution into brain tissue, the brain roof was cut along the dorsal median line. The

Fluorescence Image Enhancement Image Analyzer

Trypsin

by th.e Color

Treatment

Intact brains exposing the ventricles by cutting the dorsal median line (described above) was dipped into Holtfreter’s solution containing 0.05% trypsin (Difco, 1:250) at 24°C. The reaction was halted by removing the trypsin solution by several washings. The brains were fixed with 4% paraformaldehyde and frozen sections were prepared as described. Radioisotope

Labeling

of Tadpole Brains

Tadpole brains were biosynthetically labeled with [35S]L-methionine. After sterilization of 70% ethanol for 1 min, brains were dissected out in sterile Holtfreter’s solution and the ventricles were exposed. Fifty to one hundred brains were transferred to a half-strength of L-15 medium depleted of methionine (Nikken Seibutsu Igaku) supplemented with 0.2% horse serum (GIBCO), containing penicillin G (Meiji) (100 IU/ml), streptmycin (Meiji) (20 pg/ml), and fungizone (GIBCO) (2.5 pg/ml). [35S]L-Methionine (50 &i/O.04 nmoles Amersham) was added to the 0.5-ml culture medium. After labeling for

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FIG. 1. Indirect immunofluorescence of MAb-45 and MAb-B2 on cryostat sections of the optic tectum of Xenopus tadpoles (stage 53). (a) A coronal section of the mesencephalon stained with MAb-A5. Only the outer layers of the optic tectum (OT) show the fluorescence (arrow), whereas the tegmentum (TG) is negative (X100). (b) A section similar to (a) stained with MAb-BX. Both the optic tectum and tegmentum are stained. (c) Immunofluorescence with MAb-A5 in the optic tectum is restricted to the 8th and 9th plexiform layers (X160). (d) The section next to (c) is stained with MAb-B2. The 3rd, 5th, and 7th plexiform layers of the optic tectum show immunofluorescence with MAb-B2, but the 8th and 9th layers are practically negative. (e) The same section as (d) viewed with bright-field optics. The numbers indicate the 3rd, 5th, 7th, 8th, and 9th plexiform layers of the optic tectum, respectively.

TAKAGI

2 days at 24”C, brains were washed three Holtfreter’s solution, immediately frozen, at -30°C. Immunoadsorption

ET AL.

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times with and stored

of Labeled Antigens

Immunoadsorbents were prepared as follows. Both MAb-A5 and MAb-B2 can be purified with protein A cellulose column (MAPS kit, Bio-Rad) from the acites of mice given intraperitoneal injections of hybridomas. The eluted antibodies were dialyzed against 20 mM NaHC03 and lyophilized. Three hundred microliters of antibody solution (8 mg/ml in 0.1 M NaHC03 buffer) was coupled with 150 ~1 of activated agarose gel beads (Affi-Gel 10, Bio-Rad) according to the instruction manual of the manufacturer. The [35S]L-methionine-labeled brains were agitated vigorously with Vortex mixer in 600 ~1 of solubilizing buffer [SB: 50 mM Tris-HCl, pH 7.4, containing 0.5%

NP-40 and 200 nM phenylmethylsulfonyl fluoride (Sigma)]. After centrifugation at 10,OOOg for 30 min, the supernatant was collected. Radiolabeled supernatants were reacted with 15 ~1 of immunoadsorbent gel for 1 hr at 4°C under conditions of gentle shaking. The gel was then washed 10 times with 1 ml of SB, suspended in 30 ~1 of SDS-PAGE sample buffer containing 5% 2-mercaptoethanol, boiled for 5 min, and centrifuged. Supernatants were electrophoresed on 8% polyacrylamide gel according to Laemmli (1970). 35S-labeled protein bands were detected by fluorography, according to Laskey and Mills (1975). RESULTS

Through 10 fusions, culture supernatants from about 3000 wells were screened. Two monoclonal antibodies named A5 (MAb-A5) and B2 (MAb-B2) that bind to different regions in the optic tectum as described in detail _ below were generated from the same fusion.

FIG. 2. Indirect immunofluorescence of MAb-A5 and MAb-B2 on cryostat sections coronal section stained with MAb-A5. The nucleus Belonci shows immunofluorescence MAb-B2. The nucleus Belonci is negative. (c) A coronal section of the diencephalic The corpus geniculatum thalamicum is stained with MAb-A5 (arrow). (d) The section thalamicum is negative.

of the diencephalon of Xenopus tadpoles (stage 53). (a) A (arrow) (X130). (b) The section next to (a) stained with region that is located rostra1 to the region shown in (a). next to (c) stained with MAb-B2. The corpus geniculatum

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FIG. 3. Indirect immunofluorescence of MAb-A5 and MAb-B2 on cryostat sections of the mesencephalic region, located rostrally to the sections shown in Fig. 1. (a) A coronal section stained with MAb-A5. The pretectum shows immunofluorescence (arrow). A small region corresponding to the basal optic nucleus shows the faint fluorescence at the ventrolateral part (*) (X110). (b) The section next to (a) stained with MAb-B2. The pretectum and the basal optic nucleus are negative. (c-e) Enhanced image showing localization of MAb-A5 binding in a ventrolateral part of the mesencephalon processed by a color image analyzer: (c) integrated image (128 times) (X250), (d) specific object image extracted as the highest area which corresponds to the basal optic nucleus, and (e) image in (d) superimposed on (c).

Binding of MAb-A5 and MAb-B2 in the Mesencephalon and Diencephalon of Xenopus Tadpoles The amphibian optic tectum has a laminar structure, designated as layers 1 to 9, from the ventricle to the pia

surface (see Fig. le). The 8th and 9th plexiform layes are the termination sites of the optic nerve (Szekely and La&r, 1976; Fujisawa and Jacobson, 1980). Immunofluorescent staining showed that MAb-A5 preferentially binds to these two plexiform layers (Figs. la and lb).

TAKAGI

a

a

OT

ET AL.

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MAb-A5 binding was highest at the 8th layer and low at the superficial layer, the stratum zonale. The MAbA5 binding was practically negligible in other plexiform layers, and binding in cellular layers was nil. The MAbB2 binding pattern in the optic tectum was different and apparently complementary to that of MAb-A5. MAb-B2 binds to the 3rd, 5th, and 7th plexiform layers, but does not bind to the 9th layer (Figs. lb and Id). The 8th layer was faintly stained by MAb-B2. Binding in the cellular layers was nil. Besides the optic tectum, MAb-A5 binds to all minor visual centers in the diencephalon and mesencephalon of Xenopus tadpoles such as the nucleus Belonci, the corpus geniculatum thalamicum, and the pretectum (Figs. 2, 3, 4). MAb-A5 binding to the basal optic nucleus, the mesencephalic accessory visual center, was so weak that the fluorescent region could not be easily distinguished from the background with the conventional fluorescent microscope (Fig. 3a). However, fluorescence image enhancement by the image analyzer clearly showed the binding of MAb-A5 to a wedge-shaped region of the basal optic nucleus (Figs. 3c-3e). In contrast, MAb-B2 showed no binding to any of these visual centers, although it did bind to the surrounding neuropile fibers. To sum up, it is the general rule that MAb-A5 binds to the optic nerve termination sites, whereas MAb-B2 does not bind to them at all. In addition to the visual centers, MAb-A5 binds to the very restricted parts of the diencephalon and mesencephalon such as the habenula and the lateral neuropile of the hypothalamus. Binding to the optic nerve, optic tract, and tegmentum was nil. Expression of MAbA5 Antigen Enucleated Tadpoles

(A5-Antigen)

in

As MAb-A5 preferentially binds to the termination sites of the optic nerve fibers, one may ask whether the A5-antigen is expressed by the terminals of the optic nerve fibers. If not, the question would arise as to whether the expression of this antigen is a consequence of optic nerve innervation or is regulated independently of optic nerve innervation. In an attempt at elucidation, FIG. 4. Whole mount of the diencephalon and mesencephalon of tadpoles (stage 51). (a) Retinal central pathways in a Xenopus tadpole. All retina1 axons from the right eye were labeled with HRP to show the projection to the visual centers, i.e., the optic tectum (OT), nucleus Belonci (NB), corpus geniculatum thalamicum (CGT), and basal optic nucleus (BON). OC: optic chiasm. For the method of HRP labeling, see Fujisawa et ul. (1981). (b) The tadpole brain stained with MAb-A5 by the ABC method. The optic tectum, the nucleus Belonci, and the corpus geniculatum thalamicum are positively stained. The staining of the basal optic nucleus is obscure. (c) The tadpole brain stained with MAb-B2 by the ABC method. All visual centers are negative (X40).

96

FIG. 5. Indirect immunofluorescent which the right eye was enucleated

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analysis of the expression of A5-antigen in the optic tectum at stage 29. The left tecta (*) was not innervated by optic nerves

the left optic tectum which had never been innervated by the optic nerve (the virgin tectum) was prepared by enucleation of the right eye at stage 29 of development. The enucleated embryos were fed to stage 51 and then the optic tectum was fixed to examine the expression of ASantigen. Although the 8th and 9th layers in the virgin tectum were considerably thinner than those in the control optic tectum, MAb-A5 bound to these layers, and the intensity of immunofluorescence was not reduced compared with the control (Fig. 5). Therefore, it is con-

of eye-enucleated (X175).

tadpoles

(stage

51) in

eluded that MAb-A5 antigen is expressed by the optic tectum cells, irrespective of optic nerve innervation. E xpression

of MAbA5 Development

and MAbB2

Antigens in

In Xenopus embryos the optic tectum is innervated by the optic nerve fibers at stage 39 (Harris et al., 1985). At stage 35/36, that is, before the innervation of the optic nerve fibers, the A5-antigen is undetectable in the mes-

FIG. 6. Indirect immunofluorescence of MAb-A5 in the mesencephalic region in development. (a) A section of the rostra1 part of the optic tectum at stage 40 (X90). (b) A section caudal to the section in (a). (c) A section of the rostra1 part of the optic tectum at stage 45 (X70). (d) A section of the caudal part of the optic tectum at stage 45. Arrows indicate the stained regions.

TAKAGI

ET AL.

FIG. 7. Immunofluorescent staining of viable tadpole brains 8th. and 9th plexiform layers of theoptic tectum (X140)

Visual

with

MAb-A5

enc:ephalic region. At stage 39, a very faint staining by MAlb-A5 is detected in the neuropiles of the optic tectum. At stage 40, A&antigen is detected distinctly at the neur0I:bile beneath the pia of the optic tectum (Figs. 6a and 6b). At stage 45, the 8th and 9th layers are differentiating in 1;he rostra1 part of the optic tectum. These layers are

FIG. 8. MAb-A5 indirect immunofluorescent staining 0.05% trgpsin for 2 hr and then fixed and stained with

Centw

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(a) and MAb-B2

(b) (stage

53). Numbers

indicate

the 3rd, 5th, 7th,

stained with MAb-A5. (Fig. 6~). In the caudal part, wh:ere the neuropile has not fully differentiated, staining nTith MAb-A5 is evident (Fig. 6d). BB-antigen appeared no later than stage 36, when the first neuropiles begir 1 to form in the diencephalon and mesencephalon, and is detectable continuously thereafter.

of trypsin-digested MAb-A5 (b) Control

optic tectum nontrypsinized

(stage 53). (a) Intact optic tectum was treated optic tectum stained with MAb-A5 (X100).

with

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kDa (Fig. 9). Although the MAb-B2 antigen was trypsin resistant, three bands with apparent molecular masses of 200,160, and 80 kDa were immunoabsorbed. The 200kDa band was the densest. DISCUSSION

‘I*

FIG. 9. Fluorogram of SDS-PAGE of immunoadsorbed polypeptides. From left to right, lane 1: NP-40 extract of labeled brain. Numbers indicate the positions of molecular mass markers (kDa). Lane 2: polypeptides immunoadsorbed with MAb-B2. Arrowheads indicate the specific bands. Lane 3: polypeptides immunoadsorbed with MAb-A5. An arrowhead indicates the specific band. Lane 4: polypeptides adsorbed with control MAb which is nonreactive to Xenopus brain, as revealed by immunofluorescent staining.

Cellular

Localization

of A5- and B2-Antigens

To determine whether A5- and BZ-antigens are localized extracellularly, staining of viable brains with MAbA5 and MAb-B2 was carried out. As shown in Fig. 7, the staining pattern of the viable brains by these MAbs was similar to that of fixed brains, in both the sectioned and whole-mounted (data not shown) specimens. Thus, both A5- and BB-antigens seem to be located on the cell surface or in the intercellular spaces. Molecular

Nature

of A5- and B2-Antigens

First, the lability of these antigens to trypsin treatment was tested. Treatment of brain tissues with 0.5% trypsin weakened the intensity of the imunofluorescent staining of MAb-A5 within 15 min and abolished it 2 hr later (Fig. 8). This observation suggests that the A5antigen(s) is protein. The MAb-B2 staining was not altered by trypsin treatment (data not shown). Second, biosynthetically labeled brain extracts were analyzed with SDS-PAGE followed by fluorography. As to proteins immunoadsorbed with MAb-A5, a single band was detected around the molecular mass of 140

Recent work using monoclonal antibodies raised against crude nervous tissue reveals that cell type-specific antigens as well as unrecognized neuron subsets are present in neuronal tissues (Mckay et al., 1981). The present work clearly shows that the termination sites of a particular nerve have specific molecular characteristics. The optic nerve termination sites of all visual centers binds to MAb-A5. MAb-B2 binds to the brain surrounding all the visual centers, but was absent in the visual centers. The optic tectum is the dominant visual center in amphibian and has distinct layer structures (Szkkely and Lazar, 1976; LLzCr, 1984). MAb-A5 binds to the 8th and 9th layers, which consist of retinal and thalamic afferents and dendrites of the tectal neurons. Although the exact identification of the target of MAb-A5 in the optic tectum awaits immuno-electron microscopic examinations, several lines of observations presented in this paper suggest that the tectal dendrites are the most likely candidates. First, the eye-enucleation experiment revealed that the A5-antigens are expressed by tectal dendrites and/or the thalamic afferent fibers. The finding that A5-antigens are not expressed by the optic nerve or the optic tract suggests that the antigen is not expressed by the retinal afferents, though the possibility that only the terminal regions of the optic nerve fiber express the antigen cannot be excluded. Second, MAbA5 binding is relatively weak at the stratum zonale, the superficial layers of the optic tectum. This is apparently inconsistent with the possibility that the thalamic afferent fibers are the target of MAb-A5 since the large number of thalamic afferents terminate in this layer with only a few in the deeper layer (Lazir, 1984). The 7th plexiform layer of the optic tectum is a main afferent pathway from the optic tectum, and the 3rd and 5th plexiform layers receive afferents from the pretectal region, the contralateral tectum, the mesencephalic tegmental nuclei, and the spinal cord (Szekely and Liz&r, 1976). As the staining with MAb-B2 showed fibrous structures in the 3rd, 5th and 7th plexiform layers, some of these afferent fibers seem to be the target of MAb-B2. As a monoclonal antibody recognizes only one epitope in a molecule, the unity of antigens recognized by a particular monoclonal antibody should be examined. As to the A5-antigen, the protein nature is demonstrated by trypsin sensitivity and a single band appeared on the SDS-PAGE. Therefore, the unity of A5-antigen is highly

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ET AL.

Visual

probable, unless some antigens are not labeled with [3?3]methionine or are not solubilized using our procedures. As to B2-antigen, the results are more complicated. SDS-PAGE shows that at least three polypeptides were immunoadsorbed with MAb-B2 after solubilization with NP-40. However, the finding that the antigenesity was not abolished by trypsin treatment may suggest that lipid constitutes the antigens. It has been suggested that cellular recognitions are mediated by cell surface molecules. Bonhoeffer and Huf (1980) showed that the neurites from the chick embryonic retina grow in vitro on optic tectum cells preferentially to forebrain cells. Since optic nerve fibers show an obvious affinity to appropriate target cells under such a simple system as monolayer culture, it is highly plausible that molecular compositions of the target cell surface are distinct. Our preliminary electron microscopic studies revealed that both A5- and B2-antigens are located on the cell surface. Though functions of antigens recognized by MAb-A5 and MAb-B2 remain to be elucidated, one possibility is that these surface molecules, in particular AS-antigen, are involved in the recognition of target sites by the optic nerve. In this connection, the temporal and spatial patterns of expression of these antigens in visual centers are of interest. The observation that the expression of A5-antigen occurs in the presumptive visual centers just before or simultaneously with the optic nerve innervation and is also regulated independently of the optic nerve innervation, as revealed by the eye-enucleation experiment, is consistent with our present working hypothesis that A5-antigen plays a role as a marker molecule of the optic nerve target sites. The binding of MAb-A5 to the habenular and the hypothalamus cannot apparently be explained by this hypothesis. Further analyses are necessary to clarify this point. Initial attempts to disrupt retinotectal projections by injecting MAb-A5 into tadpole brain are under way. This work was supported by Grants-in-Aid for Special Project Research from the Ministry of Education, Science and Culture, Japan (Project No. 60105003 and No. 61131004); grants from the Ministry of Education, Science and Culture, Japan (60570029 and 61770054); and special coordination funds from the Science and Technology Agency of the Japanese Government. We thank Dr. M. Takeichi of Kyoto University for the gift of myeloma cells and for pertinent advice. We are also grateful to Dr. S. Taketani of Kansai Medical University for advice on the immunoadsorption experiment and to M. Ohara of Kyusyu University for comments on the manuscript. REFERENCES BASTIANI, M. J., DOE, C. Q., HELFAND, S. L., and GOODMAN, C. S. (1985). Neuronal specificity and growth cone guidance in grasshopper and Drosophila embryos. Trends Neurosci. 8,257-266.

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