Extracts of embryonic mouse tissues (skeletal, cardiac and smooth muscle, and brain) were analysed by Cellogel electrophoresis, for their isoenzymic distributions of three enzymes, creatine phosphokinase, aldolase and phosphoglycerate mutase. Embryonic tissues from the 12th day to the end of gestation were examined for isoenzyme transitions, and it was found that the adult forms of these enzymes appeared during gestation. Extracts from cloned teratocarcinoma cells were similarly examined in order to determine their degree of bio-chemical differentiation. Undifferentiated embryonal carcinoma cells contained only the early embryonic forms of all three enzymes, while differentiated cells formed in vivo, and in some cases in vitro, started to express the adult types of creatine phosphokinase and aldolase. Thus, biochemical parallels have been demonstrated between developing embryonic tissues and teratocarcinoma cells differentiating in vitro.

It is known that cells of embryoid bodies and derived cell lines can differentiate both in vivo and in vitro (reviewed by Martin, 1975). In most cases the differentiation has been followed by histology and they form recognizable muscle, nerve, cartilage, pigmented cells and keratinizing and glandular epithelium. Biochemical studies have shown that during the differentiation of embryoid bodies the specific activity of alkaline phosphatase and protease declines; these are enzymes characteristic of the embryonal carcinoma cells (Bernstine, Hooper, Grandchamp & Ephrussi, 1973; Hall et al. 1975). As the differentiation proceeds so the specific activity of acetylcholinesterase and creatine phosphokinase increases and one study has suggested the formation of nervous tissue (Levine, Torosian, Sarokhan & Teresky, 1974) while another showed striated muscle formation (Gearhart & Mintz, 1974). The extent to which these enzymatic properties can be taken as markers of particular cell differentiation was not established.

Here I have compared the appearance of three enzymes in developing mouse embryos and in teratocarcinomas. Particular patterns of the isoenzymic forms of these enzymes are characteristic of muscle and brain tissues in adult mouse and their appearance in developing embryonic tissues is recorded. The enzymes are creatine phosphokinase (CPK, EC 2.7.3.2), fructosediphosphate aldolase (EC 4.1.2.13), and phosphoglycerate mutase (PGM, EC 2.7.5.3). These enzymes were chosen because studies on other species have shown that the distribution of their isoenzymes changes during the development of skeletal muscle and brain.

CPK is a dimeric enzyme composed of sub-units M and B in homo-or heteropolymers MM, BB and MB. In the brains of a wide variety of species, only BB is present, while in skeletal muscle only the MM form is found. Since the type of CPK found in the early embryos of several species is usually exclusively BB, there must be a transition of isoenzymic forms during the development of skeletal muscle (Cao, de Virigilis, Lippi & Coppa, 1971; Turner & Eppenberger, 1973). PGM is a dimeric enzyme similar to CPK in the form and distribution of its isoenzymes in several mammalian species. In addition, its isoenzymic transitions are similar during human muscle development (Omenn & Hermodson, 1975).

Aldolase is a tetrameric enzyme composed of distinct sub-units A, B or C. Adult skeletal muscle usually has only one isoenzyme of aldolase, A4; both A and B sub-units are found in liver and kidney; A and C sub-units occur in brain (Rutter et al. 1968). In rat embryos the predominant aldolase isoenzyme is A4 and there is no change of type during the development of cardiac and skeletal muscle. There is, however, a change in developing brain tissue, resulting finally in approximately equal amounts of five isoenzymes, A4, A3C, A2C2, AC3 and C4 (Turner & Eppenberger, 1973).

The transitions of the isoenzymes of CPK and aldolase that occur in developing skeletal muscle in vivo also occur during the maturation and differentiation of chick myoblasts in vitro (Morris, Cooke & Cole, 1972; Turner, Maier & Eppenberger, 1974) and rat myoblasts in vitro (Yaffe & Dym, 1972). I have therefore investigated the possibility of using isoenzymic analyses to determine the type and the degree of differentiation of cultured teratocarcinoma cells.

(1) Biological materials

Several stocks of mice were used in this study and no differences were detected between them. PO mice were obtained from the Pathology department of the University of Oxford, U.K., CFLP mice from Carworth Europe, Alconbury, Hunts., U.K. The day of detection of the copulation plug was designated the first day of gestation. Pregnant female mice were killed by cervical dislocation and the embryos were dissected in ice-cold solution A of Dulbecco & Vogt (1954) (PBS). Embryonic tissues were collected into centrifuge tubes, drained free of medium and either frozen at –-20 °C, or processed immediately.

Cultured teratoma cells and solid teratomas were obtained from 129/J and C3H derived growths (Papaioannou, McBurney, Gardner, & Evans, 1975). This material was kindly provided by M. McBurney of this laboratory.

OC15 SI is a cloned line of cells derived from a transplantable teratocarcinoma of strain 129/J. Three other cloned cell lines from transplantable teratocarcinoma of strain C3H mice were also examined. These lines were maintained as homogeneous populations of embryonal carcinoma cells. All the cell lines could differentiate under suitable conditions of culture (McBurney, in preparation).

(2) Tissue extractions

Adult tissues were homogenized with four volumes of either 0·25 M sucrose, 0·025 M Tris-HCl, pH 7·4, 2·5 mM disodium salt of EDTA (for brain tissue and also for tissue culture cells), or four volumes of 0·25 M sucrose, 0·025 M Tris-HCl, pH 7·4, 0·025 M magnesium acetate, 0·2% (v/v) 2-mercaptoethanol (for all other tissues).

Embryonic tissues were suspended in a volume of the above solutions approximately equal to that of the tissue and were sonicated for two bursts of 10 sec each or until disintegrated.

Homogenates were centrifuged at 100000 g for 30 min at 2·4 °C in an M.S.E. High-Speed 65 centrifuge. Supernatants were frozen at – 20 °C in small aliquots.

(3) Electrophoretic analyses

Three to five microlitres of tissue extracts were analysed electrophoretically on Cellogel strips (Reeve Angel Scientific Ltd, Whatman Labsales Ltd, Maidstone, Kent, U.K.), using a micro-scale applicator. The electrophoretic buffer and strip soaking solution was 0·06 M barbitone buffer, pH 8·6, containing 0·1 mM 2-mercaptoethanol (and also 5 mM EDTA for aldolase determinations). Electrophoreses (at 4 °C) were run at 250 V (25 V/cm) for 60 min in the case of CPK separations, 60–75 min for aldolase and 312 h for PGM on a Shandon Model U77 electrophoresis apparatus (Shandon Scientific Company Ltd, 65 Pound Lane, London NW10).

Creatine phosphokinase isoenzymes were stained using an agar overlay essentially as described by Dawson & Eppenberger (1970). Also included in the reaction mixture was 1 mM adenosine-5′-monophosphate in order to inhibit the enzyme myokinase which also stains with this reaction mixture. Controls were performed by staining duplicate Cellogel strips in a reaction mixture which did not contain the substrate creatine phosphate.

Aldolase isoenzymes were stained similarly by the reaction mixture, essentially as described by Penhoet, Rajkumar & Rutter (1966). A modification introduced by Lebherz & Rutter (1969) was used in order to reduce alcohol dehydrogenase staining. Control strips were incubated with the same agar overlay except that fructose diphosphate was omitted. Cellogel/agar strips were incubated at 37 °C in the dark for a period of 10–30 min or until a good formazan colour had developed. The strips were processed and ‘whitened’ by the procedure recommended by the manufacturers before being photographed.

Phosphoglycerate mutase isoenzymes were made visible on Cellogel strips by a fluorescent method essentially as described by Omenn & Hermodson (1975). After incubating 1–3 h at 37°, black spots (NAD produced by the enzyme) could be seen against a background of fluorescent NADH in ultra-violet light (365 nm). These were photographed at/3–5 for 45 sec on FP 4 film (Ilford Ltd, Ilford, Essex, U.K.) using a dark green filter.

(4) Chemical materials

General chemicals were analytical reagent grade from Fisons Scientific Apparatus, Loughborough, Leics., U.K., or from British Drug Houses, Poole, Dorset, U.K. Enzymes and substrates were obtained from Koch-Light Laboratories Ltd, Coinbrook, Bucks., U.K.: from Sigma (London) Chemical Company, Kingston-upon-Thames, Surrey, U.K.; or from the Boehringer Corporation (London) Ltd, Lewes, E. Sussex, U.K.

Table 1 shows the distribution of the isoenzymes of CPK and aldolase in adult mouse tissues. It is similar to that of rat and other mammals (Masters, 1968; Turner & Eppenberger, 1973). CPK B sub-unit homopolymer is the only isoenzyme present in adult brain tissue, and is the predominant form present in very early embryonic cells (see Table 2). Thus the tissues which must transform their CPK isoenzyme type at some stage in development are skeletal (striated muscle), cardiac muscle, and the smooth muscle of the intestine and bladder. On the other hand, for aldolase the transition must occur in brain, kidney and liver tissues since the major embryonic form is A4 (see Table 3).

Table 1

Distribution of CPK and aldolase isoenzymes in various adult tissues of mouse

Distribution of CPK and aldolase isoenzymes in various adult tissues of mouse
Distribution of CPK and aldolase isoenzymes in various adult tissues of mouse
Table 2

Changes of CPK isoenzyme distribution in developing mouse muscle tissues

Changes of CPK isoenzyme distribution in developing mouse muscle tissues
Changes of CPK isoenzyme distribution in developing mouse muscle tissues
Table 3

Changes of aldolase isoenzyme distribution in developing mouse brain tissue

Changes of aldolase isoenzyme distribution in developing mouse brain tissue
Changes of aldolase isoenzyme distribution in developing mouse brain tissue

(1) CPK isoenzymes in ontogeny

Table 2 shows the results of isoenzymic analyses of four kinds of muscle at several stages of development. By the 15th day of development a significant amount of MM activity above control (see below) is detected for the first time in skeletal muscle. It is probable that both the M and B sub-units of the enzyme are being synthesized because the heteropolymer MB is visible (Fig. 1 a, track 4) and this would not appear unless either there was simultaneous production of both sub-units in the same cytoplasm or continuous dissociation and reassociation of the sub-units of the enzyme. The embryonic heart is highly developed morphologically by the 12th day, and this correlates with the very early production of MB (Table 2).

Fig. 1

Cellogel electrophoretic analysis of CPK at various stages in the development of skeletal muscle in the hind-limb of mouse, (a) Reaction strips incubated in the presence of substrate, (b) control strips corresponding to (a). Sample 7 was run at a different time to samples 1 to 6. 1, 12th day of the gestational period; 2, 13th day; 3, 14th day; 4,15th day; 5, 16th day; 6,17th day; 7, adult skeletal muscle. Note the similar amount of staining at the position of MM in (a) samples 1 to 4, and in (b) the control samples. This enzymatic activity was assumed not to be MM (see text for details). For experimental details see the Materials and Methods section.

Fig. 1

Cellogel electrophoretic analysis of CPK at various stages in the development of skeletal muscle in the hind-limb of mouse, (a) Reaction strips incubated in the presence of substrate, (b) control strips corresponding to (a). Sample 7 was run at a different time to samples 1 to 6. 1, 12th day of the gestational period; 2, 13th day; 3, 14th day; 4,15th day; 5, 16th day; 6,17th day; 7, adult skeletal muscle. Note the similar amount of staining at the position of MM in (a) samples 1 to 4, and in (b) the control samples. This enzymatic activity was assumed not to be MM (see text for details). For experimental details see the Materials and Methods section.

Notice that in Fig. 1 there is a significant amount of staining activity at the position of MM in early embryonic extracts. This activity is found in extracts of all early embryonic cells as well as in teratocarcinoma cells. However, since the control gels (with no creatine phosphate in the agar overlay reaction mixture) showed identical staining (Fig. 1,b) at this position as well as at the myokinase position, it is likely that this activity is caused by an enzyme activity other than CPK. In Table 2 therefore, this kind of stained band is denoted by an asterisk.

The necessity for control incubations made at the same time as reaction incubations is demonstrated by Fig. 1(b). The spurious stained band at the position of MM in some of the CPK reaction gels was not identified. It might have been MM which also stained in the control gel because of the presence of creatine phosphate in the cell extract. This would have been possible only if cieatine phosphate co-electrophoresed with the MM form of the enzyme. This was tested by adding increasing amounts of creatine phosphate to an adult skeletal muscle extract, electrophoresing these mixtures as usual, and staining as for a control gel. None of the samples stained at the MM position. Thus the unknown band is unlikely to be MM. Myokinase, however, which has a mobility intermediate between that of MM and MB, was present as a stained band whose intensity increased with increasing concentrations of creatine phosphate in the original sample. This suggested that creatine phosphate was co-electrophoresing with myokinase rather than with the MM form of CPK.

(2) Aldolase isoenzymes in ontogeny

Table 3 depicts the aldolase isoenzyme transition in brain tissue. By the 14th day of development well-defined heteropolymers of A and C sub-units are present, though these are faintly visible also in the 12th and 13th day brain (Fig. 2bz). About 95% of all aldolase activity in early embyonic cells is that of A4 with 5% A3C. During the development of all types of muscle, this remains about the same.

Fig. 2

Cellogel electrophoretic analysis of aldolase (a) in developing mouse brain at the increasing times in the gestational period 1, 12th day; 2, 13th day; 3, 14th day; 4, 15th day; 5, 16th day; 6, 17th day; 7, 18th day; 8, adult brain tissue. Not all samples were run at the same time, (b) Samples stained for aldolase sometimes revealed a contaminating set of stained bands. The lower two tracks are the corresponding control strips run in parallel with 1, adult brain tissue; 2, adult cardiac muscle. Note the control stips are similarly stained and that the mobilities of the spurious bands are slightly different from true aldolase isoenzymes.

Fig. 2

Cellogel electrophoretic analysis of aldolase (a) in developing mouse brain at the increasing times in the gestational period 1, 12th day; 2, 13th day; 3, 14th day; 4, 15th day; 5, 16th day; 6, 17th day; 7, 18th day; 8, adult brain tissue. Not all samples were run at the same time, (b) Samples stained for aldolase sometimes revealed a contaminating set of stained bands. The lower two tracks are the corresponding control strips run in parallel with 1, adult brain tissue; 2, adult cardiac muscle. Note the control stips are similarly stained and that the mobilities of the spurious bands are slightly different from true aldolase isoenzymes.

In a small proportion of electrophoretic separations of aldolase isoenzymes, a set of five faint bands moving faster than aldolase was visible in both the reaction gel and the control gel (Fig. 2Z>). The slower-moving bands of this set could easily be identified erroneously as A3C, A2C2, etc., and illustrates the necessity for control gels run in parallel with the reaction strips.

(3) Ontogeny of PGM isoenzymes

The data for PGM isoenzyme patterns is shown in Table 4. In the developing skeletal muscle of the hind limb, a transition from BB to MM occurs and the first appearance of MM is observed at the 15th day of gestation as it is with CPK. In contrast to the latter, the BB isoenzyme of PGM remains the predominant form to a much later time in development and the Cellogel reaction strips have to be rather over-incubated to detect the low proportion of the MM form of very early embryonic muscle (Fig. 3). As in the case of CPK, the first appearance of MM in tongue and heart muscle is earlier (14th and 13th day respectively) than in the skeletal muscle of the hind-limb. MM does not become the predominant form of PGM until after the 17th day of development in all types of muscle.

Table 4

Changes of PGM isoenzyme distribution in developing mouse muscle tissues

Changes of PGM isoenzyme distribution in developing mouse muscle tissues
Changes of PGM isoenzyme distribution in developing mouse muscle tissues
Fig. 3

Cellogel electrophoretic analysis of PGM in developing hind-limb muscle of mouse. 1, Skeletal muscle at the 12th day of gestation; 2, 13th day; 3, 14th day; 4, 15th day; 5,16th day; 6,17th day; 7, adult skeletal muscle. The adult form (MM) is just visible in 15th day muscle but is only clearly stained in the 17th day extracts. See the Materials and Methods section for experimental details.

Fig. 3

Cellogel electrophoretic analysis of PGM in developing hind-limb muscle of mouse. 1, Skeletal muscle at the 12th day of gestation; 2, 13th day; 3, 14th day; 4, 15th day; 5,16th day; 6,17th day; 7, adult skeletal muscle. The adult form (MM) is just visible in 15th day muscle but is only clearly stained in the 17th day extracts. See the Materials and Methods section for experimental details.

(4) Are these isoenzymes and their transitions detectable in teratocarcinoma cells?

Solid teratomas consisting of several different types of tissues gave the patterns of CPK and aldolase isoenzymes expected of differentiated tissues. Fig. 4(a) and (b), track 3, shows that both muscle and brain type CPK and aldolase were present in one extract. A cell line (OC15 SI) derived from this teratoma (see Materials and Methods) and cultured under conditions to maintain it in an embryonal carcinoma (undifferentiated) form, gave only the embryonic forms of CPK (BB) (together with the spurious band at MM) and aldolase (A4). When this cell line was allowed to differentiate and both nervous- and epidermal-like cells were visible in the culture dish, the aldolase pattern had changed to a 5-banded type typical of well-differentiated brain tissue (Fig. 4 a, track 2). On the other hand no change was detected in the CPK pattern (Fig. 4 a), or in the PGM pattern.;

Fig. 4

Isoenzyme analyses in teratocarcinoma cells, (a) CPK, (b) aldolase. Track 1, embryonal carcinoma cells of clone OC15 SI; 2, differentiated OC15 SI; 3, solid teratoma tissue. This was produced in vivo from the same teratocarcinoma line (OTT 6050) which was cloned to give OC15 SI.

Fig. 4

Isoenzyme analyses in teratocarcinoma cells, (a) CPK, (b) aldolase. Track 1, embryonal carcinoma cells of clone OC15 SI; 2, differentiated OC15 SI; 3, solid teratoma tissue. This was produced in vivo from the same teratocarcinoma line (OTT 6050) which was cloned to give OC15 SI.

This result has been confirmed for several cell lines which were derived from different sources of teratocarcinoma. Two lines were from embryoid bodies of a C3H-strain tumour and one was from a solid tumour of C3H origin. Embryoid bodies, like embryonal carcinoma cells in culture, contained only the BB form of CPK and A4 aldolase. In all cases, after partial differentiation to nerve-type cells during in vitro culture, the aldolase pattern had transformed into a 3- to 5-banded type typical of brain tissue. In addition, in one dish, a moderate amount of smooth muscle tissue appeared (visible by phase-contrast optics), and when the CPK isoenzyme pattern was examined a very small amount of M B heteropolymer was observed. There was no detectable MB or MM form of PGM in any of the differentiated cell line extracts.

(1) Isoenzyme transitions in development

The study of isoenzymic transitions in developing mouse tissues gave results summarized in Tables 2, 3, and 4. These are intended to serve as normal tables of some of the changing biochemical events during differentiation. The adult forms of CPK and PGM appear at different times in different muscle tissues; they are apparent in cardiac muscle at the 12th day, in tongue by the 14th day, in most skeletal muscle by the 15th day, and in some kinds of smooth muscle at the 17th day. Developing brain tissue acquires the typical adult-type isomers of aldolase on the 14th day of gestation. In summary, CPK and PGM would appear to be markers of some types of differentiating muscle cells, and aldolase of brain cells.

Although the proportion of the adult form of PGM in early embryonic muscle tissues was lower than that of CPK, the MB and MM forms of PGM appeared at the same time in development. This was equally true of hind-limb skeletal muscle, tongue and cardiac muscle, but the adult forms appeared at a different time in each tissue. The degree of morphological development of the tissues could be roughly correlated with the time of appearance of adult isoenzymic forms. Possibly the parallel appearance of the differentiated forms of CPK and PGM denotes a linked control system such as that suggested by Turner & Eppenberger (1973). These authors argue that a number of proteins such as CPK, aldolase and myosin may be co-ordinately regulated during muscle development. It is interesting that striations are not obvious in sections of 15th day hind-limb examined in the light microscope and so the above biochemical changes occur in advance of the more obvious histological signs of differentiation.

Although the appearance of adult isomers of CPK and PGM was simultaneous in all types of developing muscle, the latter enzyme could prove to be a more reliable marker of the biochemical differentiation of muscle. This is because extracts of all tissues at all stages in development showed no staining on the control gel. There is therefore no ambiguity in the interpretation of the results.

(2) Isoenzyme transitions in teratocarcinoma cells

This is the first report of isoenzyme analyses of cloned teratocarcinoma cell lines. Figure 4 shows that undifferentiated embryonal carcinoma cells are very like early embryonic cells in having only the early embryonic isoenzymes, namely, the BB form of CPK, the BB form of PGM (not shown), and the A4 form of aldolase. The patterns for embryoid bodies formed in vivo were similar. When undifferentiated embryonal carcinoma cells of four different cloned cell lines produced in this laboratory were cultured under conditions to promote their differentiation, they formed morphologically recognisable nerve, epidermal and muscle cells. Extracts from a cloned line (OC15 SI) which produces a large proportion of nerve-type cells gave a highly differentiated brain-type pattern of aldolase isoenzymes. One cloned cell line which differentiated to give some patches of smooth-muscle-like cells as well as nerve cells, produced the corresponding isoenzyme patterns, that is, a band (rather faint) of the M B form of CPK and a set of four bands of A and C aldolases.

For mouse teratocarcinomas to be useful as an alternative to embryos in the study of cell differentiation, they have to be shown to have properties which closely parallel those of embryos. The stem cells of the tumours (embryonal carcinoma cells) have been shown to be pluripotent (Kleinsmith & Pierce, 1964), and similar to early embryonic cells, both ultrastructurally (Damjanov, Solter & Skreb, 1971) and antigenically (Artzt et al. 1973). Histochemical studies have shown that the distribution of alkaline phosphatase follows the pattern in embryos (Bernstine et al. 1973; Solter, Damjanov & Skreb, 1973). Some biochemical properties of in vivo embryoid bodies allowed to differentiate in vitro have been determined. Gearhart & Mintz (1974) followed the increasing acetylcholinesterase specific activities of such cultures and identified striated muscle fibres which eventually formed in them. Levine et al. (1974) showed that specific activities of both acetylcholinesterase and CPK increase during culture of in vivo embryoid bodies, but they correlated these with the production of nerve cells on the basis of histology and the identification of the BB type of CPK. Since embryoid bodies were shown (above) to contain the BB form of CPK before differentiation, there was no transition of isoenzyme type during differentiation.

It is important to show that clonal teratocarcinoma cells differentiating under defined conditions in vitro reflect the orderly processes which occur during the development of the embryo. Martin & Evans (1975) showed that one such clonal line produced embryoid bodies in vitro, and these contained the correct distribution of alkaline phosphatase. Here I have described the distribution in embryos of the isoenzymes of three enzymes, and have shown that differentiating teratocarcinoma cell clones change their patterns similarly.

I wish to thank Miss S. E. Ayers for skilful technical assistance. I am grateful to Dr C. F. Graham for helpful discussions and criticisms, and to Dr M. McBurney for cloned teratocarcinoma cell lines. This work was supported by the Cancer Research Campaign.

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