Embryonic ectoderm (EmE), extraembryonic ectoderm (EE), ectoplacental cone diploid cells (EPC) and secondary giant cells (GC) were isolated from -day mouse embryos and their polypeptide synthetic profile assessed by fluorography of 2D polyacrylamide gels. Fifty polypeptides showed different distributions amongst the tissues, permitting characterization of each tissue by an array of polypeptide markers typical for the tissue at that developmental stage. The three tissues on the presumptive trophectoderm lineage did not show identical synthetic patterns. However, culture of EE cells in vitro resulted in conversion of their polypeptide synthetic profile to that of EPC after 2 days and of GC after 6 days, whilst culture of EPC cells converted their polypeptide synthetic profile to that of GC after only 4 days. These changes in polypeptide synthesis correlated well with the ploidy levels of the tissues at different times in culture.

The analysis of cell lineage and prospective fate in development requires the manipulation and analysis of marked populations of cells. Using such approaches it has proved possible to draw up a tentative fate map of the early mouse embryo (see Gardner & Johnson, 1975; Gardner & Papaioannou, 1975; Rossant, 1977; Rossant & Papaioannou, 1977). It is believed that the ICM of the blastocyst gives rise to the primitive ectoderm (which later forms the definitive ectoderm, mesoderm and endoderm of the foetus) and primitive endoderm of the postimplantation embryo. The trophectoderm of the blastocyst forms the ectoplacental cone and primary and secondary giant cells. There is also evidence to suggest that the extraembryonic ectoderm, which later forms the ectoderm of the chorion, arises from the trophectoderm (Gardner, Papaioannou & Barton, 1973; Gardner & Johnson, 1973, 1975; Rossant & Ofer, 1977; Frels, Rossant and Chapman, 1979) and not from the ICM as has been suggested by some authors (Robinson, 1904; Rugh, 1968).

We present here data obtained in an attempt to analyse at the molecular level the relationships between tissues of the implanted embryo. The polypeptides synthesized by embryonic ectoderm (EmE), extraembryonic ectoderm (EE), ectoplacental cone diploid tissue (EPC) and secondary giant cells (GC) have been analysed (a) to determine whether tissue-specific markers exist and (b) to discover whether associations of lineage and fate between constituent cell populations can be established on the basis of molecular identity. EPC and EE were also cultured in vitro and their polypeptide synthetic profiles analysed during the process of transformation to GCs (Rossant & Ofer, 1977).

Recovery of embryos, dissection and incubation

Random-bred mice of either CFLP (Hacking & Churchill Ltd) or PO (Pathology, Oxford) stock were used throughout this study. Previous studies have not revealed any consistent differences between preimplantation embryos from different mouse strains (Levinson, Goodfellow, Vadeboncoeur & McDevitt, 1978; Van Blerkom & Johnson, unpublished). Phosphate-buffered medium (Whittingham & Wales, 1969) + 10 % heat-inactivated foetal calf serum was used for dissection and storage of embryos. Embryos were dissected from the uteri of mice on the 8th day of pregnancy ( embryos). At this stage, the primitive streak is well established and the amniotic folds are almost complete, separating the EE from the embryonic shield. Using fine watchmaker’s forceps, Reichert’s membrane was torn away carefully, to avoid damaging the overlying secondary GCs. A cut was then made close to the origin of Reichert’s membrane to separate the embryonic and extraembryonic regions from the EPC. A solid lump of presumed diploid trophoblast cells can be seen in the core of the EPC, and this was dissected out as cleanly as possible from the overlying cells and maternal blood cells. These remaining fragments were retained for separation of GCs. The egg cylinders were cut into extraembryonic, exocoelomic and embryonic regions with glass needles (Fig. 1). The exocoelomic fragments were discarded.

Fig. 1.

Diagram of 712-day mouse embryo. The dotted lines mark the lines of dissection.

Fig. 1.

Diagram of 712-day mouse embryo. The dotted lines mark the lines of dissection.

Separation of the germ layers in the embryonic and extraembryonic fragments was achieved by incubating in 2·5 % Pancreatin and 0·5 % trypsin in Ca-, Mg-free Tyrode’s saline at 4 °C for 10–20 min, as described previously (Rossant & Ofer, 1977). After sucking up and down in flame-polished micropipettes, the EE can be separated cleanly from the endoderm and mesoderm and the EmE can be separated from the endoderm and most of the mesoderm except in the region of the primitive streak. This region was cut away and discarded so that only pure EmE tissue was retained. The presumptive diploid EPC fragments were also incubated in the Pancreatin/trypsin mixture for 10 min. This procedure clears any remaining intercellular matrix, damaged cells and contaminating maternal cells from the ectoplacental lumps. To obtain clean preparations of secondary GCs, the fragments of GCs, Reichert’s membrane and adhering maternal cells were incubated in 0·25 % trypsin in Ca-, Mg-free saline at 37 °C for 20 min. After sucking up and down in progressively smaller micropipettes, giant cells were freed from other tissues and could be sucked up and collected in groups of at least 50 in fresh medium.

For analysis of immediate protein synthesis, individual EPCs, EEs, EmEs and groups of secondary GCs were incubated in 0·1 ml RPMI (Flow Labs) containing 10 µl [S35]methionine (Radiochemicals, Amersham; specific activity approx. 1000 Ci/mmol) at 37 °C in 5 % CO2, 90 % N2 and 5 % O2 for 4 h. After this time the medium was withdrawn and the samples lysed in c. 30 μl of 2D buffer (O’Farrell, 1975), and stored at −70°C.

EEs and EPCs were also cultured in RPMI + 10 % FCS for periods of up to 6 days, by which time both fragments transformed into giant cells (Rossant & Ofer, 1977). In this case, groups of three fragments were cultured together and pooled for analysis. Incubation in the labelled amino acid was performed as above after 2, 4 and 6 days of culture.

Microdensitometry

Cell spreads were prepared from EPC and GC isolated directly from the embryos, and from EPC and EE, after various times in culture. A mouse liver imprint was also placed on each slide and all the cells were fixed in acetic alcohol. The cell spreads were stained by the Feulgen technique for DNA (Pearse, 1972). Microdensitometry measurements were made using a Leitz MPV microspectrophotometer, with the interference filter set to give peak transmission at 550 nm. Control liver readings were made for each slide. The results were expressed in the form of histograms of total absorbance measured in arbitrary units. These histograms were then calibrated in multiples of the haploid DNA values (C) by comparison with and extrapolation from the liver controls, whose cells contain 2C, 4C and 8C values of DNA. The histograms of EPC and GC isolated directly from the embryo show little overlap in DNA values between the two tissues (Fig. 2), indicating satisfactory isolation methods.

Fig. 2.

Histograms of DNA contents of (A) secondary giant-cell nuclei, (B) diploid ectoplacental cone nuclei. C values are derived by comparison with and extrapolation from the absorbance readings of control liver nuclei. Data from three different samples stained identically, were pooled for each histogram. The diploid ectoplacental cone samples contained cells with 2C and 4C DNA levels but no higher C values were found. Quite a large proportion of the cells appear to be tetraploid rather than diploid but this may simply reflect the fact that this cell population is highly proliferative and many cells will be in the tetraploid G2 phase of the cell cycle. DNA values for the secondary giant-cell samples reveal a much wider spread but few, if any, cells appear to be diploid. Most cells appear to be around the 8C level but some higher values were also observed. Thus, the isolation techniques for diploid ectoplacental cone and secondary giant cells allow very little crosscontamination between the two cell types and so comparison of their electrophoretic profiles is valid.

Fig. 2.

Histograms of DNA contents of (A) secondary giant-cell nuclei, (B) diploid ectoplacental cone nuclei. C values are derived by comparison with and extrapolation from the absorbance readings of control liver nuclei. Data from three different samples stained identically, were pooled for each histogram. The diploid ectoplacental cone samples contained cells with 2C and 4C DNA levels but no higher C values were found. Quite a large proportion of the cells appear to be tetraploid rather than diploid but this may simply reflect the fact that this cell population is highly proliferative and many cells will be in the tetraploid G2 phase of the cell cycle. DNA values for the secondary giant-cell samples reveal a much wider spread but few, if any, cells appear to be diploid. Most cells appear to be around the 8C level but some higher values were also observed. Thus, the isolation techniques for diploid ectoplacental cone and secondary giant cells allow very little crosscontamination between the two cell types and so comparison of their electrophoretic profiles is valid.

Electrophoresis

Electrophoretic separation of polypeptides was performed on twodimensional gels as described by O’Farrell (1975). Samples for two-dimensional analysis were thawed and frozen gently 3X, urea crystals were added to saturation, and 20 pl of sample were applied to preequilibrated cylindrical 4 % acrylamide gels (O’Farrell, 1975). Samples were run for a total of 6000 volt hours, the last hour of which was at 800 V. Gels were then equilibrated with two changes of SDS sample buffer for and applied to the top of gradient slab gels (7–15% acrylamide 110×150mm overlain by 4·5% acrylamide 10 × 150 mm) embedded in 1 % agarose in double-strength SDS buffer. The samples were run at 15mamps for 4–5 h. Gels were fixed in 25% TCA, processed through 7 % acetic acid, three changes of DMSO (2 h each), 20 % PPO in DMSO (2 h), water (three rinses), 7 % acetic acid (2 h) and then dried onto cards under vacuum (Bonner & Laskey, 1974). Dried gels were exposed to preflashed Fuji X-ray film (Laskey & Mills, 1975) for 1 to 8 weeks at −70 °C. Films were developed for 4 min at 20 °C in Kodak DX-80 Developer. Several films of varying exposure times were available for each preparation. The film used for the analysis was selected by examination of several reference polypeptides for roughly equivalent intensity in each film (see below).

Procedure for gel analysis

The films produced by exposure to gels were viewed on a modified X-ray viewer. A film of an EmE sample was selected as standard, and compared first with all other films from EmE separations. Most polypeptides migrated in similar relative positions and were of similar relative intensities. These polypeptides were noted and were used in comparisons with films from other tissues. A few polypeptides were variable between gels. Some of this variation appeared due to secondary modification of invariant polypeptides, but in a few cases polypeptides appeared in one or two gels but no others. In subsequent comparisons with other gels, these polypeptides were either not detected or were detected variably in these tissues also.

Sufficient similarities existed between films from different tissues for a set of reference polypeptides, common to all films, to be established. These were used first to assess the degree of any distortions of gels during processing, and then as the reference points for adjacent tissue-limited polypeptides. All comparisons between gels were made on four separate occasions at least 2 weeks apart and the results from each analysis recorded and compared subsequently. Only polypeptides which were consistently scored in the same relative position and intensity for each gel were considered to be reliable markers.

A total of 36 gels was examined (7 EmE, 6 EE, 15 EPC and 8 GC) and 50 polypeptides were found that showed consistent differences. Each of the 50 polypeptides examined in every gel was evaluated as strong, weak or not detectable relative to the reference polypeptides or as not scorable due to streaking or other local gel imperfections. When this analysis was completed, a further comparison was made with eight 2D separations of polypeptides from EPC tissue culture in vitro for 2 or 4 days and twelve from EE tissue cultured in vitro for 2, 4 or 6 days.

Each of the 50 polypeptide spots was assigned a reference code. Those polypeptides which showed a migration identical to polypeptides studied previously on preimplantation-stage embryos were assigned the same code number as used previously (Handyside & Johnson, 1978; Johnson, 1979). Other variant polypeptides were assigned a letter code. Certain of the polypeptides (codes N, h, i and 14) appear to be present as two or three spots often along a horizontal (isoelectric focusing) axis and show reciprocal changes in intensity. In the figures and tables, the components of these groups of polypeptides are given suffices 1 and 2 or 1,2 and 3 reading position from basic to acidic. On each gel a small black or white point marks the position of the trailing (left) edge of the polypeptide or its position and the adjacent number/ letter code is above or to the left of the point.

For the block summary diagrams, each square represents an aggregate score of all the gel separation patterns examined for the polypeptide and tissue in question.

Figures 36 show representative plates derived from two-dimensional electrophoretic analysis of EmE, EE, EPC and GC. A summary of the distribution of each of the tissue-marker polypeptides amongst the 36 films analysed is presented in Fig. 7 and Table 1. Table 2 summarizes the extent to which different tissues show molecular identity in regard to their synthesis of the tissue-marker polypeptides.

Table 1.

Code references of polypeptides limited to any tissue or group of tissues

Code references of polypeptides limited to any tissue or group of tissues
Code references of polypeptides limited to any tissue or group of tissues
Table 2.

Total number of polypeptides which show identical presence / absence patterns* in gel separation of different tissues ( + ve association in brackets)

Total number of polypeptides which show identical presence / absence patterns* in gel separation of different tissues ( + ve association in brackets)
Total number of polypeptides which show identical presence / absence patterns* in gel separation of different tissues ( + ve association in brackets)
Fig. 3.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 3.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 4.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 4.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 5.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 5.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 6.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 6.

Fluorographs of representative two-dimensional gels of polypeptides derived from 712-day EmE (Fig. 3), EE (Fig. 4), EPC (Fig. 5) and GC (Fig. 6). Individual polypeptides are marked with a white or black spot to the left of their position and assigned a white or black reference code above or to the left. (White and black are used for maximum contrast only and have no other significance). Fig. 3 indicates pH and M.W. (× 104) range of separations.

Fig. 7.

Summary of polypeptide synthetic profiles of 712-day embryonic ectoderm (EmE), extraembryonic ectoderm (EE), ectoplacental cone cells (EPC) and secondary giant cells (GC).

Fig. 7.

Summary of polypeptide synthetic profiles of 712-day embryonic ectoderm (EmE), extraembryonic ectoderm (EE), ectoplacental cone cells (EPC) and secondary giant cells (GC).

Two-dimensional electrophoresis was performed on samples of both EPC and EE which had been cultured in vitro for 2–6 days. During this period, both cell types transform their appearance (Rossant and Ofer, 1977) and DNA content to resemble GCs. However, the.time course of transformation was different in the two tissues (Fig. 8). EE cells remained diploid for at least 2 days in culture before beginning to endoreduplicate whereas EPC cells are already moving away from diploid DNA values by 2 days. Examination of the films derived from 2D separations shows a corresponding molecular transformation to a secondary giant-cell pattern. The results of these separations are summarized in Fig. 9, which show that (a) the final molecular pattern for both tissues is almost identical to that of secondary giant cells (b) EE takes approximately 2 days longer than EPC to transform totally its synthetic pattern (c) the earliest molecular transformations shown by EE involve primarily those polypeptides by which extraembryonic ectoderm differs from EPC (polypeptides ACEKLM3 4 16 13OPQ7allZdfgl5h1h2h3i1i2 – only polypeptides S T X N1 and W do not conform to this pattern). This observation suggests that EE transforms to GC through an EPC type profile, a conclusion confirmed by polypeptides g, h2 and ii which are weak in EE, present in EPC and weak or absent in GC. During culture of EE in vitro these three polypeptides show a transitory increase followed by a decline.

Fig. 8.

Histograms of DNA contents of EE and EPC nuclei after various times in culture. These histograms were prepared in the same way as those in Fig. 2. EE cells remain diploid for 2 days before transforming into giant cells whereas the peak DNA values for EPC are already greater than 2C by 2 days. The histograms of EE at 4 days and EPC at 2 days are almost exactly superimposable, as are the histograms for EE at 6 days and EPC at 4 days. Tn the later cultures, the largest DNA values are underestimates because the largest cells tended to be disrupted during preparation of the cell spreads.

Fig. 8.

Histograms of DNA contents of EE and EPC nuclei after various times in culture. These histograms were prepared in the same way as those in Fig. 2. EE cells remain diploid for 2 days before transforming into giant cells whereas the peak DNA values for EPC are already greater than 2C by 2 days. The histograms of EE at 4 days and EPC at 2 days are almost exactly superimposable, as are the histograms for EE at 6 days and EPC at 4 days. Tn the later cultures, the largest DNA values are underestimates because the largest cells tended to be disrupted during preparation of the cell spreads.

Fig. 9.

Summary of polypeptide synthetic profiles of 712-day extraembryonic ectoderm immediately after isolation (EE 0), after 2, 4 and 6 days in culture (EE2, EE4 and EE 6) and of 712-day ectoplacental cone cells immediately after isolation (EPC 0), after 2 and 4 days in culture (EPC 2 and EPC 4) and of secondary giant cells immediately after isolation (GC 0).

Fig. 9.

Summary of polypeptide synthetic profiles of 712-day extraembryonic ectoderm immediately after isolation (EE 0), after 2, 4 and 6 days in culture (EE2, EE4 and EE 6) and of 712-day ectoplacental cone cells immediately after isolation (EPC 0), after 2 and 4 days in culture (EPC 2 and EPC 4) and of secondary giant cells immediately after isolation (GC 0).

Examination of the two-dimensional electrophoretic profiles of polypeptides synthesized by various tissues of the mouse embryo has revealed several polypeptides that appear to be limited to one tissue or group of tissues (Table 1).

Each of the four tissues analysed is characterized by a typical array of endogenous polypeptide markers, detection of which as a group may be taken as diagnostic of the tissue at that point in its development. However, although this analysis may prove to have been useful in providing a complex of stage and tissue-specific markers, it does not provide clear and independent support at the molecular level for experimentally-derived cell-lineage relationships. Thus, as may be seen from Table 2, EE appears to occupy a position roughly midway between EmE and EPC in its communality of polypeptides. Such a result is not surprising for both technical and theoretical reasons.

First, the gel patterns observed after labelling and two-dimensional electrophoresis of embryonic polypeptides are the result of a complex sequence of events. The final gel patterns may be influenced by differences in the developmental staging of equivalent age tissues, slight differences in techniques of isolation and handling of tissues from experiment to experiment, possibility of viral or bacterial contamination of tissues during handling and culture, accessibility to and toxicity for the tissues by the label, responses of the tissue to isolation and short-term culture and post-translational modification of polypeptides in situ and during processing. All of these features might operate differently in different tissues. Whilst it is highly unlikely that all the differences between tissues are being induced experimentally, each tissue in fact synthesizing identical polypeptides in situ, it is not improbable that some of the differences observed are due to factors such as these. The problem posed by the technical artifacts can be reduced somewhat by standardizing conditions and their magnitude can be assessed by co-labelling and co-processing of different tissues. However, for tissues of such complexity, it will not be easy to eliminate these problems entirely. In this study, only a minority of the polypeptides being synthesized by the tissues are examined, the rest being either excluded from the isoelectric range analysed or too weak for detection, and of this minority, most are present in all films, some show a variable and apparently random appearance and only a group of 50 show consistent tissue patterns. This highly selected minority of polypeptides thus constitute a reliable set of markers for a tissue taken at a defined age and processed in a defined way. They do not represent the complete pattern of distinctive polypeptides synthesized in situ. Their value is primarily diagnostic rather than analytic (see also Dewey, Fuller & Mintz, 1978, for a discussion of this problem).

Second, even assuming that the gel patterns observed did represent the synthetic pattern in situ, there would be no particular reason to expect that the synthesis of those polypeptides which reflect a particular tissue’s past lineage or future potential should predominate over synthesis of those which are characteristic of its present differentiated state. Thus, although experimental studies strongly support a common lineage and potential for EE and EPC (Gardner & Papaioannou, 1975), the tissue organization of the EE into an epithelium resembles more closely the differentiated state of EmE than EPC. The polypeptide synthetic profile of EE not surprisingly reflects all of these features of its organization. General molecular comparisons of this sort seem unlikely to shed light on cell lineages.

Considerations such as these make it unprofitable to compare in any detail the results described in this paper with earlier molecular studies described previously for morulae, blastocysts and teratocarcinoma cells (Van Blerkom, Barton & Johnson, 1976; Handyside & Johnson, 1978; Dewey et al. 1978). Although the polypeptides characterized previously as trophectodermal markers (2, 3, 4, 7, 13 and 16) were detected in this study only in cells of the putative trophectodermal lineage and thus appear to conform to the putative lineage, of the three polypeptides detected in this study that co-migrate with previously described ICM marker polypeptides, number 11 is present in both EmE and EE, number 15 is absent only from EmE and number 14 is synthesized by all four tissues. For the reasons outlined above, and given the increased embryonic complexity that has occurred over the 4-day period separating the two embryonic stages analysed, little significance can be placed on this observation.

A more profitable approach to molecular studies of lineage and potential comes from the analysis of the changes in molecular expression with time by a tissue exposed to altered conditions. For example, we have previously demonstrated that groups of inside cells isolated from morulae and blastocysts show, on their isolation and culture in vitro, a molecular transition towards a blastocyst-type pattern. The completeness of this altered molecular response can be correlated with the pluripotentiality of the inside cells (Johnson, 1979). Using a comparable approach with isolated and cultured EPC and EE, a striking correlation between transformation at molecular and morphological levels has been observed. Thus, the EE cells in culture transform over the first 2 days to an EPC-type pattern during which period they remain diploid. Then over a further 4 days both EE and EPC transform together to a GC-type polypeptide pattern and both become polyploid (Figs. 8 and 9). Only 7 of 42 polypeptides (B, X, N1, S, T, V and W) do not conform strictly to this sequence, which is remarkable considering that the gels represent static sampling of a continuous transition. Thus, trophoblast giant cells generated in vivo or in vitro show identical polypeptide synthetic profiles whatever their origin, indicating a common pathway of differentiation open to all trophectoderm derivatives and raising the possibility that in the normal embryo the EE cells may act as a reservoir of cells that can be recruited to the EPC to generate new giant cells.

We would like to acknowledge the technical assistance of Debbie Eager. This work was supported by grants to M.H.J. from the MRC and the Ford Foundation. J.R. was a Beit Memorial Fellow and is now supported by the Canadian NSERC. We wish to thank Dr Y. Masui, Department of Zoology, University of Toronto for use of the microspectrophotometer.

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