Expression of the Lewis groupcarbohydrate antigens during Xenopus development

Chikako Yoshida-Noroa,2,3, Janet Heasman3,4, Kim Goldstone3, Lucinda Vickers3 and Chris Wylie3,4

2Cell & Information,Precursory Research for Embryonic Science and Technology (PRESTO),Japan Science and Technology Corporation (JST), Tsukuba 305, Japan, 3Wellcome/CRC Institute,University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK,and 4Institute of Human Genetics,University of Minnesota, Minneapolis, MN 55455, USA

Received on February 10, 1999. revisedon April 22, 1999; accepted on April 22, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We have examined the pattern of expression of the Lewis groupcarbohydrate antigens during the development of African toad Xenopus laevis. One of these antigens, Lewis x (Lex,also known as SSEA-1), was previously shown to be involved in cell–celladhesion in early mouse embryos and teratocarcinoma stem cells.Recently another member of these antigens, sialyl-Lex,was found to be one of the major ligands for the selectin familyof cell–cell adhesion molecules. In order to study therole of carbohydrate-mediated cell adhesion during Xenopus development,we first studied the expression pattern of the Lex. Wefound that Lex was not expressed in early embryos, startedto be expressed at the tail bud stage in anterior regions of thebody such as the cement gland or head skin, and was gradually showedmore posterial expression at later stages. At tadpole stage, itwas also expressed on specific cell bodies in brain, and in axon regionin brain and neural retina. Antibodies against Lex blockedneurite outgrowth in the explant culture of tadpole brain. One ofthe candidates for Lex carrier protein in the tadpolebrain is a 200 kDa glycoprotein detected by Western blotting. Inadult tissues, it was expressed in brain, testis, and gut, but notin kidney, lung, spleen, ovary, or muscle. We also examined theexpression patterns of other Lewis group antigens. Among them, sialyl-Lex was expressedon endothelial cells and on leukocytes, suggesting the possibilitythat it functions as a ligand for selectin in Xenopus.


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Cell surface carbohydrates have important roles for cell interactionsand cell recognition. For example, virus or bacteria recognizesthe cell surface carbohydrates and attaches to them at the firststep of infection. Some hormones or toxins have effects throughbinding to cell surface carbohydrates. Carbohydrates on the eggsurface are important for recognition by sperm at fertilization.Also, carbohydrates are important in cell adhesion. The carbohydratesof laminin or proteoglycan molecules are important in cell–extracellularmatrix adhesion. It is shown that carbohydrates are involved inneural crest cell adhesion and migration during development. Thus,it is conceivable that studying cell–cell interaction orcell adhesion by taking an approach from the side of carbohydratesis important.

In particular, a group of carbohydrates which is known as the Lewisblood group antigens in humans have been shown to be important incell adhesion. One of these carbohydrates is the Lewis x antigen(Lex), the structure of which was identified as Gal ß (1–4) [Fuc {alpha}(1–3)] GlcNAc(15GoGooi et al., 1981),known as lacto-N-fucopentaose III (LNFIII) or leukocyte antigen CD15.It is also called SSEA-1 (stage-specific embryonic antigen-1) andknown to have an important role for cell adhesion of mouse embryosor teratocarcinoma cells. SSEA-1 was originally defined by a monoclonalantibody raised against the murine teratocarcinoma cell line F9(40GoSolter and Knowles, 1978). Itis expressed on the surfaces of undifferentiated but not differentiated,teratocarcinoma cells (41GoSolter and Knowles,1979; 20GoKnowles et al.,1980). In the mouse embryo, it appears at the late eight-cellstage, at the onset of compaction and is highly expressed on thesurface of trophectoderm, primitive ectoderm, primitive endoderm,and primordial germ cells (PGCs) during migration (12GoFenderson et al., 1990; 14GoGomperts et al., 1994).

The biological functions of Lex/SSEA-1 havebeen examined by several groups using either monoclonal antibodiesor purified carbohydrates. 5GoBird and Kimber (1984) found that LNFIII inhibited the compaction ofmouse morulae. 9GoEggens et al.(1989) or 21GoKojima etal. (1994) observed that free LNFIII inhibited intercellularadhesion of teratocarcinoma cells. They showed that Lex isa homophilic adhesion molecule and proposed the model that carbohydrate-carbohydrateinteraction is important in cell adhesion. Lex can becarried either on glycolipids or glycoproteins as seen by Westernblotting and thin layer chromatography (19GoKannagi et al., 1982; 6GoChilds et al., 1983; 32GoOzawa et al., 1985; 9GoEggenset al., 1989; Rosenman et al.,1989; 43GoStreit et al., 1996).

Recently, some of the Lewis group carbohydrates have been identifiedas potential ligands for selectins (47GoVarki,1994, for review). Selectins are a class of transmembranecell–cell adhesion proteins composed of three extracellulardomains; Ca2+-dependent lectin domain, epidermalgrowth factor (EGF)-like domain and a variable number of complementbinding protein repeats. They bind to carbohydrates on the complementarycell mainly via lectin domain (see Stoolman, 1989, for review).So far three types (L-,E-,P-) of selectins were identified. Though sialyl-Lex andsialyl-Lea are known as major ligands for all three typesof selectins, it showed that Lex is also one of the ligandsfor P-selectin (23GoLarsen et al.,1990). In these years, selectins have only been foundand studied extensively in mammalian blood vascular systems. However,recently similar molecule was identified in Drosophila (24GoLeshko-Lindsay and Corces, 1997) and itrevealed that it is necessary for development of the eye and mechanosensorybristles. Also, as shown in a separate paper (Yoshida-Noro etal., unpublished observations), we have succeeded in cloningof a Xenopus selectin gene. Though we have no informationof the ligands for these newly found selectins at the moment, celladhesion and cell recognition through selectin and its ligand carbohydrateseem to be conserved mechanism in animal kingdom.

We have been interested in role of carbohydrates in cell adhesionand cell recognition during Xenopus development.We have previously shown (46GoTurner et al., 1992) that the carbohydrate antigen M4B isinvolved in cell adhesion in early Xenopus embryos.M4B is carried on a group of neutral glycolipids, and antibodiesagainst it cross-react with early blastomere cell surfaces as wellas migrating primordial germ cells. Though the sugar structure ofM4B has not yet been identified, M4B is probably different fromLex from the result of antibody staining, sugar blockingexperiments, and thin layer chromatography.

Though Lex has been identified in mice (teratocarcinoma cellsand embryo; Solter and Knowles, 1978: embryo, fetus, and adult; 13GoFox et al., 1981: embryonicbrain; 48GoYamamoto et al.,1985), humans (embryonal carcinoma cells; 2GoAndrews et al., 1989, 1990: embryonic brain; 36GoSchwarting et al., 1989; 34GoSatoh and Kim, 1994: embryonic spinalcord; 1GoAloisi et al., 1992:lens; 29GoOgiso et al., 1992:blood cells; 19GoKannagi et al.,1982; 25GoMacher and Beckstead, 1990),rats (primary sensory neuron; 18GoJessell andDodd, 1986), bovine (brain; 7GoDasgupta et al., 1996), chicken (embryonic brain; 43GoStreit et al., 1996: Bcell; 26GoMasteller et al.,1995), quails (spinal sensory neuron lineage; 37GoSieber-Blum, 1989ab; 33GoRacheland Sieber-Blum, 1989), it has not been studied in Xenopus.In this work we examined the expression pattern of Lex aswell as other Lewis group antigens during Xenopus developmentin order to get information for studying these important carbohydratesin cell adhesion.

Our result show that unlike mice, Lex expressionin Xenopus was not detected in early embryos, insteadit started at later stages, i.e., from tail bud stage, in anteriorstructures. Expression could be detected more posteriorly in skinor gut as development proceeded. At the tadpole stage, Lex wasexpressed in axons and specific neuron cell bodies in brain andan axon layer of neural retina but not in the spinal cord or ganglia.In explant cultures of tadpole brain, we showed that it was expressedon the surfaces of specific cell bodies as well as axons, and thatantibodies against Lex perturbed neurite outgrowth suggestingthe role of Lex or its carrier molecule in neurite elongationor adhesion. A candidate for the major carrier of Lex ontadpole brain was revealed to be a 200 kDa glycoprotein.

Sialyl-Lex which is known to be one of the majorligands for selectins is expressed on the endothelial cells, leukocytesand also on cells in gut and mesentery, suggesting the possibility forfunctioning as a ligand for Xenopus selectin.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Lex expression starts at tail bud stage
Lex expression was first detected at the tail budstage (st.34), in the cement gland by immunostaining (Table Go). No expression was observed from egg toneurula stages. At st.37 (tadpole), skin of the head and pharyngealepithelium were also positive. Expression in these tissues was localizedto particular cells. From st.39, positive staining was detectedin neuronal tissues such as forebrain, neural retina and some cellsin the olfactory pits. Some anterior coils of gut were also positiveat this stage. After st.44, expression in hindbrain and posteriorgut became extensive. At the early stages of metamorphosis (st.49–53), expressionpatterns were similar to earlier tadpole stages (st.44–46).Overall, expressions of the Lex antigen started from theanterior end of the larva and became expanded to the posterior partas development proceeded. Some tissues such as muscle, liver, kidney,or lung, were completely negative throughout development. UnlikeM4B antigen, Lex was negative on migrating primordialgerm cell surface (at st.44–46) as well as blastulae (st.9)cell surfaces.


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Table I. Expression of Lewis x antigen during Xenopus development
 
Expression in the developing brain and retina
At st.45, Lex showed a quite specific pattern of expressionin the brain, including axons and some specific cell bodies in the hindbrain(Figure 1A.). The optic nerves and gangliawere negative. In contrast, HNK-1, another carbohydrate antigen(carried on N-CAM) known to be a marker for neuronal tissues, stainedalmost all of brain cells and axons (Figure 1C)as well as ganglia. Lea was completely negative in allneuronal tissues (Figure 1B).



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Fig. 1. Expression of LeX inbrain and eye of st.45 tadpole. LeX was expressed onaxon and some specific cell bodies in brain (A)and inner plexiform layer of neural retina (D)while Lea showed no expression in neural tissues (B, E). HNK-1 was expressed in all neural tissues (C, F). Asterisks indicate skinpigments (AC) and pigmentretina (DF). Magnification,100x.

 
In neural retina, Lex was expressed only in the innerplexiform layer where axons are connected with each other (Figure 1D), while HNK-1 was expressed all over theretina (Figure 1F). Lea was negativein the retina (Figure 1E).

In tadpole forebrain, Lex also exists on the axonsof ventrolateral side. In contrast, HNK-1 expressed all over inforebrain, while Lea showed negative results (data notshown).

Expression of Lex on brain cells inculture
Fragments of brain from stage 32/33 tail bud stage embryo weredissected and cultured. At this stage, no expression of Lex wasdetected in sectioned material. After culturing for 48 h, by whichtime the sibling embryos reached stage 44/45, cultures werefixed for immunohistochemistry. As seen in Figure 2, some populations of cultured brain cellsexpressed Lex antigen. The expression was detected bothon axons (Figure 2A) and on the surfacesof some cell bodies (Figure 2B). However,other populations of neuron cell bodies or glial cells did not express Lex,confirming the result seen in the sections.



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Fig. 2. Expression of LeX incultured brain. Lex was expressed on axons (A,arrows) and specific neuronal cell bodies (B, arrowheads).No expression was detected on underlying glia cells. Magnification,200x.

 
Anti-Lex antibodies inhibit axon outgrowthin brain culture
In the same culture conditions as above, antibody against Lex (SignetBG-7; final 5 µg/ml) or Lea (SignetBG-5; final 5 µg/ml) was addedto the culture at 48 h and incubated further 24 h (Figure 3). The antibodies showed no general toxic effectsand cells looked healthy throughout the experiments. Cultures werephotographed before and after the 24 h incubations with antibodies.As shown in Figure 3, before addition ofantibody (A, B, C, and D), axons were well spreaded. Following antibodyincubation, anti-Lex antibody showedan inhibitory effect on neurite outgrowth (Figure 3E,F) while anti-Lea showed no effect(Figure 3G) compare to the control (withoutantibody) (Figure 3H). The same effect couldbe seen at the antibody concentration range of 2.5–10 µg/ml or using other clonesof anti-Lex monoclonal antibodies (data not shown). Thus,this effect seemed to be specifically caused by blocking Lex determinanton axons. Antibodies had no effect on the glial cells. Consideringfrom the result shown in Figure 2 and 3,Lex is expressed on the axon and specific neuron cellbodies in brain and antibody against Lex affect on theadhesion between axons or axon–substrate interactions.However, free oligosaccharide (25 µg/ml;Oxford Glycosystems), or trisaccharide–biotin polymer complex(200 µg/ml; Seikagaku: affinityis 102–105 higher than free sugar)of Lex or Lea showed no inhibitory or promotoryeffect on neurite outgrowth (data not shown). Thus, it is also possiblethat not Lex itself but its carrier macro­moleculeis involved in axon adhesion.



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Fig. 3. Effect of anti LeX antibodyon neurite outgrowth. Anti LeX (E, F) or Lea (G) or no(H) antibody was added to the culture of siblingst.44/45 brain. Comparing before (AD) and 24 h after (EH) the addition of antibodies, inhibition of neuriteoutgrowth was observed only in the culture with anti LeX antibody. Magnification,200x.

 
Lex in adult tissues
In adult Xenopus, brain, retina, and gut epitheliumexpressed Lex (Table Go). Lex expressionwas also seen in the adult spinal cord, though it was negative atthe tadpole stages as discussed above. This also suggests that expressionproceeds from anterior to posterior during development. Low levelsof expression were also seen in the spleen. Most extensive expressionwas found in the adult testis. During spermatogenesis, Lex expressionwas only detected on differentiated cells, i.e., secondary spermatocyte,spermatid, and mature sperm (Figure 4A)while Lea showed no expression in testis. In contrast,in ovary there was no expression of Lex either on oocytesor follicle cells (data not shown).



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Fig. 4. Lex expression inadult testis (A). Lex was expressedon differentiated cells such as secondary spermatocyte (s), spermatid(t), or mature sperm (m), but not on undifferentiated spermatogonia(g) or primary spermatocyte (p), while Lea showed noexpression in testis (B). Magnification 100x.

 
A similar pattern of expression in testis was obtained using M4Bantibodies (data not shown). 10GoFenderson et al. (1984) studied the expression ofcarbohydrates in mouse testis. They showed three monoclonal antibodiesagainst carbohydrates bind to early and late pachytene spermatocyteand round spermatids. One of the antibodies later revealed to beagainst Lex. It is not clear whether these various surfacecarbohydrates have functions in gametogenesis or in fertilization.On the other hand, an cell adhesion molecule called basigin, whichwas isolated as Lex carrier protein in teratocarcinomacells (27GoMiyauchi et al.,1990) exists in mouse testis and is expressed by differentiatedcells after spermatocytes (Maekawa et al., personalcommunication). Basigin is demonstrated to be involved in spermatogenesisusing knockout mouse. Furthermore, 8GoD'Cruz et al. (1997) showed CD15/Lex isexpressed at the acrosomal region on the surface of human spermhead and that antibody against CD15 disturbed sperm–egginteractions. Thus, Lex or its carrier molecule couldbe involved in cell recognition at fertilization.

Molecular carrier of Lex
Whole proteins from adult liver and testis, or tadpole brainand gut, were separated by SDS–PAGE, and Western blottedusing anti-Lex and Lea antibodies. A bandof ~200 kDa, which reacted to anti-Lex antibodies, wasfound in tadpole brain (Figure 5). A wideband of ~220 kDa in tadpole gut and a weak band around 205 kDa inadult testis were detected in the blot with anti-Lex antibodies.No reaction was seen with the anti-Lea antibody. However,this antibody was active, as shown by the reaction with proteinextracted from F9 cells, used as a positive control. Some nonspecificcross-reactions were seen with proteins less than 130 kDa. Thesewere also seen in controls lacking primary antibodies (not shown).We tried thin layer chromatography to see if there are also glycolipidcarriers of Lex in tadpole. The result was negative (datanot shown). Thus, the major carrier of Lex in tadpolebrain is considered to be a 200 kDa protein. Whether Lex antigenin 200 kDa glycoprotein is involved in O-linked or N-linked glycanis under investigation.



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Fig. 5. Western blot analysis for Lex carrierprotein. Proteins from various tissues were separated, blotted,and detected by anti Lex antibody (left) or anti-Lea antibody(right). Te, Adult testis; Td, tadpole; Br, brain; Gu, gut; Lv,adult liver; F9, murine teratocarcinoma stem cell line as positivecontrol *, As F9 was strongly reacted to anti Lex antibody,shorter exposure was used for this lane to show the bands clearly.Approximately 200 kDa and 220 kDa glycoprotein bands reacting toanti-Lex antibody was detected in tadpole brain and gutrespectively. Weak band reacting to Lex was detectedin adult testis. These bands were not reacted to anti Lea antibody.The band above the 116 kDa marker (approximate size 130 kDa) whichwas seen in every lane seemed to be nonspecific as it was also seenin the blot without primary antibody.

 
Expression patterns of other Lewis group antigens
The expression of other Lewis group antigens (LeY,Lea, Leb, sialyl-Lex, and sialyl-Lea)was also examined. None of them were expressed at blastula stage(data not shown).

At tadpole stage (st.45), most of them except sialyl-Lex did notshow any remarkable results though Leb was expressedon some of the coils of gut and LeY was expressed weaklyon some cells of pharynx. In contrast, sialyl-Lex, whichis one of the major ligands for selectins, was expressed on endothelial cells,epithelial cells in lung, liver, and kidney, and also on cells inthe gut and mesentery. Especially at the mesonephric region, cellsin the area surrounded by the dorsal aorta, the vena cava, and theWolffian ducts showed extensive staining (Figure 6A). Cells exist in this area were shown tobe leukocyte (30GoOhinata et al.,1989). Sialyl-Lex was also expressed on ependymalcells (Figure 6B) and lateral line cells.Sialyl-Lea was expressed weakly at the ventrolateralregion of forebrain at st.45. At later tadpole stages (st.47, 48,53), similar expression patterns of Lewis group carbohydrates wereobtained though Lea was expressed at pharynx and theanterior part of the gut. In adult testis, Lea, Leb,and sialyl-Lea were negative while LeY wasweakly expressed on some differentiated sperm. Sialyl-Lex wasexpressed on endothelial cells.



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Fig. 6. Sialyl-Lex expressionin st.45 tadpole. Sialyl-LeX was expressed on endothelialcells (arrow), cells in the gut and mesentery, leukocytes (arrowhead)at the mesonephric region (A), and ependymal cellsin hindbrain (B). D, dorsal aorta; V, vena cava;W, Wolffian ducts; G, gut; M, mesentery; N, notochord; B, brain;Mu, muscle. Magnification, 100x.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We report here the pattern of expression of the Lewis group carbohydrate antigensduring Xenopus development. Though 39GoSlack et al. (1985) investigated the expressionof several glycoconjugates in early embryos, this is the first reportto show systematically the expression of an carbohydrate duringdevelopment of Xenopus. It provides a clue to studythe role of carbohydrate in development. Unlike mice, expressionof Lex was not seen in early embryos but starts at thelater tail bud stage and proceeds from anterior to posterior ofthe body. We previously found that the M4B glycolipid antigen isexpressed instead at the earlier developmental stages. Since M4Bis expressed on primordial germ cells and early blastomeres in Xenopus and anti-M4B shows an inhibitory effecton blastomere adhesion (46GoTurner et al., 1992), it may be M4B, which performs a functioncorresponding to SSEA-1 in mouse. Presumably Lex in Xenopus has different role from SSEA-1.

We also show specific patterns of expression of Lex inneuronal tissues. In tadpole brain, Lex expression wasfound on axons and specific neuronal cell bodies. One of the candidate forLex carrier in tadpole brain was 200 kDa glycoprotein. 48GoYamamoto et al. (1985) andSchwarting and Yamamoto (1988) investigated expression of glycoconjugatesduring development of the murine nervous system. Using monoclonal antibody7A, which recognizes Lex, they studied expression of thiscarbohydrate in the central nervous system. As a result, specificregion of embryonic (11d to 17d postcoitum) cerebral cortex expressedthis carbohydrate. Culture experiments revealed that it existedon cell membrane. Lex expressed on the proliferatingcell, which is a precursor of neuron and glial cells, but not onthe migrating or later stage cells. Besides mice, it exists on rat,rabbit, bovine, and human embryonic brain. They showed that theLex antigen is carried on membrane glycolipids in theembryonic cerebral cortex, whereas it is carried preferentiallyon glycoproteins in postnatal brain (48GoYamamoto et al., 1985). Recently, other groups(Satoh and Kim 1994; 7GoDasgupta et al., 1996; 16GoGotz et al., 1996) also studied Lex or sialyl-Lex expressionin human, mouse, chick, rat, and bovine embryonic brain, and discussedthe roles of these carbohydrates in cell–cell interactionor recognition of neuronal cells and development of central nervoussystem. Though these groups suggested the important roles of Lex innervous system, so far there have been little functional experimentsto demonstrate its direct contribution to cell adhesion or axonalguidance.

To examine the function of Lex during developmentof nervous system, we tried explant culture experiments. Anti-Lex antibodyreacted with the surfaces of cell bodies and axons in the culturedtadpole brain explants. Addition of anti-Lex to the cultureperturbed neurite outgrowth while addition of anti-Lea, whichdoes not react in tissue sections, showed no effect. This is thefirst evidence to suggest that Lex may play a role inneurite adhesiveness. We do not know yet whether cell–cellor cell–substrate adhesiveness is being affected. SinceLex could function via carbohydrate–carbohydrateor carbohydrate–lectin interactions, we tried to add Lex carbohydrateto the culture. It showed no promotory or inhibitory effect on neuriteextension. Therefore it has not clarified that Lex determinantitself provides an active site for adhesion or carrier moleculeof Lex is involved in cell adhesion. However, as 44GoSudou et al. (1995) reportedthat transfection of fucosyltransferase to L cell leads expressionof Lex and as a result cell–substrate adhesionpromoted, there is a possibility that Lex itself is involvedin adhesion to substrate and extension of axon. On the other hand,as in the case of E-selectin, not only ligand carbohydrate but also apart of carrier protein is necessary for recognition and binding.More precise molecular study is necessary for understanding thedeterminant. Identification of 200 kDa carrier protein and Lex receptorprotein in brain may help it. As so far Xenopus selectinis not expressed on neuronal cells (Yoshida-Noro etal., unpublished observations), it is unlikely that receptor proteinis selectin.

We studied the expression of other Lewis group carbohydratesand found that sialyl-Lex showed interesting patternof expression. It was not expressed in early embryo. In tadpole,it is expressed in endothelial cells, cells in gut and gut mesentery, andleukocytes at the mesonephric region suggesting the possible roleas a selectin ligand. In normal human tissue, sialyl-Lex isexpressed only in blood system. Most of granulocytes and 6–7% oflymphocytes expressed sialyl-Lex. Expression of sialyl-Lex divergedepends on the type or differentiation stage of human lymphocyte (31GoOhmori et al., 1993).Also in human fetus, it showed temporal expression in epithelialcells in lung, pancreas, and kidney. These results are partly consistentwith the expression patterns in Xenopus. Expressionof sialyl-Lex during chicken B-lymphocyte developmentwas studied by 26GoMasteller etal. (1995). They showed that preB cell expresses sialyl-Lex butafter reaching bursa of Fabricius, cells ceased the expression ofsialyl-Lex and instead began to express Lex.It is interesting that the expression of this carbohydrate changed duringdifferentiation. The paper does not mentioned about the expressionin blood vessel though there is a description of expression in medullaof bursa. In our study, at st.45 Xenopus tadpole,though lymphocyte has not well differentiated at this stage, expressionof sialyl-Lex in leukocytes was observed. To see if sialyl-Lex isa ligand for Xenopus selectin, we are trying bindingassay using recombinant protein. We are also trying to identifysialyl- Lex carrier protein in Xenopus.

These results show the expression patterns of Lewis group carbohydratesduring development and suggest the role of Lex in neuronalcell adhesion. Further study is necessary for clarifying their precisefunctions. It is useful to compare their expression in differentspecies to examine their conserved roles and species specific roles.These study leads to find a clue to investigate the roles of carbohydratesin cell–cell interaction, cell recognition and cell adhesion.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Materials
Xenopus embryos and tadpoles were obtained byin vitro fertilization (46GoTurner et al., 1992) and staged according to Nieuwkoop andFaber (1967).

Anti Lex/SSEA-1 monoclonal antibody TG-1(4GoBeverley et al., 1980)was obtained as serum-free culture supernatant by Dr. Miranda Gompertsin our laboratory. Monoclonal antibodies against Lex,Lewis Y (LeY), Lewis a (Lea), Lewis b (Leb) werepurchased from Signet Laboratories Inc. (USA). Monoclonal anti-sialyl-Lea waspurchased from Seikagaku Corp. (Japan). Monoclonal anti-sialyl-Lex (FH-6)antibodies were gifts from Dr. Reiji Kannagi (Aichi Cancer Center,Japan). HNK-1 (Sigma) antibody was a gift from Dr. Colin Sharpein our laboratory.

Histology and immunostaining
Embryos or tissues were fixed in either 2% (w/v)TCA (trichloroacetate) or Bouin’s solution (0.9% picricacid, 9% formaldehyde and 5% acetic acid, Sigma)overnight, embedded in polyethylene glycol 400 diesterate (PEDS,Koch Chemicals Ltd.) wax containing 1% (w/v) cetylalcohol and sectioned at 10 µm.

Immunohistological staining was carried out at room temperatureusing VECTASTAIN ABC kit (DAKO). Sections were incubated with eitherserum-free culture supernatant of TG-1 hybridoma or 1/50dilution of anti-Lex, LeY, Lea,Leb (original concentration; 0.125–0.5 mg/ml)for 60 min after blocking of nonspecific binding using 10% goatserum. Sections were washed with phosphate-buffered saline (PBS)for 30 min, incubated in biotinylated second antibody (original0.5 mg/ml, 1/200 dilution in PBS, ABC kit) for30 min, washed again for 30 min with PBS, and then incubated inavidin–alkaline phosphatase complex in PBS (1/100dilution, ABC kit) for 30 min. After a final 30 min wash with PBS,color reactions were carried out using X-phosphate (5-bromo-4-chloro-3-indolyl-phosphateat 50 mg/ml in 70% dimethylformamide in the stocksolution) and NBT (4-nitroblue-tetrazolium-chloride at 75 mg/mlin dimethylformamide in the stock solution) as substrates for alkalinephosphatase. 35 µl of X-phosphate solutionand 45 µl of NBT solution were addedto 10 ml of reaction buffer (100 mM Tris–HCl, pH 9.5, 100mM NaCl, 50 mM MgCl2).

Cell culture
Small pieces of brain were dissected from stage 32 tail bud embryosand cultured on laminin-coated plastic dishes in modified L-15 medium(60% L-15, 10% fetal calf serum supplemented withpenicillin and streptomycin) for 2 days at 20°C untilsibling stage 44/45 tadpole. The culture was photographedand the medium was then changed to new medium with or without antibodies(5 µg/ml). The cultures wereincubated for another 24 h (sibling stage 46/47) and thenphotographed. For antibody staining, cultured tissues were fixedin MEMFA buffer (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4,and3.7% formaldehyde; Harland 1991).

Western blotting and thin layer chromatography
Western blotting was carried out according to 45GoTowbinet al. (1979) with some modifications includinguse of semidry blotting system (ATTO, JAPAN) (Kyhse-Andersen 1984).All procedures were performed according to the ECL system manual(Amersham) at room temperature. Blots were incubated with 3% (w/v)bovine serum albumin (BSA), 0.1% (v/v) Tween 20in PBS (T-PBS) for more than 2 h to block nonspecific binding. Thenthe blots were incubated with the primary antibodies (~10 µg/ml)in T-PBS for overnight. Following the 30 min washingby T-PBS, blots were incubated with the second antibodies (anti-mouseIgM biotinylated; VECTOR, 2.5 µg/ml)for 1 h. After 30 min washing in T-PBS, blots were incubated withstreptavidin-biotinylated horseradish peroxidase complex (Amersham,1/5000 dilution) in T-PBS. Detection was performed after30 min washing by T-PBS, using ECL western blotting detection reagents(Amersham) and blots were exposed to x-ray films (Fuji, Japan).Crude lipid extraction and high performance thin layer chromatography(HP-TLC) were carried out according to 46GoTurner et al. (1992). For the detection of antigenson the Western blots or TLC plates, blocking for the nonspecificbinding was performed by using 3% BSA instead of nonfatmilk powder in PBS because Lewis group antigen carbohydrates areknown to exist in milk.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
We thank Prof. Hajime Fujisawa in Nagoya University and Prof.Shin-ichi Abe for their useful histological comments. We thank Dr.Reiji Kannagi in Aichi Cancer Center for anti sialy-Lex antibody.This work was supported by grants from Wellcome Trust, ERATO JSTand PRESTO JST.


    Footnotes
 
a Towhom correspondence should be addressed at: Division of Experimental AnimalResearch, Life Science Tsukuba Research Center, The Institute of Physicaland Chemical Research (RIKEN), Tsukuba, Ibaraki 305-0074, Japan Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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3 Andrews,P.W.,Nudelman,E., Hakomori,S. and Fenderson,B.A. (1990)Different patterns of glycolipid antigens are expressed followingdifferentiation of TERA-2 human embryonal carcinoma cells inducedby retinoic acid, hexamethylene bisacetamide (HMBA) or bromodeoxyuridine(BUdR). Differentiation, 43, 131–138.[ISI][Medline]

4 Beverley,P.C.,Linch,D. and Delia,D. (1980) Isolation of human haematopoieticprogenitor cells using monoclonal antibodies. Nature, 287, 332–333.[ISI][Medline]

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