Development 99, 155-162 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

155

Cell- and tissue-specific monoclonal antibodies in eggs and embryos of

the ascidian Halocynthia roretzi*

IZUMI MTTA-MIYAZAWA, TAKAHITO NISHIKATA and NORIYUKI SATOH

Department of Zoology, Kyoto University, Kyoto 606 and Asamushi Marine Biological Station of Tohoku University, Aomori 039-34, Japan

' All the monoclonal antibodies are available for studies of ascidian developmental biology on request from I.M.-M.

Summary

To obtain specific immunological probes for studyingmolecular mechanisms involved in the early embry-onic development of ascidians, we have producedmonoclonal antibodies directed against a homogenateof larvae of the ascidian Halocynthia roretzi. Amongthese, we have screened monoclonal antibodies thatspecifically recognize cells and/or tissues of theembryo. ..Characterization of six epidermis-specificmonoclonal antibodies (including larval tunic-specificand larval fin-specific), three muscle-specific anti-bodies, two endoderm-specific antibodies, onenotochord-specific antibody and two monoclonalantibodies that specifically recognize trunk-lateral

cells suggests that these monoclonal antibodies may beuseful as markers for analysing molecular mechan-isms involved in specification of these cells. Sevenmonoclonal antibodies characteristically stain inter-cellular materials of the developing embryo and maytherefore be valid for studying cellular construction ofthe embryo. Furthermore, monoclonal antibodies thatrecognize components of follicle cells, perivitellinespace and sperm have also been established.

Key words: ascidian embryos, monoclonal antibodies,differentiation markers, intercellular'materials, folliclecells, perivitelline space, sperm.

Introduction

Eggs of ascidians (subphylum Urochordata, classAscidiacea) offer advantages for studying cellularand molecular mechanisms involved in specificationof embryonic cells (see reviews by Reverberi, 1971;Whittaker, 1979; Jeffery, 1984): (1) an egg developsvery rapidly into a tadpole larva which consists ofa comparatively small number (about 2500) of cellsbut of several distinct types of differentiated cells;(2) embryonic cell lineages are well known; thedevelopmental pattern is 'mosaic' and there isaccumulating evidence for the existence of egg cyto-plasmic determinants which are differentially segre-gated by a determinate cleavage pattern into certaincell lineages where they appear to play a crucial rolein programming the differentiation pathways of thecells and (3) although a certain number of DNAreplications may be an essential prerequisite, neithercytokinesis nor nuclear division is required for tissue-specific enzyme development.

However, despite such advantages, the number ofdifferentiation markers is rather limited; myofila-

ments and acetylcholinesterase for the muscle cells,melanin and tyrosinase for the melanocytes, vacuo-late inclusions for the notochordal cells, extracellu-lar secretions for the epidermal cells and alkalinephosphatase for the endoderm cells. In addition,these morphological and histochemical markers arenot always used directly in molecular studies on cellspecification. This situation therefore seems to re-strict an overall understanding of mechanisms under-lying differentiation in the mosaic eggs.

Immunological probes, particularly those withmonoclonal antibodies, have successfully been intro-duced in various fields of developmental biology. Inorder to facilitate a study of the molecular mechan-isms underlying early development of this mosaic eggand also to investigate the functions of accessory cells(i.e. follicle cells and test cells), we have attemptedto produce monoclonal antibodies that specificallyrecognize cells and tissues of developing ascidianembryos. Results obtained are described here anddiscussed with respect to the validity for their use infurther investigations.

156 /. Mita-Miyazawa, T. Nishikata and N. Satoh

Materials and methods

EmbryosNaturally spawned eggs of the ascidian, Hahcynthia roretzi,were fertilized artificially with a dilute suspension of nonselfsperm and raised in filtered seawater at 13-15 °C.

Antigen preparation and immunizationNewly hatched larvae were collected by centrifugation,washed with an ice-cold physiological saline solution (PSS)for mouse and suspended in an equal volume of PSS. Thesuspension was homogenized by several vertical strokes ofa Teflon pestle fitted into a Potter glass homogenizer andcentrifuged at 3000 revs min"1 for lOmin, then at 10000revs min"1 for 5min. The supernatant was used as anantigen. Female BALB/c mice were injected intraperito-neally with 0-5 ml supernatant and boosted intraperito-neally once or twice at about 3 week intervals with 0-5 mlinjection of the immunogen.

Production of monoclonal antibodiesThree days after the last intraperitoneal hyperimmuniz-ation, the mice were killed and their spleens removed.Spleen cells-myeloma hybridomas were generated accord-ing to the protocol of Galfre, Howe, Milstein, Butcher &Howard (1977); about lxlO8 spleen cells were fused with2-5xlO7 P3U-1 myeloma cells (purchased from How Lab.)by using 50% (w/v) polyethylene glycol 4000 (NakaraiChem. Co. Ltd, Kyoto). Hybridomas were raised in HATmedium according to Littlefield (1964). The fused cellswere separated into 96 multiwells (106 cells/well) and,when cell growth was apparent (7-10 days after fusion),samples of supernatant of hybridoma culture medium fromeach well were assayed by immunofluorescence mi-croscopy. Hybrid cells producing antibodies of interestwere cloned once or twice by picking up a single cell with asmall pipette under an inverted microscope.

Fixation and immunofluorescence staining of eggs andembryosFor screening the hybridoma supernatant medium, largequantities of Halocynthia eggs and embryos at variousstages were fixed for 10 min in methanol (—20 °C), followedby ethanol (—20 °C), and embedded in polyester wax(Steedman, 1957; BDH Chem. Ltd). Sectioned specimenswere mounted on small coverslips. After removal of poly-ester wax with absolute ethanol, specimens were washedwith phosphate-buffered saline (PBS), then immersed in100/d of hybridoma culture fluid for lh at room tem-perature. Coverslips were then washed in PBS at roomtemperature for 30min, and each coverslip incubated for30 min with 6 [A of fluorescein isothiocyanate-conjugatedrabbit anti-mouse IgG serum (Miles-Yeda, Ltd) diluted1:60 in PBS. Coverslips were then washed in PBS atroom temperature for 30 min, mounted in 80% glyceroland observed with a Nikon Labophoto equipped with anepifluorescence optic unit (EFD). Photomicrographs weretaken with Kodak Tri-X Pan film.

Results

To obtain specific immunological probes for studyingmolecular mechanisms involved in cell specificationof ascidian embryos, we immunized mice with ahomogenate of newly hatched larvae, fused themouse spleen cells to mouse myeloma cells to estab-lish hybridoma cell lines and screened individualhybridoma culture fluid samples by immunofluor-escence microscopy. In four series of productions,more than 800 samples were tested, from which weselected and cloned hybridoma cell lines that secretedantibodies that specifically recognize certain cells.

Epidermis-specific, larval tunic-specific and/'or larvalfin-specific monoclonal antibodies

The outermost part of an ascidian embryo consists ofa layer of epidermal cells and a developing tadpole-like embryo is surrounded by two acellular layers, thelarval tunic and larval fin (Fig. 1). As summarized inTable 1, six hybridoma clones, 3A7D5 (Fig. 2),4C5B7 (Fig. 3), 5D3E8, 7H6B6, 12N1D2 and 5F1F8(Fig. 4), were found to secrete monoclonal antibodieswhich specifically recognized epidermal cells, larvaltunic, and/or larval fin. For example, the antigenrecognized by each of 3A7D5 and 4C5B7 first appearsat the early tailbud stage, initially within epidermalcells. Progressively with developmental time, a form-ing larval tunic becomes stained very intensely(Figs 2,3). 3A7D5 stains the boundary of neighbour-ing epidermal cells more intensely than the insideof cells (Fig. 2), whereas the staining with 4C5B7appears punctate and intracellular (Fig. 3). In con-trast to the five other antibodies that recognizeepidermal cells and larval tunic, 5F1F8 recognizeslarval fin (Fig. 4). The antigen is first expressed at themiddle tailbud stage, when formation of the larval finbegins. In tadpole larvae of this species, fin-ray-likestriped structures running very regularly through thelarval fin are easily observed with differential inter-ference contrast microscopy. As development pro-ceeds, 5F1F8 actually recognizes the component ofthis fin-ray-like structure (Fig. 4).

Muscle-specific monoclonal antibodiesA tadpole larva of H. roretzi contains 21 striated butmononucleate muscle cells on each side of the tail(Fig. 1). We have produced three muscle-specificmonoclonal antibodies, 6F2D3 (Fig. 5), 3M1F11(Fig. 6) and 3M3A7 (Table 1). As clearly shown inFigs 5 and 6, the monoclonal antibodies recognizecomponents exclusively present in muscle cells. Theantigenicity of each of them first appears at the earlytailbud stage, but is not detected at the neurula andearlier stages. 3M1F11 and 3M3A7 react with muscle

bs n

Monoclonal antibodies against ascidian embryos 157

mu

tic

IfSC ep

A en B

mu

es It

Fig. 1. Schematic representation of structures of the tailbud-stage H. roretzi embryo. (A) Midsagittal section of theembryo; (B) sagittal section; (C) cross section through the middle part of the tail, b, brain; bs, brain stem; en,endoderm; ep, epidermis; es, endodermal strand; //, larval fin; It, larval tunic; mch, mesenchyme; mu, muscle; n,notochord; sc, spinal cord; tic, trunk-lateral cell.

Table 1. Cell- and tissue-specific monoclonal antibodies in eggs and embryos of the ascidian Halocynthia roretzi

Monoclonalantibody

Regional distribution of antigens*

ep V tic

Developmental stageat which theantigenicityfirst appears

Cross reactivitywith embryonic

tissues ofCiona intestinalis

Epidermis-specific3A7D5 + +4C5B7 + +5D3E8 + +7H6B6 + +12N1D2 +5F1F8 +

Muscle-specific3M3A7 +4M1F11 +6F2D3 +

Trunk-lateral cell-specific5A4E1 +5A4A3 +5A4A6 +5A11C11 +

Notochord-specific5F1D5

Endoderm-specific4B3C36C9D1

* ep, epidermis; It, larval tunic; //, larval fin; mu, muscle; tic, trunk-lateral cell; n, notochord; en, endoderm.

+

± +± +

early tailbudearly tailbudearly tailbudmiddle tailbudlate tailbudmiddle tailbud

early tailbudearly tailbudearly tailbud

middle tailbudmiddle tailbudmiddle tailbudmiddle tailbud

early tailbud

early gastrulafertilized egg

+ (larval tunic)+ (larval fin)

+ (muscle)+ (muscle)

1 1

1 1

-

-

components of another ascidian species, Ciona intesti-nalis, and also recognize body wall muscle cells ofthe adult animal. Therefore, the antigens may becommon components of ascidian muscle cells.

Monoclonal antibodies that specifically identify trunk-lateral cellsA recent study of cell lineages in ascidian embryoshas shown that if the A6.3 cell (one of the anterior

vegetal blastomeres) is marked with horseradish per-oxidase at the 32-cell stage, the dorsal endoderm ofthe head region and undefined cells lying lateral tothe brain, stem are stained (Nishida & Satoh, 1985).Further analyses have revealed that the property ofthe undefined cells, designated as 'trunk-lateral cells',is inherited by the A7.6 cell of the 64-cell embryo; i.e.the A7.6 cell gives rise to only trunk-lateral cells(Nishida, unpublished).

158 /. Mita-Miyazawa, T. Nishikata and N. Satoh

We have produced four monoclonal antibodies,5A4E1 (Fig. 7), 5A4A3, 5A4A6 and 5A11C11, whichspecifically identify trunk-lateral cells (Table 1). Theantigen recognized with 5A4A3 or 5A4E1 is highlyrestricted to trunk-lateral cells. The antigens areexpressed from the middle tailbud stage onwards andthe entire cell, except for the nucleus, stains verystrongly with FITC-labelled monoclonal antibodies(Fig- 7).

Notochord-specific monoclonal antibodyIn a developing H. roretzi larva, 40 notochord cellsrun anteroposteriorly through the centre of the tail(Fig. 1). A monoclonal antibody 5F1D5 has beenidentified to be almost specific to notochord cells(Fig. 8), although a faint staining is sometimes seenin endodermal cells. The antigen is expressed fromthe initial tailbud stage onwards. It initially appearspunctate and intracellular but as development pro-ceeds, the border of notochord cells or the noto-chordal sheath stains very strongly (Fig. 8).

Endoderm-speciflc monoclonal antibodiesThe central region of the head of a developing tailbudembryo is occupied by a group of endodermal cellswhich give rise to the gut of adult animals (Fig. 1).Two monoclonal antibodies, 4B3C3 (Fig. 9) and6C9D1 (Fig. 10), recognize components of endo-dermal cells (Table 1). The antigen of 4B3C3 is firstexpressed in endodermal cells at the early gastrula

Figs 2—8. Regional distribution of antigens recognizedwith tissue-specific monoclonal antibodies on polyesterwax sections of middle to late tailbud-stage embryos (Figs2, 3, 5-8) and on a whole mount of swimming tadpolelarvae (Fig. 4) of the ascidian Halocynthia roretzi. Scalebar, 50 um.Fig. 2. An epidermis-specific monoclonal antibody3A7D5 stains the boundary of the epidermal cells andlarval tunic very strongly.Fig. 3. The staining with an epidermis-specific antibody4C5B7 appears punctate and intracellular.Fig. 4. The antigen of an epidermis-specific 5F1F8 isexpressed in the fin-ray-like structure of the larval fin (If).The opaque appearance of the larval body (Ib) is not dueto immunofluorescence staining.Fig. 5. A section of a late tailbud embryo stained with amuscle-specific 6F2D3 clearly shows that muscle cells(mu) of each side of the elongating tail are recognizedwith this antibody. B is an enlargement of A.Fig. 6. A muscle-specific 3M1F11 stains exclusivelymuscle cells.Fig. 7. Staining with 5A4E1 indicates that the antigenrecognized with this antibody is expressed in only trunk-lateral cells (arrow).Fig. 8. A monoclonal antibody 5F1D5 intensely stainsthe boundary of notochord cells (n) or notochordalsheath.

Monoclonal antibodies against ascidian embryos 159

stage. A weaker staining is sometimes seen in noto-chord (Fig. 9).

Monoclonal antibodies against components ofintercellular materials

Seven monoclonal antibodies summarized in Table 2have been found to identify intercellular materials.The antibodies recognize components that firstappear at the neural plate (12N2D4, Fig. 11), neurula(4B4D2, 3M2A11, 3M5A10) and early tailbud stages(5D7E7,6A1F2), respectively. The component recog-nized with 6B12C3 (Fig. 12), however, is presentfrom the unfertilized egg through to embryogenesis.Staining patterns of the antibodies that recognizenewly synthesized extracellular materials differ indetail from each other (Table 2). For example, theantigen identified with 12N2D4 is expressed at theboundary between notochord, muscle, endodermalstrand, brain and spinal chord, but not at the borderof epidermal cells (Fig. 11), whereas 3M5A10 stainsepidermal cells and larval tunic of the head regionvery strongly.

Follicle cell-specific monoclonal antibodiesAn ascidian egg is enclosed by the outer follicle cellsand the inner test cells, with an acellular vitellinemembrane or the chorion separating these cells. Twomonoclonal antibodies, 4D9D3 (Fig. 13) and 5F3H2,stain follicle cells very markedly. The antigenicity ofboth monoclonal antibodies is already detected infollicle cells of the gonad. The reactivity of 5F3H2 isexclusively found in follicle cells, whereas a faintstaining with 4D9D3 is also detected in the yolk areaof a developing embryo (Fig. 13). These monoclonalantibodies do not stain Ciona embryonic tissues.

Monoclonal antibodies for components of theperivitelline space

The perivitelline space of an ascidian egg is thought tocontain not only water and inorganic substances butalso mucus-like components whose function has notyet been defined. Three monoclonal antibodies,3PvA6, 3PvC2 and 3F2F10, recognized componentsof the perivitelline space. No crossreactivity of thesemonoclonal antibodies with Ciona tissue is observed.

Sperm-specific monoclonal antibodiesAlthough the antigen used to immunize mice wasa homogenate of larvae, two sperm-specific mono-clonal antibodies, 4D5A10 and 3M2G8 (Fig. 14),were obtained. The antigen recognized by 4D5A10 isseen on the overall surface of sperm including its headand tail: It is evident that an oviform structure nearthe head is intensely stained with 3M2G8 (Fig. 14). Ithas been shown that an ascidian sperm contains onelarge mitochondrion, which moves during activationfrom its original position at the middle piece to the

160 /. Mita-Miyazawa, T. Nishikata and N. Satoh

Monoclonal antibodies against ascidian embryos 161

Table 2. Regional distribution of antigens recognized with seven monoclonal antibodies against components ofintercellular materials

Monoclonalantibody

Regional distribution of antigens*

ep // sc mu mch

Developmental stageat which the

antigenicity firstappears

Cross reactivitywith Ciona

embryonic tissues

12N2D44B4D23M2A113M5A105D7E76A1F26B12C3

neural plateneurulaneurulaneurulaearly tailbudearly tailbudunfertilized eggs

+ (epidermis)

'ep, epidermis; It, larval tunic; //, larval fin; b, brain; sc, spinal cord; mu, muscle; mch, mesenchyme; n, notochord; e, endoderm; es,endodermal strand.

tip of the tail and is then finally discarded (Lambert& Epel, 1979). Judging from its position and mor-phology, 3M2G8 is likely to recognize some anti-gen localized at this sperm mitochondrion. Thesperm-specific antibodies do not stain sperm of Cionaintestinalis.

Discussion

The objective of this study was to establish hybri-doma clones that secrete monoclonal antibodiesspecific to cells or tissues of ascidian embryos. Asdescribed, we have obtained many monoclonal anti-bodies fulfilling this objective. Characterization ofsix epidermis-specific monoclonal antibodies, threemuscle-specific antibodies, two endoderm-specificantibodies, one notochord-specific antibody and twomonoclonal antibodies that specifically recognize

Fig. 9. The antigen of 4B3C3 is expressed on the surfaceof endoderm cells (en). This antigen is first expressed atthe early gastrula stage, although a faint staining issometimes seen in notochord cells. Scale bar, 50 fim.Fig. 10. The antigen of 6C9D1 is present in fertilizedeggs. As development proceeds, the antigen becomesmainly distributed in endoderm cells (en). Scale bar,50/mi.Fig. 11. 12N2D4 stains the borders of the notochord (n),muscle (mu), endodermal strand (es), brain (b), andspinal cord (sc), but does not stain the border of theepidermis. A shows a midsagittal section of a late tailbud-stage embryo, while B is a cross section of the middlepart of the tail of the same stage. Scale bar, 50 /an.Fig. 12. The antigen of 6B12C3 is detected at the surfaceof an unfertilized egg. As development proceeds, theboundary of every cell stains with this antibody.Fig. 13. A monoclonal antibody 4D9D3 specificallyrecognizes follicle cells (fc). em, embryo. Scale bar,50 /mi.Fig. 14. A sperm-specific monoclonal antibody 3M2G8stains mitochondria (arrows) intensely. Scale bar, 10/an.

trunk-lateral cells suggests that these monoclonalantibodies may be useful as markers for analysingmolecular mechanisms involved in specification ofthese cells. However, prior to using these monoclonalantibodies as immunological probes, we have tocharacterize antigenic polypeptides recognized bythese antibodies. Although all the monoclonal anti-bodies are different in their temporal appearanceand spatial localization, there remains the possibilitythat two or more antibodies recognize an identicalantigenic polypeptide.

Most developmental biological studies of ascidianembryos have focused on the specification ofembryonic cells and studies of morphogenesis orconstruction of a particular form of the embryo arerather scarce. We have produced seven monoclonalantibodies specific to intercellular materials whichmay be used as immunological probes for furtherstudies of mechanisms involved in the construction ofthe embryonic form. In addition, we have obtainedseveral antibodies specific to components of folliclecells, the perivitelline space and sperm. These anti-bodies may also be useful for further analysis of theirfunctions.

In this study we have attempted to produce mono-clonal antibodies that can be used as differentiationmarkers. The results obtained are not always com-plete, since it was not possible to produce monoclonalantibodies specific to mesenchyme, brain or spinalcord. However, we envisage the monoclonal anti-bodies established in this study as being useful asimmunological probes for further studies of ascidiandevelopmental biology.

We thank Dr M. Takeichi and Mr K. Hatta for theirtechnical advice and help during the course of mono-clonal antibody production. Thanks are also due to Dr T.Numakunai and all other members of Asamushi MarineBiological Station for their kind hospitality. We are also

162 /. Mita-Miyazawa, T. Nishikata and N. Satoh

grateful to Dr A. Rossiter for his help in the preparation ofthe manuscript. This study was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture,Japan (No. 60105001) and by Kudo Academic Foundation.

References

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LAMBERT, C. C. & EPEL, D. (1979). Calcium-mediatedmitochondria movement in ascidian sperm during ferti-lization. Devi Biol. 69, 296-304.

LITTLEFIELD, J. W. (1964). Selection of hybrids frommatings of fibroblasts in vitro and their presumed re-combinants. Science 145, 709-710.

NiSfflDA, H. & SATOH, N. (1985). Cell lineage analysis inascidian embryos by intracellular injection of a tracerenzyme. II. The 16- and 32-cell stages. Devi Biol. 110,440-454.

REVERBERI, G. (1971). Ascidians. In Experimental Embry-ology of Marine and Fresh-Water Invertebrates (ed. G.Reverberi), pp. 507-550. Amsterdam: North-Holland.

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WHTTTAKER, J. R. (1979). Cytoplasmic determinants oftissue differentiation in the ascidian egg. InDeterminants of Spatial Organization (ed. S. Subtelny &I. R. Konigsberg), pp. 29-51. New York: AcademicPress.

(Accepted 29 August 1986)