The expression of Forssman antigen on the surface of cells of post-implantation mouse embryos between 5 and days old and of cells of the gonads from days has been followed using the monoclonal antiserum Ml/22.25. In the early post-implantation embryo a lineage-related distribution is found.

The inner cell mass of the blastocyst was previously shown to be Forssman antigen positive and its derivative tissues the epiblast of the 5-day embryo and the primary embryonic endoderm are also positive. The endoderm cells remain positive both over the embryonic and extraembryonic portions of the embryo but the epiblast becomes Forssman antigen negative as it differentiates into embryonic ectoderm. The extraembryonic ectoderm which is derived from the Forssman negative trophectoderm remains negative throughout. The primordial germ cells are Forssman antigen positive from their first appearance in the germinal ridge until day 14 when they become negative but after that time it is other cells not related by direct lineage which become Forssman antigen positive. These are tentatively identified as Sertoli cells precursors as it is the Sertoli cells which are the antigen-positive population in the adult testis.

There is now abundant evidence for cell-surface phenotypic diversity within a single organism. This diversity can be recognized in a number of different ways and must reflect the varied functional properties of distinct cell types. In particular individual plasma membrane components can be recognized immunologically by specific antisera and one can use such antisera to identify and separate cells even though the structure and function of these components is largely unknown.

Many antisera which recognize antigens shared between embryonal carcinoma cells (EC) (the stem-cell line of mouse teratocarcinoma) and early mouse embryo cells have been described (Erickson, 1977). The pattern of reactivity reported for such antisera falls into two classes: those that recognize antigens expressed on ‘undifferentiated’ cells in embryo, EC, and only the germ-cell line in the adult (Artzt et al. 1973); and those that share essentially the same embryo-EC distribution of reactivity but also react with cells of other adult tissues apart from the germ line, for example brain and/or kidney (Stern et al. 1975).

Antisera are complex mixtures of antibodies with multiple specificities and the two above classes may represent overlapping sets of reactivities. It is difficult to define the shared and unique specificities of such conventional antisera precisely. Monoclonal antibody technology allows the production of large quantities of antibodies to single antigenic determinants (Kohler & Milstein, 1976). This avoids the problems associated with the interpretation of the reactions of conventional heterogeneous antisera.

Two monoclonal antibodies have recently been described which belong to the second class because they react with early embryo cells and EC and some tissues of the adult mouse (Stern et al. 1978; Willison & Stern, 1978; Solter & Knowles, 1978). One of these, a rat monoclonal antibody MI/22.25 was directed against a Forssman antigenic determinant (FA). This determinant was shown in previous studies to be first expressed on the trophectoderm of the early blastocyst (Willison & Stern, 1978). It disappeared from this tissue at the time of implantation but continued to be present on the cells of the inner cell mass and primary endoderm. This paper is concerned with the expression of FA in the post-implantation phase of mouse embryogenesis with particular emphasis on its expression by the cells of the germ line through to the adult. The pattern of expression of FA differs markedly from other EC-embryo antigens, including that of F9 antigen(s) (Buc-Caron, Condamine & Jacob, 1978) and demonstrates a complex expression in the genital tissues. Some of these results have been previously described in part. (Evans, Lovell-Badge, Stern & Stinnakre, 1979).

Mice

All embryos were taken from an inbred stock of 129 Sv Ev C P mice in which heterozygosity for the steel gene Slj was maintained. They were caged in pairs in ambient daylight conditions and examined for mating plugs each morning. The day when a plug was detected was considered as day 0 of pregnancy and conception was assumed to have occurred midway through the dark period. This was not always the case as litters varying in their development by up to ± day were found. Allowance was made for this in the interpretation of the results. Litters examined were either from wild type or from Slj/+ x Slj+ matings. The Wv gene was on the H x 101 stock (Heath, 1978) at F7 and adult Wv/ Wv males were used for tissue absorption studies.

Isolation and dissection of embryos

Four-day embryos were flushed from the uterus in calcium- and magnesium-free phosphate-buffered saline (PBS) and 5- to embryos were dissected from their decidua in PBS. Reicherts membrane was opened, most of the trophoblastic cone discarded and the embryo divided by a transverse cut into embryonic and extraembryonic regions. These portions were incubated in cold 0·1 % pancreatin in PBS or 0·25% trypsin for a few minutes, foetal calf serum added and the ectodermal and endodermal layers dissected apart either with tungsten needles or by pipetting through a drawn-out, siliconized pasteur pipette. The isolated tissue layers were further disaggregated when required by a second treatment with trypsin alone.

Ten and a half-day and later embryos were dissected from their decidual tissue and extraembryonic membranes. Liver, brain and spinal cord were isolated and disaggregated by trypsin treatment. The germinal ridges were dissected and freed from the accessory ducts and blood vessel. Twelve and a half-day and older embryonic gonads were sexed by their appearance and the presence of the spermatic blood vessel. Earlier embryos were sexed by orcein staining of the amnion and examination for the presence or absence of Barr Bodies (Farias, Kajii & Gardner, 1967). The two methods were crosschecked with each other on representative litters of embryos. In order to identify Sl/Sl embryos of between and days one genital ridge or half of one ridge was stained for the presence of alkaline phosphatase activity by squashing and incubation at room temperature in staining solution (0·075 M-Tris/HCl buffer pH 8·6 containing 0·8 % NaCl, 0·05 % disodium napthol ASMX phosphate, 2% dimethylformamide and (freshly added) 0·01% fast blue BB diazo salt). By this method unfixed cells containing alkaline phosphatase are stained deep blue within 5 min and the germ cells are readily seen (Chiquoine, 1954). The germ ridges of Sl/Sl embryos are almost entirely lacking in germ cells (Bennett, 1956; McCoshen & McCallion, 1975) and are identified by this technique. The contralateral ridge was used for further examination. Such germ-cell-deficient ridges were not found in wild-type litters and their correspondence with the Sl/Sl genotype was confirmed in preliminary experiment by grafting pieces of dorsal embryo skin to adult hosts and examining the graft tissue after 3 weeks for hair pigmentation (Bennett, 1956; Stevens, 1967).

Cells from germinal ridges were disaggregated by trypsin treatment and suspended in Dulbecco-modified Eagles medium containing 18mM-HEPES, 0·1 % sodium azide and 10% heat-inactivated foetal calf serum. Preparation of cell suspensions from adult testes and of other tissues for the absorption assays were as previously described (Stern et al. 1978).

Immunological assays

The monoclonal antibody preparation was the same as previously described (Stern et al. 1978) and a single batch of antiserum directly conjugated to fluorescein was used for all the fluorescence studies. Cells were suspended in -diluted direct-coupled Ml/22.25 (M1/22.25-F1) for h at 0 °C, washed twice in suspension medium and examined with a Zeiss photomicroscope equipped with epifluorescent illumination. Cells were observed by phasecontrast and fluorescence microscopy and only live cells scored. In some cases cells were also stained for alkaline phosphatase (as above), or for Δ 5–3 β hydroxysteroid-dehydrogenase (Wattenberg, 1958). The deposition of the blue stain did not obliterate the very bright fluorescence obtainable with the M1/22.25-F1 reagent. For cytotoxicity assays, the cells were suspended in -diluted Ml/22.25 at 0 °C for h. The cells were washed in suspension medium and then incubated at 37 °C in -diluted Guinea-pig complement GPC (L.I.P. Ltd, U.K.) for h. The cells resuspended in PBS containing 0·1% trypan blue and examined immediately. Killed cells take up the blue stain. Control incubations without Ml/22.25 treatment but with complement treatment were done in parallel.

1. Expression of FA in - to 6-day embryos

Inner-cell-mass cells isolated from - to -day embryos express Forssman antigen (Willison & Stern, 1978). The inner cell mass differentiates into embryonic ectoderm and endoderm at about the time of implantation. When these two tissues are isolated from - to -day embryos they are strongly Forssman antigen positive. The extraembryonic ectoderm, which is derived from the trophoblast (Gardner & Papaioannou, 1975), is Forssman antigen negative during this period. By -days, the intensity of FA labelling of the embryonic ectoderm is reduced but both the parietal and visceral endoderm is still brightly fluorescent.

2. Expression of FA in 6- to -day embryos

When 6- to -day egg-cylinder stages are dissected into embryonic and extraembryonic regions and the germ layers further dissected and examined either as tissue sheets or cell suspensions by Ml/22.25 immunofluorescence or cytotoxicity, those dissects containing endodermal cells have significantly greater proportions of antigen-positive cells. The majority of antigen-expressing cells have an endodermal morphology; these cells being generally larger and with more granular cytoplasms than the ectodermal cells. An example of such an experiment is shown in Table 1. Separated disaggregated embryonic ectoderm from 7·5-day embryos has no antigen-positive cells whereas embryonic endoderm has 76 % FA-positive cells. The cells which are antigennegative in embryonic endoderm preparations have a morphology consistent with their being contaminating embryonic ectoderm. Table 1 shows a similar result for extraembryonic ectoderm and endoderm. Both parietal and visceral endoderm continue to express the Forssman antigen.

Table 1.

Direct immunofluorescence with Ml/22.25 antibodies conjugated to fluorescein on disaggregated cells of separated germ layers of 712-day embryos

Direct immunofluorescence with Ml/22.25 antibodies conjugated to fluorescein on disaggregated cells of separated germ layers of 712-day embryos
Direct immunofluorescence with Ml/22.25 antibodies conjugated to fluorescein on disaggregated cells of separated germ layers of 712-day embryos

Cytotoxicity experiments confirm the results obtained by immunofluorescence with MI/22.25 antibodies. In the experiment shown in Table 1, for example, 70 % of the embryonic ectoderm cells were alive after incubation with M 1/22.25 and guinea-pig complement whereas only 13% of the embryonic endoderm cells survived. All the latter were small cells with a smooth cytoplasm -a morphology suggesting that they were contaminating embryonic ectodermal cells. When portions of a 7-day egg cylinder with both embryonic ectoderm and endodermal cells were treated with Ml/22.25 antibodies and GPC only the embryonic ectoderm cells remain viable, as judged by exclusion of trypan blue. A similar dissect treated only with GPC shows few cells stained. Figure 1 shows the same result by immunofluorescence. In summary, FA appears to be expressed on the embryonic ectoderm and endoderm before day 6. After this time the majority of embryonic ectoderm cells no longer have detectable antigen expression whereas both types of endodermal cells continue to express it strongly.

Fig. 1.

A portion of the embryonic region of a 7-day egg cylinder stained with fluorescein-conjugated M1/22.25 showing (a) phase-contrast appearance, (b) fluorescence. A sheet of embryonic ectodermal cells are seen protruding from the edge of the fragment and they are totally unstained in contrast to the brightly stained embryonic endodermal cells.

Fig. 1.

A portion of the embryonic region of a 7-day egg cylinder stained with fluorescein-conjugated M1/22.25 showing (a) phase-contrast appearance, (b) fluorescence. A sheet of embryonic ectodermal cells are seen protruding from the edge of the fragment and they are totally unstained in contrast to the brightly stained embryonic endodermal cells.

3. Expression of FA in liver and brain of 10- to 13-day embryos

From 8 days because of the rapidly increasing complexity of the embryo it can no longer be easily dissected into component germ layers. In the adult, FA is detectable in the brain, kidneys and testes, although not in liver. Table 2 shows that cell suspensions from day-10 to -13 brain and spinal-cord preparations contain increasing proportions of Ml/22.25 reactive cells, whereas those from liver contain less than 0·1% antigen-positive cells. It appears that cells in brain/spinal cord begin to express FA at about day 11. Other tissues, with the exception of genital ridges (see below), were not examined. The antigenpositive cells in brain remain to be identified.

Table 2.

Tissue distributions

Tissue distributions
Tissue distributions

4. Expression, of FA in genital tissues

Genital ridges contain increasing proportions of FA-positive cells from day 11 to about day 14 (Fig. 2). It is known that germ cells migrate from the allantois to hind-gut endoderm, the dorsal mesentery, the coelomic angles and finally start to enter the genital ridges at day 10 (Everett, 1943; Chiquoine, 1954; Mintz & Russell, 1955 & 1957; Bennett, 1956; Ozdzenski, 1967). These cells are easily identified histochemically because they have high levels of alkaline phosphatase and a distinctive morphology. Figure 2 shows that there is reasonable correspondence in the proportions of cells which are FA positive and the proportion with high levels of alkaline phosphatase until day 13. Figure 3 (a, b) shows FA-positive cells from a 12-day genital ridge which have typical germ-cell morphology; the cells are large with clear pseudopodia. A double-labelling experiment with M1/22.25-FL and histochemical stain for alkaline phosphatase (Table 3) shows that at day 12 the proportion of doublestaining cells can reach 83%. After this time it is known that alkaline phosphatase is a less specific marker for germ cells because other cells in the genital ridge show slight activity of this enzyme. The proportion of FA-positive cells in the genital ridges increases until day 14 (Figure 2). Before day 14 there is a greater proportion of FA-positive cells in the female gonad than in the male gonad ; the relative percentages are reversed after this time (see Figure 2).

Table 3.

Double labelling of genital-ridge cell suspensions for alkaline phosphatase (AP) and Forssman antigen (FA)

Double labelling of genital-ridge cell suspensions for alkaline phosphatase (AP) and Forssman antigen (FA)
Double labelling of genital-ridge cell suspensions for alkaline phosphatase (AP) and Forssman antigen (FA)
Fig. 2.

A histogram showing the percentage of Forssrnan-antigen-positive and alkaline-phosphatase-positive cells found in disaggregates of genital ridges from the embryos resulting from both wild-types 129 matings and from Sl/ + × Sl/+ matings. The numbers in brackets show the number of litters of each class examined. (++× ++- and Sl/+Sl/ + respectively) Where male and female embryos were separated the results are shown separately.

Key – open bars represent % F.A. 4-ve cells from +/+ × +/+ litters.

– hatched bars represent % F.A. + ve cells from Sl/ + × Sl/ + litters.

– horizontal lines represent % alkaline phosphatase + ve cells.

For each individual sample > 200 cells of each class were counted. The percentage values have been averaged between litters.

Fig. 2.

A histogram showing the percentage of Forssrnan-antigen-positive and alkaline-phosphatase-positive cells found in disaggregates of genital ridges from the embryos resulting from both wild-types 129 matings and from Sl/ + × Sl/+ matings. The numbers in brackets show the number of litters of each class examined. (++× ++- and Sl/+Sl/ + respectively) Where male and female embryos were separated the results are shown separately.

Key – open bars represent % F.A. 4-ve cells from +/+ × +/+ litters.

– hatched bars represent % F.A. + ve cells from Sl/ + × Sl/ + litters.

– horizontal lines represent % alkaline phosphatase + ve cells.

For each individual sample > 200 cells of each class were counted. The percentage values have been averaged between litters.

Fig. 3.

Isolated primordial germ cells from 12-day embryos stained with Ml /22.25-F1 (a) phase contrast (b) fluorescence.

Fig. 3.

Isolated primordial germ cells from 12-day embryos stained with Ml /22.25-F1 (a) phase contrast (b) fluorescence.

Up to day 15, the majority of the FA-positive cells have distinctive germ-cell morphology (Figure 4). After this time FA is expressed by cells other than germ cells. These FA-positive non-germ cells are small granular cells and in the testes could be either Sertoli cells, Leydig or other interstitial cells. Doublelabelling experiments with M1/22.25-F1 and the histochemical stain for Δ5–3β hydroxysteroid dehydrogenase activity (specific for Leydig cells; Niemi & Ikonen, 1961) on day-16 testicular cells shows that Leydig cells are not the antigen-positive non-germ cell component. This enzyme activity is already present in mouse Leydig cells by 13 days (Scheib & Lombard, 1971). After day 16 some of the antigen-positive cells are found in close contact with germ cells and there are increasing numbers of antigen-negative germ cells (Figure 4).

Fig. 4.

Histogram of the percentage of Forssman-antigen-positive cells showing typical germ-cell morphology when isolated from the gonad of progressively older embryos.

Fig. 4.

Histogram of the percentage of Forssman-antigen-positive cells showing typical germ-cell morphology when isolated from the gonad of progressively older embryos.

Leydig cells, identified histochemically, from new born, post-partum, day-8, day-23 and adult mice were FA-negative. In two experiments, new-born testes contained 41 % antigen-positive cells; 3% of the cells were Leydig cells and these were FA-negative. All stages of gonocyte differentiation in the postpartum mouse testes are negative; spermatozoa are not labelled with Ml/22.25 antibodies conjugated to fluorescein. Thus it appears that FA is expressed by the germ cells at least until day 16 of embryonic development but not in the germ line of the new-born or of the adult male mouse. Leydig cells are negative and some other cell type(s) is antigen-positive.

5. Sl and W loci

The Sl and W are complex loci; they are a series of semi-dominant alleles, some of which, when homozygous, lead to an almost complete absence of germ cells (reviewed by Heath, 1978). Figure 2 shows that there are consistently reduced proportions of FA-positive cells in genital ridge preparations from Sl/+ × Sl/ + litters compared to +/+×+/+ at days 12 to 16. Analysis of the individual embryos from Sl/ + × Sl/+ crosses shows three classes of embryo with respect to the proportions of FA-positive cells in the genital ridge : those with virtually no FA-positive cells (Sl/Sl), those with normal ( + / + ) and an interfnediate class (Sl/ + ). (Table 4) These SI/SI embryos do not survive longer than about 14 days in utero.

Table 4.

Effect of SlJ locus on % FA cells in genital ridge

Effect of SlJ locus on % FA cells in genital ridge
Effect of SlJ locus on % FA cells in genital ridge

There are viable alleles, e.g. Wv, which will survive to term but are sterile. The expression of Forssman antigen in Wv/ Wv homozygote male mice was examined by absorbtion of Ml/22.25 cytotoxicity for sheep erythrocytes (which express FA (Stern et al. 1978)). A normal tissue distribution of Forssman antigen was found (Figure 5). The Wv/ Wv testes which have virtually no germ cells (Coulombre & Russell, 1954) had only a slightly reduced amount of FA when compared with other tissues.

Fig. 5.

Tissue distribution of Forssman antigen in tissues of an adult Wv/ Wv mouse. The antigen is present in high levels in brain, kidney and testis as it is in wild-type animals. Increasing amounts of tissue homogenate were used to absorb an aliquot of Ml/22.25 whose ability to cause complement mediated lysis of 51Cr-labelled sheep red blood cells was then tested.

Fig. 5.

Tissue distribution of Forssman antigen in tissues of an adult Wv/ Wv mouse. The antigen is present in high levels in brain, kidney and testis as it is in wild-type animals. Increasing amounts of tissue homogenate were used to absorb an aliquot of Ml/22.25 whose ability to cause complement mediated lysis of 51Cr-labelled sheep red blood cells was then tested.

Neither the Slj nor Wv alleles affect the expression of FA per se since 26/27 isolated ICM’s from Slj/ + × Slj/+ crosses and 40/40 ICM’s from Wv/ + × Wv/ + crosses were found to be FA-positive.

Serological reagents used in the study of embryonic development may identify antigens which will give further insight into the heterogeneity of the cells in the developing embryo and in consequence be useful as cell phenotype markers. Their use to define cell surface markers has, moreover, led to the supposition that these themselves may provide markers of cell lineage within the developing embryo. In the case of FA the distribution in the preimplantation embryo might have suggested that this antigen would be expressed on derivatives of the ICM. The surprising result was that its expression was not detected on embryonic ectoderm but on the embryonic endoderm.

The distribution of expression of FA on the cells of the preimplantation and early post-implantation embryo (to days) seems, however, to follow a recognizable pattern. This is depicted schematically in Figure 6. At days the morula has no detectable antigen-positive cells. Both cell types derived from this stage are expressing FA by days. The trophectoderm loses the FA just before implantation but it can still be detected on the ICM. Extraembryonic ectoderm derived from the trophectoderm overlying the inner cell mass continues to have no detectable FA throughout the period studied. The ICM gives rise to two cell lineages, the embryonic ectoderm and the endoderm. Up to and including 5 days both tissues are antigen-positive but after this time only one of the ICM derivatives, the endoderm, retains antigen expression. Within the period of embryonic development to day , FA may be a useful marker of various different lineages. It is clear that FA differs from the embryonic antigen(s) defined by mouse anti-F9 serum (Buc-Caron et al. 1978) by being found on the surface only of the endodermal cells in the 6- to post-implantation embryo. What is not clear is whether mouse anti-F9 and other similar antisera might contain some anti-FA-like activity in addition to other specificities.

Fig. 6.

Summary of ontogenetic distribution of cell surface Forssman antigen during early development of the mouse.

Fig. 6.

Summary of ontogenetic distribution of cell surface Forssman antigen during early development of the mouse.

The difficulty with a lineage-related view of FA continues in its expression in later embryos and adults. At day 11 expression of this antigen is first detected in neural tissues and in the genital ridges. In the latter it is clear that the antigen-positive cells are germ cells as identified by morphology and histochemically for alkaline phosphatase. The round shape and characteristic staining of the primordial germ cells -particularly their alkaline phosphatase histochemistry have previously allowed their lineage to be traced by Ozdzenski (1967) to a region at the caudal end of the primitive streak in embryos, where the embryonic ectodermal cells form part of the allantoic rudiment. It is important to remember, however, that the immunological assays used here do not detect small subpopulations of cells and it cannot be certain that the embryonic ectoderm after day 6 has absolutely no FA-positive cells.

The view that the antigen-positive cells of the gonadal ridge include a major contribution from the germ cells is supported by the consistent reduction in the proportions of FA-positive cells in genital ridges from 129 Sl/+ × 129 Sl/ + crosses compared with wild type. Also crosses which produce Sl/Sl homozygotes have individual embryos which contain few or no germ cells. The trimodal distribution of numbers of FA-positive cells found in the germinal ridges of embryos from such crosses is also seen by alkaline phosphatase staining (McCoshen & McCallion, 1975, and our own observations). The detection of antigen-positive germ cells in the genital ridge and the increase in the percentage of positive cells in this tissue until day 14 is entirely consistent with the known migratory and multiplicative phases of mouse germ cells. The changes in the relative proportions of positive cells in female versus male genital ridges after day 14 probably reflect a number of events in the differentiation of testis and ovary respectively. In the testis at day 15 a separation of the gonocytes and the supporting cells takes place ; this might increase the relative yield of gonocytes from the trypsinized genital ridge. At day 15 in the ovary, most of the germ cells have already entered meiotic prophase.

It is likely that until day 14 the majority of antigen-positive cells are germ cells but thereafter the picture becomes more complicated. There are definitely non-germ-cell FA-positive cells present by day 16 and the primary candidates for this cell type in the testes would be Leydig or Sertoli cells. The Leydig cells can be identified histochemically and double-labelling experiments showed this cell type is not the cell which expresses the FA in the embryonic or adult testes. Although in the previous study (Stern et al. 1978) spermatozoa from adults were found to be FA-positive by absorption, no evidence of FA expression on the germ line after birth could be found. The previous positive results for sperm can easily be explained by the presence of small numbers of contaminating cells. It seems likely that by birth the major cellular component contributing to the absorptive capacity of the testes is non-germ line. This is supported by the near normal absorptive capacity of Wv/Wv homozygote testes for Ml/22.25. A small change with respect to other tissues could be expected since the testes of Wv/Wv animals are much smaller than normal and relative amounts of contributing tissues within the testes are different. The number of positive cells in the adult mouse testes and the morphology of the antigen-positive cells in culture (unpublished) makes it likely that this cell type is a Sertoli cell. In all these experiments with genital tissues it is difficult to detect small subpopulations of a given cell type which are FA-positive.

Thus although some germ cells are FA-positive in the embryo until at least day 16, by birth this lineage no longer expresses the antigen; another cellular component of the testes, probably Sertoli cells, accounts for the positivity of this tissue.

Considering these results together with those for the early embryo, the conclusion must be that either it is not possible to interpret FA as a lineage-related marker, or that our preconceived ideas about the lineages are incorrect. If FA were to be a lineage-related cell marker it might suggest a derivation of germ cells either via endoderm or from a subpopulation of ICM cells before they become committed to ectoderm formation at day 6. It might also suggest that some accessory gonadal cells could be derived from the primordial germ cells. This latter possible interpretation is, however, probably ruled out by the development of FA-positive cells in Wv/Wv testes. Nevertheless, at a given stage of development FA expression is clearly delineating different subpopulations. Their relationship is unclear but M1/22.25 antibodies have given a different view of the cells in the developing embryo.

Although cell antigens shared by different types of cell may not imply a lineage relationship they might well imply a functional homology of shared, possibly biologically important, cell-surface molecules. Since there is no apparent lineage relationship between the different cell types which express this antigen in the embryo or in the adult it may be useful to look for a functionally common denominator. The problem is where to start looking. It is known that the antigen recognized (at least on EC cells and sheep red blood cells) is a glycolipid, and there is some evidence that glycolipids may enhance hormone binding (Fishman & Brady, 1976), and cell adhesion (Huang, 1978). Both of these areas might merit further investigation.

The important conclusions from this study are that FA does not give a readily predictable pattern of expression in the embryo with respect to cell lineage. Thus using monoclonal antibodies has not really simplified the situation. However, if such a complex picture emerges from using antibodies against a single defined antigenic determinant then interpretation of the results with conventional antisera must be treated with a great deal more caution.

The financial support of the Cancer Research Campaign, the Wellcome Foundation and the Medical Research Council are very gratefully acknowledged.

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