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.
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Abstract |
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Introduction |
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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 (14) [Fuc (13)] GlcNAc(15
Gooi 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(40
Solter and Knowles, 1978). Itis expressed on the surfaces of undifferentiated but not differentiated,teratocarcinoma cells (41
Solter and Knowles,1979; 20
Knowles 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 (12
Fenderson et al., 1990; 14
Gomperts et al., 1994).
The biological functions of Lex/SSEA-1 havebeen examined by several groups using either monoclonal antibodiesor purified carbohydrates. 5Bird and Kimber (1984) found that LNFIII inhibited the compaction ofmouse morulae. 9
Eggens et al.(1989) or 21
Kojima 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 (19
Kannagi et al., 1982; 6
Childs et al., 1983; 32
Ozawa et al., 1985; 9
Eggenset al., 1989; Rosenman et al.,1989; 43
Streit et al., 1996).
Recently, some of the Lewis group carbohydrates have been identifiedas potential ligands for selectins (47Varki,1994, for review). Selectins are a class of transmembranecellcell 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 (23
Larsen 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 (24
Leshko-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 (46Turner 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; 13Fox et al., 1981: embryonicbrain; 48
Yamamoto et al.,1985), humans (embryonal carcinoma cells; 2
Andrews et al., 1989, 1990: embryonic brain; 36
Schwarting et al., 1989; 34
Satoh and Kim, 1994: embryonic spinalcord; 1
Aloisi et al., 1992:lens; 29
Ogiso et al., 1992:blood cells; 19
Kannagi et al.,1982; 25
Macher and Beckstead, 1990),rats (primary sensory neuron; 18
Jessell andDodd, 1986), bovine (brain; 7
Dasgupta et al., 1996), chicken (embryonic brain; 43
Streit et al., 1996: Bcell; 26
Masteller et al.,1995), quails (spinal sensory neuron lineage; 37
Sieber-Blum, 1989ab; 33
Racheland 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.
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Results |
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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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Molecular carrier of Lex
Whole proteins from adult liver and testis, or tadpole brainand gut, were separated by SDSPAGE, 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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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 (30Ohinata 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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Discussion |
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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. 48Yamamoto 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 (48
Yamamoto et al., 1985). Recently, other groups(Satoh and Kim 1994; 7
Dasgupta et al., 1996; 16
Gotz 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 cellcell 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 cellcellor cellsubstrate adhesiveness is being affected. SinceLex could function via carbohydratecarbohydrateor carbohydratelectin 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 44Sudou et al. (1995) reportedthat transfection of fucosyltransferase to L cell leads expressionof Lex and as a result cellsubstrate 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 67% oflymphocytes expressed sialyl-Lex. Expression of sialyl-Lex divergedepends on the type or differentiation stage of human lymphocyte (31Ohmori 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 26
Masteller 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 cellcell interaction, cell recognition and cell adhesion.
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Materials and methods |
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Anti Lex/SSEA-1 monoclonal antibody TG-1(4Beverley 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 Bouins 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; 0.1250.5 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 inavidinalkaline 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 TrisHCl, 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 45Towbinet 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 46
Turner 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.
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Footnotes |
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References |
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