Cell, Vol. 14, 785-793,
August
1978, Copyright
0 1978 by MIT
Expression of a Forssman Antigenic Specificity in the Preimplantation Mouse Embryo Keith R. Willison and Peter L. Stern MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England
Summary A monoclonal antibody recognizing a Forssman antigenic specificity has been shown to react with cells of the preimplantation mouse embryo. The antigen is believed to be carried on glycolipid molecules on teratocarcinoma stem cells. This antigen is first detected on the trophectoderm of the early blastocyst. The topography of the expression on the trophectoderm is striking and novel. The antigen is no longer found on these cells after the blastocyst has hatched from the zona pellucida in utero. Inner cell masses are antigen-positive at all times. This is the first study of the distribution of a single antigenic determinant in early mouse embryogenesis. Introduction It seems probable that the cell surface has an important role in development (Bennett, Boyse and Old, 1971); the generation of cell surface diversity may allow differential recognition of interacting cells and may therefore be a key factor in morphogenesis. Immunological approaches have provided powerful tools to investigate this idea. A variety of immunization procedures, using a number of species and immunogens, have produced antisera which define antigens that are expressed by particular cell types in the early mouse embryo (Solter and Schachner, 1975; Erickson, 1977; Jacob, 1977). Some of the best studied antisera have been raised in mice and rabbits using murine teratocarcinoma cells (Edidin et al., 1971; Artzt et al., 1973; Gooding, Hsu and Edidin, 1976; Gachelin et al., 1977). The temporal and cellular specificity of the expression of these various antigens differs considerably. F9 antigen(s) (Artzt et al., 1973) is first detected on the fertilized egg and increases to a maximum on the morula; it remains expressed, at a lower level, on the trophectoderm and inner cell mass (ICM) of the blastocyst (Jacob, 1977). Antigen(s) II (Gooding et al., 1976) first appears on the trophectoderm and ICM cells. PCC4 (Gachelin et al., 1977) is only detected on the ICM of the implanting blastocyst. Even with syngeneic immunization (F9 and PCC4), massive absorptions are required to remove uninteresting antibody specificities. In addition, normal mouse serum is known to contain naturally occurring antibodiesfor example, to embryonal carcinoma cells (Artzt
and Jacob, 1974)-and there is the possibility of cross-reaction of such antibody specificities on embryo cells. This problem, and the complexity of the antibodies evoked by immunization, make it very difficult to monitor the expression of a single molecule on the surface of the cells of the mouse embryo. In the accompanying paper (Stern et al., 1978), we describe a monoclonal antibody which recognizes a Forssman antigenic specificity. This antigenie determinant is expressed by certain cells in brain, kidney, testes and some lymphoid tissues, although not in liver or thymus. It is not expressed by a variety of mouse tumor cell types with the exception of embryonal carcinoma stem cells. As with other antisera to embryonal carcinoma, the M1122.25 antigen is expressed by cells of the mouse embryo. The availability of monoclonal antibodies has allowed a description of the expression of a single defined antigenic determinant during mouse preimplantation development. Using indirect immunofluorescence, this antigen is first shown to be expressed by cells of the morulaearly blastocyst stage. The trophectodermal cells show novel patterns of expression. The antigen is no longer detected on the trophectodermal cells of the blastocyst when it has hatched from its zona pellucida in utero. ICMs isolated from both early and late blastocysts are positive. Results Expression on the Trophectoderm Embryos at stages of development from the unfertilized egg to the hatched blastocyst were assayed for the presence of the antigen recognized by Ml/ 22.25 monoclonal antibodies using indirect immunofluorescence with rabbit anti-rat immunoglobulin conjugated to fluorescein (R.anti-rat.lg-Fl). Morulae and blastocysts were obtained either by culturing from 2- or 8-cell stages, or by flushing from the uterus. The antigen was not detected on unfertilized or fertilized eggs, or 2-, 4- and 8-cell embryos. A proportion of the embryos were labeled at the morula stage (Table 1). The embryos were generally those which had begun to make a blastocoel. This is illustrated by Figures 1A and 1 B. The expression of Forssman antigen increases to a maximum on the trophectoderm of the early blastocyst (for example, in Figures 1C and 1D). The percentage of embryos labeled also increases at the blastocyst stage (Table 1). Smaller proportions, however, are positive in the late stages or hatched from the zona pellucida stages shown in Table 1. In vivo hatched blastocysts flushed from the uterus just before implantation lack the antigen on their trophectoderm or express it very weakly (see Fig-
Cell 706
Table
1. Expression
of a Forssman
Specificity Number
in Preimplantation of Embryos”
Mouse
Labeled
Embryos
by Indirect
lmmunofluorescence
Monoclonal Antibody
Unfertilized Egg
Fertilized Egg
2-Cell
4-Cell
E-Cell
Morulae
Early Blastocystsb
Late Blastocysts’
Hatched Blastocystsd
Isolated ICM
ICM on Outgrowths
Ml 122.25
o/9
O/18
O/58
O/57
017.5
19/79
72192
45177
31167
46148
21121
0%
0%
0%
0%
0%
24%
78%
58%
46%
100%
100%
o/9
o/10
NT’
O/6
NT
o/15
0125
o/37
O/16
O/21
NT
0%
0%
0%
0%
Mll9.3
0%
a Data are pooled from over 700 embryos. Later stages b 3-3.5 day old embryos. c 3.5-4 day old embryos. * 4-5 day old embryos (or their in vitro equivalents). c NT = not tested.
0% were
obtained
ures 1 E and 1F). The specificity of labeling with M1122.25 culture supernatant on the embryos is shown by controls using another monoclonal antibody M119.3 (Table 1). This recognizes an antigen on mouse lymphocytes (Springer et al., 1978); no labeling is seen on any embryo stage tested. In many embryos, the antigen is not detected on all the cells of the trophectoderm of the expanded blastocyst, even when expression appears maximal. Two examples of blastocysts showing this differential distribution are given in Figure 2. There are groups of negative cells surrounded by other brightly staining trophectodermal cells. No consistent topography of positive and negative cells was seen with respect to the position of the inner cell mass. In some embryos, only a single cell was seen to be fluorescent. It seemed possible that some of the cells failed to label because the pronase, used to remove the zona pellucida, destroyed the antigen. Since some blastocysts which hatched in vitro with no pronase treatment had both antigen-positive and -negative cells, this is improbable. Furthermore, blastocysts cultured for several hours after zona removal with pronase showed similar patterns of fluorescence. The presence of azide throughout the labeling procedure should prevent pinocytosis or redistribution of the antigen. The lack of labeling is also not due to insufficient antibody; the antibodies were used at a concentration 4 times greater than that which gives plateau labeling with embryonal carcinoma cells. The period of time at which the antigen is expressed was examined more precisely during the Figure All (A) (8) (C) (D) (E) (F)
1. Expression
of a Forssman
Specificity
on Mouse
0% from
in vitro
0% culture
and by flushing.
in vitro development of 8-cell eggs to hatched blastocysts (data not included in Table 1). This is to circumvent the problem of asynchrony in groups of embryos obtained from different females. 8-cell eggs were recovered from several mice, mixed and cultured in groups of 20 in microdrops of Whitten’s (1970) medium. The embryos were assayed for the Forssman specificity over 3.5 days of culture. Scoring the embryos is a complicated procedure because there are variable numbers of positive cells and because the total number of trophectodermal cells increases during the experiment. The following system was devised which divides the labeled embryos into four classes. The first includes embryos with greater than IO positive cells, the second 6-10, the third 1-5, and finally embryos showing no fluorescent labeling. The data from two separate experiments are presented in the form of histograms in Figure 2. During the first day in culture, the embryos reach the morula-early blastocyst stage. At this time, there are few embryos with any positive trophectodermal cells. In the next 24 hr, the percentage of blastocysts with increasing numbers of positive cells rises. At the later time points, there are still embryos with some fluorescent cells. Whitten’s medium may not provide optimal conditions for hatching, since on the average, only 60% escaped from the zona pellucida. Blastocysts were therefore cultured in enriched medium containing serum. Under these conditions, they will hatch and attach to the substratum, and the trophectodermal cells will develop well and maintain many biochemical properties in common with in
Embryos
embryos are labeled with M1122.25 and R.anti-rat.lg-Fl. Magnification -300x Phase-contrast microscopy of four morulae, one with a blastocoel forming (arrow). Fluorescence microscopy; embryo with blastocoel is positive. Phase-contrast microscopy of an expanded blastocyst. Fluorescence microscopy; trophectoderm is strongly positive. Phase-contrast microscopy of two blastocysts hatched from the zona pellucida in utero. Fluorescence microscopy showing that hatched blastocysts are essentially antigen-negative.
Forssman 707
Specificity
in Preimplantation
Mouse
Embryo
Cell
788
Figure
2. Patterns
of Expression
Fluorescence microscopy of two surface. Magnification -460x.
blastocysts
labeled
with
M1122.25
and R.anti-rat.lg-Fl
showing
positive
and negative
cells
on the upper
Forssman
Specificity
in Preimplantation
Experiment
1
Mouse
Embryo
709
cells of the ICM at both 3.5 and 4.5 days are labeled with M1/22.25, but not with M119.3 (Table 1); the fluorescence is extremely bright (Figures 5A and 5B). Individual 3.5 day blastocysts co-express the antigen on the trophectoderm and the ICM. This was demonstrated by labeling the trophectoderm with M1/22.25 and R.anti-rat.lg-Fl. These were examined under the fluorescence microscope, and positive embryos were then transferred to GPC. The trophectodermal cells lyse and the ICM can be isolated (as above). These ICMs were also positive when labeled with M1/22.25 and R.anti-rat.lg-Fl. The trophoblast outgrowth experiments suggest that the ICM is still antigen-positive at implantation. On day 4.5, those cells on the surface of the ICM which are exposed to the blastocoel have formed a layer of primitive endoderm; the rest are becoming embryonic ectoderm. If ICMs are isolated and placed in culture for 2 days, they form a complete layer of endoderm over their surface (Solter and Knowles, 1975; Hogan and Tilley, 1977; Pedersen, Spindle and Wiley, 1977). The outer cells of such ICMs show bright ring fluorescence (Figures 5C and 5D). Thus the Forssman specificity is expressed strongly by the cells of the ICM and on its first differentiated product, endoderm, at least soon after its formation.
100 72hr 50-
55hr
53hr
39hr
Discussion Number of fluorescent
Figure
3. Time
of Appearance
cells per embryo
of Forssman
Hours in culture from the &cell stage both experiments are positive within Solid bars are negative embryos.
Specificity
in Vitro
are shown. All embryos 43 or 39 hr, respectively.
in
vivo trophoblast (Sherman, 1975). These outgrowths were examined for expression of the Forssman specificity, and only one had any positive trophectodermal cells (l/25 outgrowths). In contrast, some of the ICM cells were very brightly stained (Figures 4A and 48). In summary, there appears to be a transient expression of this Forssman specificity on the trophectodermal cells of the preimplantation embryo. It is first detected at the time of blastocoel formation and apparently disappears at the time of implantation. Expression on the Inner Cell Mass The trophectoderm of the blastocyst encloses a fluid-filled cavity called the blastocoel, at one end of which lies the ICM. The ICM consists of the pluripotential cells which will give rise to the embryo proper. ICMs were isolated from 3.5 and 4.5 day blastocysts by immunosurgery (Solter and Knowles, 1975), using a rabbit anti-mouse serum and guinea pig complement (GPC). All the outer
Antisera have been widely used to investigate the role of the cell surface in early mouse embryogenesis (Erickson, 1977; Jacob, 1977). The complexity of conventional antisera, however, makes it difficult to follow the expression of a single antigenic determinant. The availability of monoclonal antibodies recognizing cell surface molecules in the developing embryo will greatly facilitate this approach. The identification of a particular embryonic antigen, whether specific for embryos or not, does not necessarily mean that it is carried by molecules of any functional significance. A description of the temporal and topographical expression of such an antigen, however, is an important prerequisite to the investigation of any functional role. Using the M1/22.25 monoclonal antibodies, we have examined the expression of a single antigenic determinant (a Forssman specificity) during mouse preimplantation development. The antigen is first detected around the time of differentiation of trophectoderm (Burgoyne and Ducibella, 1977). There are interesting patterns of expression, both temporal and spatial. The significance of the observation that some cells express the antigen while others do not is unclear. This could be due to the fact that cells in certain periods of the cell cycle may fail to express surface antigens (Isersky, Metz-
Cdl 790
Figure
4. Trophoblast
Outgrowths
(A) Phase-contrast microscopy showing (8) Fluorescence microscopy showing rat.lg-Fl. Magnification -460x.
an attached blastocyst with that trophoblast is negative,
trophoblast and that
outgrowth and ICM after 3 days in culture. some of the ICM is positive with M1/22.25
and
R.anti-
Forssman 791
Figure (A) (B) (C) (D)
Specificity
5. inner
Phase-contrast Fluorescence Phase-contrast Fluorescence
in Preimplantation
Mouse
Embryo
Cell Masses microscopy microscopy microscopy microscopy
of three isolated ICMs. showing labeling with W/22.25 and Ranti-rat.lg-Fi. of ICM from a chimaeric blastocyst after 43 hr in culture. An endodermal showing endodermal cells labeled with W/22.25 and R.anti-rat.lg-Fl.
layer
is visible.
Cell 792
ger and Buell, 1975). A more interesting possibility is that the negative cells are those first committed to transformation into giant cells (Barlow and Sherman, 1972). The first differentiated product of the ICM, primary endoderm, also expresses this Forssman specificity. This is in contrast to the endoderm formed by teratocarcinoma simple embryoid bodies, which is antigen-negative (Stern et al., 1978). These structures are believed to be analogous to the embryonic portion of the 5 day old embryo (Martin, 1975). This latter endoderm may represent a type of endoderm (parietal) different from that which is present on the blastocoelic surface of the ICM (visceral) (Zetter and Martin, 1978). Since it has been suggested that the surface properties of the trophectoderm may prevent premature implantation of the embryo into the uterine wall, the absence of the antigen on the tropectoderm of the in vitro hatched blastocyst may be relevant (Burgoyne and Ducibella, 1977). H2 antigens are also transiently expressed by the trophectoderm (Searle et al., 1976). Perhaps it is not surprising that surface antigen expression is altered if one considers the extensive alterations in the physiology and membrane properties of the trophoblast. These include increasing polyploidy, the acquisition of phagocytic properties and changes in membrane and cell ultrastructure, and in membrane glycoproteins (Searle et al., 1976; Jenkinson and Searle, 1977). The mechanism by which these antigens disappear is unknown, but the in vitro trophoblast outgrowth experiments suggest that it is not necessarily dependent upon maternal factors. However, in preliminary experiments with uterine blastocysts, prevented from implanting by ovariectomy (Van Blerkom and Brockway, 1975), the Forssman specificity is still expressed at day 6. It is possible that this represents a hormonal influence of the ovary in the uterine environment. There is evidence that the antigenic specificity recognized by M1/22.25 monoclonal antibodies on embryonal carcinoma cells is carried by a glycolipid (Stern et al., 1978). It has been shown that gangliosides will interact with thyrotropin and human chorionic gonadotropin, and it has been suggested that such glycolipids may be part of the membrane receptor for these hormones (Fishman and Brady, 1976). It is possible that the molecule bearing the Forssman specificity on the trophectoderm is involved in the general response of the embryo to hormonal influences (Raff, 1976). The functional significance of the molecule carrying the Forssman determinants in embryogenesis might also be further investigated by attempts to disrupt development in vitro. This approach has been used by Kemler et al. (1977). They have shown
that monovalent rabbit anti-F9 antibodies will reversibly block the development in vitro of the mouse embryo at the 8-cell stage. The interpretation of this result is difficult since there is no definitive evidence that the observed block is due to antibodies direct against F9 antigen(s). The advantage of monoclonal antibody is that precise concentrations of a single antibody can be used and its effect assayed unequivocally. Experimental
Procedures
Mice Eggs and embryos were obtained from Swiss mice bred in the animal house of the Laboratory of Molecular Biology or MFI mice purchased from Olac Southern Ltd. (Oxon). 129/Sv mice were a gift from M. J. Evans. Preparation of Preimpiantation Embryos Swiss and MFI females were superovulated with 7.5 IU of folliclestimulating hormone intraperitoneally (Foiligon Intervet, London) in the evening. 48 hr later, they received an injection of 7.5 IU of human chorionic gonadotrophin (Chorulon Intervet, London) (Gates, 1965). They were then caged with males and examined for vaginal plugs the following morning (day zero of pregnancy). 1297 Sv mice were not superovulated. Unfertilized and fertilized eggs were flushed from oviducts on day zero. 2-4-cell eggs were flushed late on day 1. 6-16-cell morulae were obtained by flushing oviducts and uteri on day 2. Blastocysts were flushed from uteri on days 3 and 4. Unfertilized and recently fertilized eggs were freed of cumulus cells by incubation in 0.5% hyaiuronidase (Sigma) in phosphatebuffered saline (PBS). To remove the zona pellucida. eggs and embryos were incubated in 0.5% pronase (CalbiochemlB grade) in PBS for 6-12 min at room temperature (Mintz, 1962). Eggs were cultured to the biastocyst stage in 50 ~1 microdrops of Whitten’s medium under preequilibrated paraffin oil (Boots Pure Drug Co.) in Nunclon 35 mm plastic tissue culture dishes. Blastocyst outgrowths were produced by culturing blastocysts in RPM1 1640 containing 10% fetal calf serum (Wudl, Sherman and Hillman, 1977). isolation and Culture of inner Ceil Masses ICMs were isolated from early and late blastocysts by immunosurgery (Solter and Knowles, 1975). Blastocysts were incubated in unabsorbed rabbit anti-mouse serum for 30 min at 37°C and then for 30 min in unabsorbed guinea pig complement (GPC) at 3PC. ICMs, isolated from late blastocysts, were made to form a complete layer of endoderm in culture as described by Hogan and Tilley (1977) and Pedersen et al. (1977). indirect immunoftuorescence Staining Embryos and ICMs were washed in Earle’s balanced salt solution (BSS) containing 0.6% bovine serum albumin (BSA), 10 mM HEPES and 0.1% sodium azide. lo-20 embryos were placed in a 10 ~1 drop of M1/22.25 or M179.3 culture supernatant containing 10 mM HEPES, 0.1% Na azide for 30 min at room temperature. They were washed in a large volume of Earie’s medium and then incubated in a 20 ~1 drop of rabbit anti-rat immunoglobulin conjugated to fluorescein (R.anti-rat.lg-Fl; Miles-Yeda), diluted l/5 in Earle’s medium for 30 min at room temperature. For fluorescence microscopy, labeled embryos were placed in a 5 ~1 drop of Earle’s medium on a cavity slide and covered with paraffin oil. Biastocyst outgrowths were stained in situ by covering individual outgrowths with 10 ~1 of antibody for 30 min at 3PC. After washing, 10 ~1 of R.anti-rat.ig-Fl were added for 30 min at 3PC. The plastic dish was cut up and mounted on a microscope slide for fluorescence microscopy. A Zeiss photomicroscope with phase-contrast and incident fluorescence optics
Forssman
Specificity
in Preimplantation
Mouse
Embryo
793
and an Osram HBO-200 mercury lamp was used. Photographs were taken with Kodak Tri-X b/w film or Kodak EktachromeTungsten 50 color film (50 ASA). In some experiments, a Zeiss microscope with inverting phase and epifluorescence was used. Culture Supernatants Clones M1122.25 and Ml/g.3 were grown, and culture supernatants were harvested as described by Stern et al. (1978) and Springer et al. (1978). A single batch of M1122.25 and M119.3 supernatants was used neat in all experiments. Acknowledgments We thank Martin Evans and John Gurdon for useful discussions; Ken Harvey and his colleagues for photographic work; and Miss S. Wilby for typing the manuscript. K.R.W. is an MRC postgraduate scholar. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
March
24.1978
References Artzt, K. and Jacob, F. (1974). H-2 on primitive teratocarcinoma 7 7, 632-634.
Absence cells
of serologically detectable in culture. Transplantation
Artzt. K., Dubois, P.. Bennett, D.. Condamine, Jacob, F. (1973). Surface antigens common embryos and primitive teratocarcinoma cells Acad. Sci. USA 70, 2988-2992.
H.. Babinet, C. and to mouse cleavage in culture. Proc. Nat.
Barlow, P. W. and Sherman, M. I. (1972). The biochemistry of differentiation of mouse trophoblast: studies on polyploidy. J. Embryol. Exp. Morphol. 27, 447-465. Bennett, D.. Boyse. E. A. and Old, L. J. (1971). Cell surface immunogenetics in the study of morphogenesis. In Cell Interactions, L. G. Silvestri. ed. (Amsterdam: North-Holland), pp. 247263. Burgoyne. P. S. and Ducibella, T. (1977). Changes in the properties of the developing trophoblast of preimplantation mouse embryos as revealed by aggregation studies. J. Embryol. Exp. Morphol. 40, 143-157. Edidin, M., Patthey. H. L.. McGuire, E. J. and Sheffield, W. D. (1971). An antiserum to “embryoid body” tumor cells that reacts with normal mouse embryos. In Conference and Workshop on Embryonic and Fetal Antigens in Cancer, N. G. Anderson and J. H. Coggin, Eds. (Springfield: NTIS Conference 71-0527), pp. 239248. Erickson, R. P. (1977). Differentiation and other alloantigens of spermatozoa. In lmmunobiology of Gametes, M. Johnson and M. Edidin. eds. (London: Cambridge University Press), pp. 85-114. Fishman, P. H. and Brady, R. 0. (1976). of gangliosides. Science 794, 906-915.
Biosynthesis
and function
Gachelin, G.. Kemler, R., Kelley, F. and Jacob, F. (1977). PCC4, a new cell surface antigen common to multipotential embryonal carcinoma cells, spermatozoa, and mouse early embryos. Dev. Biol. 57, 199-209. Gates, A. H. (1965). Rate of ovular development as a factor in embryonic survival. In Ciba Foundation Symposium on Preimplantation Stages of Pregnancy, G. E. W. Wolstenholme and M. O’Connor, eds. (London: Churchill), pp. 270-262 Gooding. L. R., Hsu, Y. C. and Edidin, M. (1976). Expression of teratoma-associated antigens on murine ova and early embryos. Dev. Biol. 49, 479-466. Hogan, B. and Tilley, R. (1977). In vitro culture and differentiation of normal mouse blastocysts. Nature 265, 626-629.
Iserksy, C., Metzger, H. and Buell. D. N. (1975). Cell cycle associated changes in receptors for IgE during growth and differentiation of a rat basophilic leukaemia cell line. J. Exp. Med. 141, 1147-1162. Jacob, F. (1977). Mouse teratocarcinoma gens. Immunol. Rev. 33, 3-32. Jenkinson, the mouse 390.
and
embryonic
anti-
E. J. and Searle, R. F. (1977). Cell surface changes on blastocyst at implantation. Exp. Cell Res. 106, 386-
Kemler, R., Babinet, C., Eisen, antigen in early differentiation. 4449-4452.
H. and Jacob, F. (1977). Surface Proc. Nat. Acad. Sci. USA 74.
Martin, G. R. (1975). Teratocarcinomas study of embryogenesis and neoplasia. Mintz, B. (1962). lian egg: removal
Experimental of the zona
as a model system Cell 5, 229-243.
for the
study of the developing mammapellucida. Science 736, 594-595.
Pedersen. R. A., Spindle, A. I. and Wiley, L. M. (1977). Regeneration of endoderm by ectoderm isolated from mouse blastocysts. Nature 270, 435-437. Raff, M. C. (1976). 259, 265-266.
Self regulation
of membrane
receptors.
Nature
Searle, R. F.. Sellens, M. H., Elson. J., Jenkinson, E. J. and Billington. W. D. (1976). Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labelling. J. Exp. Med. 143. 348-359. Sherman, blastocysts.
M. I. (1975). The Cell 5, 343-349.
culture
of cells
derived
Solter, D. and Knowles, B. 8. (1975). lmmunosurgery blastocyst. Proc. Nat. Acad. Sci. USA 72, 5099-5102.
from
mouse
of mouse
Solter, D. and Schachner. M. (1975). Brain and sperm cell surface antigen (NS-4) on preimplantation mouse embryos. Dev. Biol. 52, 98-104. Springer, T., Galfre, G., Secher, D. and Milstein, C. (1978). Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur. J. Immunol., in press. Stern, P. L., Willison, Secher, D.. Ziegler, antibodies as probes antigens: a Forssman Cell 14, 775-783.
K. R.. Lennox, E.. Galfri?, G., Milstein. C., A. and Springer, T. (1978). Monoclonal for differentiation and tumor-associated specificity on teratocarcinoma stem cells.
Van Blerkom. J. and Brockway, G. 0. (1975). Qualitative patterns of protein synthesis in the preimplantation mouse embryo. II. During release from facultative delayed implantation. Dev. Biol. 46, 446-451. Whitten, W. K. (1970). Nutrient requirements for the culture preimplantation embryos in vitro. In Advances in Biosciences, G. Raspe, ed. (New York: Pergamon Press), pp. 129-141.
of 6,
Wudl, L. R., Sherman, lethality oft mutations
of
M. I. and in embryos.
Hillman. N. (1977). Nature Nature 270, 137-140.
Zetter. B. R. and Martin, G. R. (1978). molecular weight cell surface glycoprotein tation mouse embryos and teratocarcinoma Acad. Sci. USA, in press.
Expression of a high (LETS) by preimplanstem cells. Proc. Nat.
August
1978, Copyright
0 1978 by MIT
Expression of a Forssman Antigenic Specificity in the Preimplantation Mouse Embryo Keith R. Willison and Peter L. Stern MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH, England
Summary A monoclonal antibody recognizing a Forssman antigenic specificity has been shown to react with cells of the preimplantation mouse embryo. The antigen is believed to be carried on glycolipid molecules on teratocarcinoma stem cells. This antigen is first detected on the trophectoderm of the early blastocyst. The topography of the expression on the trophectoderm is striking and novel. The antigen is no longer found on these cells after the blastocyst has hatched from the zona pellucida in utero. Inner cell masses are antigen-positive at all times. This is the first study of the distribution of a single antigenic determinant in early mouse embryogenesis. Introduction It seems probable that the cell surface has an important role in development (Bennett, Boyse and Old, 1971); the generation of cell surface diversity may allow differential recognition of interacting cells and may therefore be a key factor in morphogenesis. Immunological approaches have provided powerful tools to investigate this idea. A variety of immunization procedures, using a number of species and immunogens, have produced antisera which define antigens that are expressed by particular cell types in the early mouse embryo (Solter and Schachner, 1975; Erickson, 1977; Jacob, 1977). Some of the best studied antisera have been raised in mice and rabbits using murine teratocarcinoma cells (Edidin et al., 1971; Artzt et al., 1973; Gooding, Hsu and Edidin, 1976; Gachelin et al., 1977). The temporal and cellular specificity of the expression of these various antigens differs considerably. F9 antigen(s) (Artzt et al., 1973) is first detected on the fertilized egg and increases to a maximum on the morula; it remains expressed, at a lower level, on the trophectoderm and inner cell mass (ICM) of the blastocyst (Jacob, 1977). Antigen(s) II (Gooding et al., 1976) first appears on the trophectoderm and ICM cells. PCC4 (Gachelin et al., 1977) is only detected on the ICM of the implanting blastocyst. Even with syngeneic immunization (F9 and PCC4), massive absorptions are required to remove uninteresting antibody specificities. In addition, normal mouse serum is known to contain naturally occurring antibodiesfor example, to embryonal carcinoma cells (Artzt
and Jacob, 1974)-and there is the possibility of cross-reaction of such antibody specificities on embryo cells. This problem, and the complexity of the antibodies evoked by immunization, make it very difficult to monitor the expression of a single molecule on the surface of the cells of the mouse embryo. In the accompanying paper (Stern et al., 1978), we describe a monoclonal antibody which recognizes a Forssman antigenic specificity. This antigenie determinant is expressed by certain cells in brain, kidney, testes and some lymphoid tissues, although not in liver or thymus. It is not expressed by a variety of mouse tumor cell types with the exception of embryonal carcinoma stem cells. As with other antisera to embryonal carcinoma, the M1122.25 antigen is expressed by cells of the mouse embryo. The availability of monoclonal antibodies has allowed a description of the expression of a single defined antigenic determinant during mouse preimplantation development. Using indirect immunofluorescence, this antigen is first shown to be expressed by cells of the morulaearly blastocyst stage. The trophectodermal cells show novel patterns of expression. The antigen is no longer detected on the trophectodermal cells of the blastocyst when it has hatched from its zona pellucida in utero. ICMs isolated from both early and late blastocysts are positive. Results Expression on the Trophectoderm Embryos at stages of development from the unfertilized egg to the hatched blastocyst were assayed for the presence of the antigen recognized by Ml/ 22.25 monoclonal antibodies using indirect immunofluorescence with rabbit anti-rat immunoglobulin conjugated to fluorescein (R.anti-rat.lg-Fl). Morulae and blastocysts were obtained either by culturing from 2- or 8-cell stages, or by flushing from the uterus. The antigen was not detected on unfertilized or fertilized eggs, or 2-, 4- and 8-cell embryos. A proportion of the embryos were labeled at the morula stage (Table 1). The embryos were generally those which had begun to make a blastocoel. This is illustrated by Figures 1A and 1 B. The expression of Forssman antigen increases to a maximum on the trophectoderm of the early blastocyst (for example, in Figures 1C and 1D). The percentage of embryos labeled also increases at the blastocyst stage (Table 1). Smaller proportions, however, are positive in the late stages or hatched from the zona pellucida stages shown in Table 1. In vivo hatched blastocysts flushed from the uterus just before implantation lack the antigen on their trophectoderm or express it very weakly (see Fig-
Cell 706
Table
1. Expression
of a Forssman
Specificity Number
in Preimplantation of Embryos”
Mouse
Labeled
Embryos
by Indirect
lmmunofluorescence
Monoclonal Antibody
Unfertilized Egg
Fertilized Egg
2-Cell
4-Cell
E-Cell
Morulae
Early Blastocystsb
Late Blastocysts’
Hatched Blastocystsd
Isolated ICM
ICM on Outgrowths
Ml 122.25
o/9
O/18
O/58
O/57
017.5
19/79
72192
45177
31167
46148
21121
0%
0%
0%
0%
0%
24%
78%
58%
46%
100%
100%
o/9
o/10
NT’
O/6
NT
o/15
0125
o/37
O/16
O/21
NT
0%
0%
0%
0%
Mll9.3
0%
a Data are pooled from over 700 embryos. Later stages b 3-3.5 day old embryos. c 3.5-4 day old embryos. * 4-5 day old embryos (or their in vitro equivalents). c NT = not tested.
0% were
obtained
ures 1 E and 1F). The specificity of labeling with M1122.25 culture supernatant on the embryos is shown by controls using another monoclonal antibody M119.3 (Table 1). This recognizes an antigen on mouse lymphocytes (Springer et al., 1978); no labeling is seen on any embryo stage tested. In many embryos, the antigen is not detected on all the cells of the trophectoderm of the expanded blastocyst, even when expression appears maximal. Two examples of blastocysts showing this differential distribution are given in Figure 2. There are groups of negative cells surrounded by other brightly staining trophectodermal cells. No consistent topography of positive and negative cells was seen with respect to the position of the inner cell mass. In some embryos, only a single cell was seen to be fluorescent. It seemed possible that some of the cells failed to label because the pronase, used to remove the zona pellucida, destroyed the antigen. Since some blastocysts which hatched in vitro with no pronase treatment had both antigen-positive and -negative cells, this is improbable. Furthermore, blastocysts cultured for several hours after zona removal with pronase showed similar patterns of fluorescence. The presence of azide throughout the labeling procedure should prevent pinocytosis or redistribution of the antigen. The lack of labeling is also not due to insufficient antibody; the antibodies were used at a concentration 4 times greater than that which gives plateau labeling with embryonal carcinoma cells. The period of time at which the antigen is expressed was examined more precisely during the Figure All (A) (8) (C) (D) (E) (F)
1. Expression
of a Forssman
Specificity
on Mouse
0% from
in vitro
0% culture
and by flushing.
in vitro development of 8-cell eggs to hatched blastocysts (data not included in Table 1). This is to circumvent the problem of asynchrony in groups of embryos obtained from different females. 8-cell eggs were recovered from several mice, mixed and cultured in groups of 20 in microdrops of Whitten’s (1970) medium. The embryos were assayed for the Forssman specificity over 3.5 days of culture. Scoring the embryos is a complicated procedure because there are variable numbers of positive cells and because the total number of trophectodermal cells increases during the experiment. The following system was devised which divides the labeled embryos into four classes. The first includes embryos with greater than IO positive cells, the second 6-10, the third 1-5, and finally embryos showing no fluorescent labeling. The data from two separate experiments are presented in the form of histograms in Figure 2. During the first day in culture, the embryos reach the morula-early blastocyst stage. At this time, there are few embryos with any positive trophectodermal cells. In the next 24 hr, the percentage of blastocysts with increasing numbers of positive cells rises. At the later time points, there are still embryos with some fluorescent cells. Whitten’s medium may not provide optimal conditions for hatching, since on the average, only 60% escaped from the zona pellucida. Blastocysts were therefore cultured in enriched medium containing serum. Under these conditions, they will hatch and attach to the substratum, and the trophectodermal cells will develop well and maintain many biochemical properties in common with in
Embryos
embryos are labeled with M1122.25 and R.anti-rat.lg-Fl. Magnification -300x Phase-contrast microscopy of four morulae, one with a blastocoel forming (arrow). Fluorescence microscopy; embryo with blastocoel is positive. Phase-contrast microscopy of an expanded blastocyst. Fluorescence microscopy; trophectoderm is strongly positive. Phase-contrast microscopy of two blastocysts hatched from the zona pellucida in utero. Fluorescence microscopy showing that hatched blastocysts are essentially antigen-negative.
Forssman 707
Specificity
in Preimplantation
Mouse
Embryo
Cell
788
Figure
2. Patterns
of Expression
Fluorescence microscopy of two surface. Magnification -460x.
blastocysts
labeled
with
M1122.25
and R.anti-rat.lg-Fl
showing
positive
and negative
cells
on the upper
Forssman
Specificity
in Preimplantation
Experiment
1
Mouse
Embryo
709
cells of the ICM at both 3.5 and 4.5 days are labeled with M1/22.25, but not with M119.3 (Table 1); the fluorescence is extremely bright (Figures 5A and 5B). Individual 3.5 day blastocysts co-express the antigen on the trophectoderm and the ICM. This was demonstrated by labeling the trophectoderm with M1/22.25 and R.anti-rat.lg-Fl. These were examined under the fluorescence microscope, and positive embryos were then transferred to GPC. The trophectodermal cells lyse and the ICM can be isolated (as above). These ICMs were also positive when labeled with M1/22.25 and R.anti-rat.lg-Fl. The trophoblast outgrowth experiments suggest that the ICM is still antigen-positive at implantation. On day 4.5, those cells on the surface of the ICM which are exposed to the blastocoel have formed a layer of primitive endoderm; the rest are becoming embryonic ectoderm. If ICMs are isolated and placed in culture for 2 days, they form a complete layer of endoderm over their surface (Solter and Knowles, 1975; Hogan and Tilley, 1977; Pedersen, Spindle and Wiley, 1977). The outer cells of such ICMs show bright ring fluorescence (Figures 5C and 5D). Thus the Forssman specificity is expressed strongly by the cells of the ICM and on its first differentiated product, endoderm, at least soon after its formation.
100 72hr 50-
55hr
53hr
39hr
Discussion Number of fluorescent
Figure
3. Time
of Appearance
cells per embryo
of Forssman
Hours in culture from the &cell stage both experiments are positive within Solid bars are negative embryos.
Specificity
in Vitro
are shown. All embryos 43 or 39 hr, respectively.
in
vivo trophoblast (Sherman, 1975). These outgrowths were examined for expression of the Forssman specificity, and only one had any positive trophectodermal cells (l/25 outgrowths). In contrast, some of the ICM cells were very brightly stained (Figures 4A and 48). In summary, there appears to be a transient expression of this Forssman specificity on the trophectodermal cells of the preimplantation embryo. It is first detected at the time of blastocoel formation and apparently disappears at the time of implantation. Expression on the Inner Cell Mass The trophectoderm of the blastocyst encloses a fluid-filled cavity called the blastocoel, at one end of which lies the ICM. The ICM consists of the pluripotential cells which will give rise to the embryo proper. ICMs were isolated from 3.5 and 4.5 day blastocysts by immunosurgery (Solter and Knowles, 1975), using a rabbit anti-mouse serum and guinea pig complement (GPC). All the outer
Antisera have been widely used to investigate the role of the cell surface in early mouse embryogenesis (Erickson, 1977; Jacob, 1977). The complexity of conventional antisera, however, makes it difficult to follow the expression of a single antigenic determinant. The availability of monoclonal antibodies recognizing cell surface molecules in the developing embryo will greatly facilitate this approach. The identification of a particular embryonic antigen, whether specific for embryos or not, does not necessarily mean that it is carried by molecules of any functional significance. A description of the temporal and topographical expression of such an antigen, however, is an important prerequisite to the investigation of any functional role. Using the M1/22.25 monoclonal antibodies, we have examined the expression of a single antigenic determinant (a Forssman specificity) during mouse preimplantation development. The antigen is first detected around the time of differentiation of trophectoderm (Burgoyne and Ducibella, 1977). There are interesting patterns of expression, both temporal and spatial. The significance of the observation that some cells express the antigen while others do not is unclear. This could be due to the fact that cells in certain periods of the cell cycle may fail to express surface antigens (Isersky, Metz-
Cdl 790
Figure
4. Trophoblast
Outgrowths
(A) Phase-contrast microscopy showing (8) Fluorescence microscopy showing rat.lg-Fl. Magnification -460x.
an attached blastocyst with that trophoblast is negative,
trophoblast and that
outgrowth and ICM after 3 days in culture. some of the ICM is positive with M1/22.25
and
R.anti-
Forssman 791
Figure (A) (B) (C) (D)
Specificity
5. inner
Phase-contrast Fluorescence Phase-contrast Fluorescence
in Preimplantation
Mouse
Embryo
Cell Masses microscopy microscopy microscopy microscopy
of three isolated ICMs. showing labeling with W/22.25 and Ranti-rat.lg-Fi. of ICM from a chimaeric blastocyst after 43 hr in culture. An endodermal showing endodermal cells labeled with W/22.25 and R.anti-rat.lg-Fl.
layer
is visible.
Cell 792
ger and Buell, 1975). A more interesting possibility is that the negative cells are those first committed to transformation into giant cells (Barlow and Sherman, 1972). The first differentiated product of the ICM, primary endoderm, also expresses this Forssman specificity. This is in contrast to the endoderm formed by teratocarcinoma simple embryoid bodies, which is antigen-negative (Stern et al., 1978). These structures are believed to be analogous to the embryonic portion of the 5 day old embryo (Martin, 1975). This latter endoderm may represent a type of endoderm (parietal) different from that which is present on the blastocoelic surface of the ICM (visceral) (Zetter and Martin, 1978). Since it has been suggested that the surface properties of the trophectoderm may prevent premature implantation of the embryo into the uterine wall, the absence of the antigen on the tropectoderm of the in vitro hatched blastocyst may be relevant (Burgoyne and Ducibella, 1977). H2 antigens are also transiently expressed by the trophectoderm (Searle et al., 1976). Perhaps it is not surprising that surface antigen expression is altered if one considers the extensive alterations in the physiology and membrane properties of the trophoblast. These include increasing polyploidy, the acquisition of phagocytic properties and changes in membrane and cell ultrastructure, and in membrane glycoproteins (Searle et al., 1976; Jenkinson and Searle, 1977). The mechanism by which these antigens disappear is unknown, but the in vitro trophoblast outgrowth experiments suggest that it is not necessarily dependent upon maternal factors. However, in preliminary experiments with uterine blastocysts, prevented from implanting by ovariectomy (Van Blerkom and Brockway, 1975), the Forssman specificity is still expressed at day 6. It is possible that this represents a hormonal influence of the ovary in the uterine environment. There is evidence that the antigenic specificity recognized by M1/22.25 monoclonal antibodies on embryonal carcinoma cells is carried by a glycolipid (Stern et al., 1978). It has been shown that gangliosides will interact with thyrotropin and human chorionic gonadotropin, and it has been suggested that such glycolipids may be part of the membrane receptor for these hormones (Fishman and Brady, 1976). It is possible that the molecule bearing the Forssman specificity on the trophectoderm is involved in the general response of the embryo to hormonal influences (Raff, 1976). The functional significance of the molecule carrying the Forssman determinants in embryogenesis might also be further investigated by attempts to disrupt development in vitro. This approach has been used by Kemler et al. (1977). They have shown
that monovalent rabbit anti-F9 antibodies will reversibly block the development in vitro of the mouse embryo at the 8-cell stage. The interpretation of this result is difficult since there is no definitive evidence that the observed block is due to antibodies direct against F9 antigen(s). The advantage of monoclonal antibody is that precise concentrations of a single antibody can be used and its effect assayed unequivocally. Experimental
Procedures
Mice Eggs and embryos were obtained from Swiss mice bred in the animal house of the Laboratory of Molecular Biology or MFI mice purchased from Olac Southern Ltd. (Oxon). 129/Sv mice were a gift from M. J. Evans. Preparation of Preimpiantation Embryos Swiss and MFI females were superovulated with 7.5 IU of folliclestimulating hormone intraperitoneally (Foiligon Intervet, London) in the evening. 48 hr later, they received an injection of 7.5 IU of human chorionic gonadotrophin (Chorulon Intervet, London) (Gates, 1965). They were then caged with males and examined for vaginal plugs the following morning (day zero of pregnancy). 1297 Sv mice were not superovulated. Unfertilized and fertilized eggs were flushed from oviducts on day zero. 2-4-cell eggs were flushed late on day 1. 6-16-cell morulae were obtained by flushing oviducts and uteri on day 2. Blastocysts were flushed from uteri on days 3 and 4. Unfertilized and recently fertilized eggs were freed of cumulus cells by incubation in 0.5% hyaiuronidase (Sigma) in phosphatebuffered saline (PBS). To remove the zona pellucida. eggs and embryos were incubated in 0.5% pronase (CalbiochemlB grade) in PBS for 6-12 min at room temperature (Mintz, 1962). Eggs were cultured to the biastocyst stage in 50 ~1 microdrops of Whitten’s medium under preequilibrated paraffin oil (Boots Pure Drug Co.) in Nunclon 35 mm plastic tissue culture dishes. Blastocyst outgrowths were produced by culturing blastocysts in RPM1 1640 containing 10% fetal calf serum (Wudl, Sherman and Hillman, 1977). isolation and Culture of inner Ceil Masses ICMs were isolated from early and late blastocysts by immunosurgery (Solter and Knowles, 1975). Blastocysts were incubated in unabsorbed rabbit anti-mouse serum for 30 min at 37°C and then for 30 min in unabsorbed guinea pig complement (GPC) at 3PC. ICMs, isolated from late blastocysts, were made to form a complete layer of endoderm in culture as described by Hogan and Tilley (1977) and Pedersen et al. (1977). indirect immunoftuorescence Staining Embryos and ICMs were washed in Earle’s balanced salt solution (BSS) containing 0.6% bovine serum albumin (BSA), 10 mM HEPES and 0.1% sodium azide. lo-20 embryos were placed in a 10 ~1 drop of M1/22.25 or M179.3 culture supernatant containing 10 mM HEPES, 0.1% Na azide for 30 min at room temperature. They were washed in a large volume of Earie’s medium and then incubated in a 20 ~1 drop of rabbit anti-rat immunoglobulin conjugated to fluorescein (R.anti-rat.lg-Fl; Miles-Yeda), diluted l/5 in Earle’s medium for 30 min at room temperature. For fluorescence microscopy, labeled embryos were placed in a 5 ~1 drop of Earle’s medium on a cavity slide and covered with paraffin oil. Biastocyst outgrowths were stained in situ by covering individual outgrowths with 10 ~1 of antibody for 30 min at 3PC. After washing, 10 ~1 of R.anti-rat.ig-Fl were added for 30 min at 3PC. The plastic dish was cut up and mounted on a microscope slide for fluorescence microscopy. A Zeiss photomicroscope with phase-contrast and incident fluorescence optics
Forssman
Specificity
in Preimplantation
Mouse
Embryo
793
and an Osram HBO-200 mercury lamp was used. Photographs were taken with Kodak Tri-X b/w film or Kodak EktachromeTungsten 50 color film (50 ASA). In some experiments, a Zeiss microscope with inverting phase and epifluorescence was used. Culture Supernatants Clones M1122.25 and Ml/g.3 were grown, and culture supernatants were harvested as described by Stern et al. (1978) and Springer et al. (1978). A single batch of M1122.25 and M119.3 supernatants was used neat in all experiments. Acknowledgments We thank Martin Evans and John Gurdon for useful discussions; Ken Harvey and his colleagues for photographic work; and Miss S. Wilby for typing the manuscript. K.R.W. is an MRC postgraduate scholar. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
March
24.1978
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Iserksy, C., Metzger, H. and Buell. D. N. (1975). Cell cycle associated changes in receptors for IgE during growth and differentiation of a rat basophilic leukaemia cell line. J. Exp. Med. 141, 1147-1162. Jacob, F. (1977). Mouse teratocarcinoma gens. Immunol. Rev. 33, 3-32. Jenkinson, the mouse 390.
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Searle, R. F.. Sellens, M. H., Elson. J., Jenkinson, E. J. and Billington. W. D. (1976). Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labelling. J. Exp. Med. 143. 348-359. Sherman, blastocysts.
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Solter, D. and Schachner. M. (1975). Brain and sperm cell surface antigen (NS-4) on preimplantation mouse embryos. Dev. Biol. 52, 98-104. Springer, T., Galfre, G., Secher, D. and Milstein, C. (1978). Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur. J. Immunol., in press. Stern, P. L., Willison, Secher, D.. Ziegler, antibodies as probes antigens: a Forssman Cell 14, 775-783.
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Van Blerkom. J. and Brockway, G. 0. (1975). Qualitative patterns of protein synthesis in the preimplantation mouse embryo. II. During release from facultative delayed implantation. Dev. Biol. 46, 446-451. Whitten, W. K. (1970). Nutrient requirements for the culture preimplantation embryos in vitro. In Advances in Biosciences, G. Raspe, ed. (New York: Pergamon Press), pp. 129-141.
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Expression of a high (LETS) by preimplanstem cells. Proc. Nat.
