Saccharide structures of the mouse embryo during the ... - Development

J. Embryol. exp. Morph. 90, 335-361 (1985)

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Saccharide structures of the mouse embryo during the first eight days of development Inferences from immunocytochemical studies using monoclonal antibodies in conjunction with glycosidases J. E. PENNINGTON, Applied Immunochemistry Research Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, U.K.

S. RASTAN, Comparative Medicine Division, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, U.K.

D.ROELCKE Institute for Immunology and Serology, University of Heidelberg, Heidelberg 1, Federal Republic of Germany ANDT.

FEIZI

Applied Immunochemistry Research Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ, U.K.

SUMMARY

Monoclonal anti-carbohydrate antibodies have been used in conjunction with glycosidases in immunofluorescence studies to derive information about the structures and in situ distribution of saccharides of the mouse embryo during the first 8 days of development. The salient findings are as follows: (a) Branched poly-N-acetyllactosamine sequences of L-antigen type are detectable from the first day onwards and are widely distributed in cells of the endoderm, ectoderm and mesoderm. (b) Linear poly-N-acetyllactosamine sequences of i-antigen type are detectable from the fifth day onwards in cells of all three lineages, but have a more restricted distribution than the sequences of I-type. (c) Poly-N-acetyllactosamine sequences that are susceptible to digestion with endo-/3galactosidase are the main carriers of the SSEA-1, C14 and the blood group B-like antigens, which have the following structures Galj81-4GlcNAc, I 1,3 Fucor

Gal/31-4GlcNAc and Galarl-3Gal, I 1,2 I 1,3 Fucor Fuca

respectively

and are found in endoderm and ectoderm but not in mesoderm cells. In the trophoblast however, these antigens are borne on saccharides that are resistant to endo-j8-galactosidase. Key words: carbohydrate structures, carbohydrate antigens, embryonic antigens, differentiation antigens, mouse embryo, saccharides, immunocytochemistry, monoclonal antibodies, glycosidases.

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J. E . P E N N I N G T O N , S. R A S T A N , D . R O E L C K E AND T.

FEIZI

(d) A proportion of the poly-N-acetyllactosamine structures in the endoderm and the ectoderm of the 5- and 6-day embryos may contain the following novel structures: Galarl-Gal/31-4GlcNAc and Galorl-Galj81-4GlcNAc I 1,3 | 1,2 | 1,3 Fucar Fuca Fucar in which antigenicities of SSEA-1 and C14 determinants are masked. (e) There are several types of sialyl-oligosaccharides: (1) those reactive with anti-Gd, which has a specificity for NeuAccv2-3Galj31-4GlcNAc sequence in the extraembryonic mesoderm and the heart; (2) those reactive with anti-Pr2 but not with anti-Gd, which may correspond to other N-acetylneuraminic acid containing sequences such as NeuAcar2-3Gal/31-3GalNAc or NeuAca2-6Gal in preimplantation embryos and in the yolk sac, neural ectoderm and mesenchyme of the 8-day embryo; (3) those with other sialic acid forms or linkages that do not react with anti-Gd and Pr2; among these are sialosyl-i sequences in the extraembryonic ectoderm, and sialosyl-I sequences in most cell types during the first 8 days. The latter are the main poly-N-acetyllactosamine structures in the neural ectoderm of the 8-day embryo. (f) The sequence Galj31-3GlcNAc/31-3GaljSl-4Glc/GlcNAc, or cross-reactive structures, which bind FC10.2 antibody occur in the extraembryonic endoderm and yolk sac. The roles of specific carbohydrate structures as receptors during embryonic development and cell growth are important topics of current research. The cytochemical approach with monoclonal antibodies in conjunction with glycosidases, as in the present study, provides a unique opportunity to visualize the in situ disposition of specific carbohydrate sequences in individual cells of the whole organism, and should facilitate systematic investigations of their functions.

INTRODUCTION

Early mouse embryos and embryonal carcinoma cells are rich in glycans with poly-N-acetyllactosamine (Gal/31-4GlcNAc/31-3)n sequences (Muramatsu et al. 1978). Immunocytochemical studies with the natural monoclonal antibodies, anti-I and anti-i have shown that in the developing mouse embryo and in embryonal carcinoma cells which are induced to differentiate in vitro, stage-specific changes occur in the branching patterns of these saccharides (Kapadia, Feizi & Evans, 1981; Feizi, Kapadia, Gooi & Evans, 1982). Branched type 2 backbone structures (I-antigen type) are detectable at the zygote stage and throughout the first 6 days of gestation, while linear structures of i-antigen type are first detectable at the onset of differentiation of primary endoderm cells. The hybridoma-defined stagespecific embryonic antigen, SSEA-1 (Solter & Knowles, 1978) consists of 3fucosyl-N-acetyllactosamine (Gooi et al. 1981; Hounsell, Gooi & Feizi, 1981). All three antigens (I, i and SSEA-1) are masked in the presence of the blood group Hassociated arl-2 linked fucose (Feizi et al. 1971; Gooi et al. 1981; Gooi et al. 1983b) or in the presence of sialic acid (Gooi et al. 1983b). Thus some of the sequential changes in antigenicity that occur during embryogenesis may result from changes in glycosylation of glycoproteins and. glycolipids (Feizi, 1981a), analogous to the developmentally regulated changes that occur on human erythrocytes (Feizi, 1981b; Hakomori, 1981). Several other hybridoma-defined saccharide antigens of the blood group series change during the development of mouse embryos (Blaineau et al. 1983) or the differentiation of human embryonal carcinoma cells (Gooi et al. 1983d), as do the major blood group antigens A, B and H in various

Saccharide structures of the mouse embryo

2>2>1

organs of the human foetus (Szulman, 1980), and the B-like structures reactive with B. simplicifolia lectin in the mouse embryo (Wu, Wan & Damjanov, 1983). However, such developmental changes are not confined to the blood group family. Studies with monoclonal antibodies have shown that during mouse embryonic development, changes also occur on carbohydrate chains of glycolipids of the globo series which express the Forssman (Stern et al. 1978; Willison et al. 1982), SSEA-3 and SSEA-4 (Kannagi et al. 1983a,b) antigens. Thus, monoclonal antibodies have been invaluable in providing structural information on the surface carbohydrates of embryonic cells which cannot be readily characterized structurally on account of their limited amounts and marked heterogeneity. In view of the association of several of the carbohydrate differentiation antigens with receptor systems (reviewed by Feizi & Childs, 1985), it is of considerable interest to visualize their in situ disposition in individual cells and tissues during embryogenesis. In the present studies several hybridoma antibodies against blood group related oligosaccharides have been used in conjunction with monoclonal human autoantibodies anti-I, i and glycosidases to derive new information on the backbone and peripheral regions of saccharides of the cells of pre- and postimplantation mouse embryos up to the eighth day of development. In addition, two human monoclonal autoantibodies recognizing N-acetylneuraminic-acid-containing structures have provided information on changes in sialylation during development. MATERIALS AND METHODS

Antibodies Four monoclonal antibodies with anti-I or I-like specificities were used (Table 1). Anti-I Ma, anti-I Step and anti-i Den are human antibodies (Feizi, 198LZ?) and the anti-I-like antibodies M39 and M18 (Gooi et al. 1983c) are mouse hybridoma antibodies [Foster, Edwards, Dinsdale & Neville (1982) gifts of Dr P. A. W. Edwards, Ludwig Institute for Cancer Research, Sutton, U.K.]. IgM w o ° (gift of Dr E. Osserman, Columbia Medical Center, New York), is a Waldenstrom macroglobulin with specificity for the type 1 (Gal/81-3GlcNAc)-based backbone sequence (Kabat, Liao, Shyong & Osserman, 1982). Anti-Pr2 (designated LTh) and anti-Gd (designated Kn) are human monoclonal antibodies which recognize the NeuAc-Gal sequence (Table 1). The former reacts with both the