/ . Embryol. exp. Morph. Vol. 34, 1,pp. 113-123, 1975
Printed in Great Britain
\\3
Immunofluorescence studies on amphibian
myoblast differentiation
BY A. M. DUPRAT, 1 A. ROMANOVSKY, 2
D. HURYCHOVA, 2 AND J. MACHA2
From the Laboratoire de Biologie generate, Universite Paul-Sabatier,
Toulouse, France, and the Department of Experimental Zoology,
Charles University, Praha, Czechoslovakia
SUMMARY
Differentiating myoblasts from urodele amphibians were cultivated in vitro and treated
by immunofluorescence techniques and (or) inhibitors of RNA and protein syntheses. These
experiments lead to the conclusion that the differentiation of these cells is directed by longlived m-RNAs which are already present in morphologically undifferentiated cells and may
act for at least 6 days.
Contrary to observations on cultivated myogenic cells from other groups, the fusion of
urodele myoblasts is not a prerequisite for a normal and functional differentiation (contractions) since isolated myoblasts differentiate quite normally.
The appearance and further evolution of myosin and actin occur synchronously in this
biological system.
INTRODUCTION
The decisive role of proteins in the process of determination and differentiation of eukaryotic cell is generally admitted. Substances known to possess
inductive capacity are proteins (Yamada, 1961, 1962; Tiedemann, 1968; Tiedemann, Born & Tiedemann, 1972; Born, Tiedemann & Tiedemann 1972 a, b) and
although it remains to be demonstrated that normal inductors themselves are
proteins, several authors believe that proteins at least act as carriers for induction. Morphological differentiation itself is often associated with production of
specific proteins (myosin, haemoglobin, etc.). The synthesis of specific proteins
associated with morphological differentiation in one special category of cells is
probably the final link in a chain of events, the beginning of which is located at
the gene level. It is difficult, within a given biological system, to understand the
biochemical mechanisms taking place in the course of this process because of
the complexity of the experimental model where the appropriate information is
often not at hand.
1
Author's address: Laboratoire de Biologie generate, Universite Paul-Sabatier, 118, Rte de
Narbonne, 31077 Toulouse-Cedex, France.
2
Authors' address: Department of Experimental Zoology, Faculty of Science, Charles
University, Vinicna 7, Praha 2, Czechoslovakia.
8-2
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A. M. DUPRAT AND OTHERS
The myoblast is one such system. Its morphological differentiation and the
development of its contractile proteins are relatively well known thanks to a number of works which need not be cited here and which use different technical
approaches (optical and electron microscopy, immunochemistry, electrophysiology, etc.) However, few are devoted to the study of the RNA directing
this differentiation (Duprat, 1965; Yaffe & Feldman, 1965; Duprat, Zalta &
Beetschen, 1966; Yaffe & Fuchs, 1967; Duprat, 1970; Luzzati & Drugeon, 1972;
Luzatti, Loomis, Drugeon & Wahrmann, 1973; Yaffe, Lavie & Dym, 1973;
Buckingham, Caput, Cohen & Gros, 1973).
Comparative studies of the differentiation of myoblasts and neuroblasts in
vitro in the presence of inhibitors of RNA synthesis have enabled us to state
that these two categories of cells exhibit different reactions. For example,
actinomycin D blocks the morphological differentiation of neuroblasts, but does
not affect the development of myoblasts. Inhibitors of protein synthesis interfere with the cytodifferentiation of both cellular types (Duprat et al. 1966;
Duprat, 1970). Several hypotheses have been put forward to explain these
differences. According to Yaffe (Yaffe & Feldman, 1965; Yaffe & Fuchs, 1967)
differentiation of the myoblasts of mammals is directed by the presence of
messenger RNA already synthesized in the undifferentiated cells. The purpose
of this paper is to demonstrate that the message for actin and myosin appears to
be also present in undifferentiated cells of Urodela as demonstrated by immunofluorescence studies.
MATERIALS AND METHODS
Preparation of myosin
Muscles of Rana temporaria were homogenized in the cold ( - 2 0 °C) in 3 vol.
of 0-37 M-KCI and the pH was adjusted to 6-8 by the addition of solid Tris
HC1. The homogenate was left for 20 min at 4 °C and then centrifuged for
30 min at 3000 g. The supernatant was poured into 10 vol. of ice-cold distilled
water which was, again, adjusted to pH 6-8 by addition of solid Tris HC1. One
hour later the resulting precipitate was centrifuged for 15 min at 3 000 g,
transferred to distilled water in a cold measuring cylinder and solid KCl added
to distilled water in a cold measuring cylinder and solid KCl added to give
a final concentration of 0-3 M. After the myosin had dissolved the solution was
centrifuged for 20 min at 12000 g and again precipitated by pouring in 10 vols.
of water. Reprecipitation was repeated three times. If greater impurities of
actomyosin were present, ATP was added to the solution to make its concentration 0-005 M: contraction of actomyosin then takes place and it is centrifuged
more easily. Centrifugation is necessary immediately after addition of ATP.
Contamination with actomyosin is detectable by means of viscosity measurements on the myosin solution in 0-6 M-KCI after addition of ATP to a final
concentration 0-005 M. The viscosity of myosin does not change, whereas the
viscosity of actomyosin is substantially lowered. This effect may be used to
Amphibian myoblast differentiation
115
check the final preparation of myosin and (or) actin. After mixing the solution
of myosin with small amounts of concentrated actin a substantial rise of viscosity
takes place, which drops after addition of ATP. The concentration of myosin
required will depend on the type of viscosimeter used.
2. Preparation of actin
Muscles of Rana temporaria were homogenized in the cold ( - 2 0 °C) in
4-5 volumes of 0-5 M-KCI and 0-05 M Tris HC1 was added to bring the pH to
7-0. The homogenate was left at + 4 °C overnight and centrifuged at 3000 g
for 45 min. If the homogenate was too thick and the muscle debris was sedimenting badly, it was possible to dilute it with extraction solution. The centrifuged extract was slowly poured into 12 vol. of ice-cold distilled water at pH 7-0
(Tris) and after 1 h the precipitate of actomyosin was centrifuged. Afterwards
the precipitate was transferred to a measuring cylinder and solid KC1 was added
to give a fixed concentration of 0-6 M. The solution was centrifuged for 15 min
at 12000 g and again precipitated with water. Reprecipitation was repeated
once more. The last precipitate was centrifuged, the actomyosin homogenized
in 10 vol. of acetone at room temperature. The suspension was filtered by
suction and the filter cake was washed with acetone, desiccated and stored in
a desiccator. Before use actin was extracted with 10 vol. of distilled water at
pH 7-5 (NaHCO3). After centrifugation the pH was adjusted to 4-8 and the
precipitated actin collected by centrifugation. The precipitate was mixed with
an equal volume of distilled water and after the addition of a small amount
of NaHCO 3 , the actin was dissolved. Eventual precipitates were removed by
centrifugation and the solution used for immunization.
The actin preparation showed a single zone in electrophoresis in 8 % acrylamide gel in a continuous buffer system of 0-0192 M glycine + 0-0025 M Tris
(pH 8-9).
3. Preparation of antisera
Anti-myosin. The final product obtained after third precipitation was collected,
dissolved in 0-3 M-KCI, diluted 1:1 with distilled water and an equivalent of
approximately 1 mg of myosin was injected subcutaneously into rabbits daily
for 3 weeks. The collected serum was absorbed with frog kidney extract (0-15
M-NaCl), tested for specificity and stored at — 25 °C. The rabbits were repeatedly
injected after 3-4 weeks rest with a single dose of fresh preparation for several
months and sera collected as above.
Anti-actin. An equivalent of 30-40 mg of actin was given subcutaneously to
rabbits weighing 4-5 kg at weekly intervals for 3 months. Serum was collected
in the usual way, absorbed with frog kidney extract (0-15 M-NaCl) and stored
at - 2 5 ° C
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A. M. DUPRAT AND OTHERS
4. Tests for specificity of antisera
Tests according to Ouchterlony were performed (1) in 0-15 M-NaCl and (2)
in 0-5 M-KCI at pH 7-4 (0-05 M Tris-HCl).
Anti-myosin. A single major component was found to react against the myosin
preparation. Often a very weak additional line was present at the side closer to
the antiserum well. This line was not due to the reaction with actin, as the antiserum did not react with the actin preparation. In reaction with an 0-5 M-KCI
extract of frog muscle two lines usually appeared, one of which was unspecific,
appearing in the reaction of muscle extracts with normal rabbit or frog serum.
As control fluorescence experiments with normal rabbit or frog serum showed
no positive reaction, it seems certain that non-specificity in the agar diffusion
test is not reflected in the experiments described.
Anti-actin. The antiserum gave only one weak precipitin line in the Ouchterlony test in reaction both with the actin preparation and with muscle extract
(0-15 M-NaCl or 0-5 M-KCI). Myosin (0-5 M-KCI) gave no reaction.
5. Preparation of embryonic material
Cells for cultures were prepared from the neural plate and from chordomesoblast of neurulas at stage 13 of Pleurodeles waltlii and Ambystoma< mexicanum
according to the technique already described (Duprat et al. 1966).
Cells cultured on slides were fixed in cold (0-2 °C) absolute ethyl alcohol
overnight, and after 1-2 additional changes of absolute ethyl alcohol at room
temperature were transferred to terpineol where they were stored until used for
immunofluorescence. Before being so used the terpineol was removed in several
changes of absolute alcohol, the slides were transferred to 96 % and 80 % alcohols and then to buffered saline for at least 10 mins.
6. Immunofluorescence
The indirect immunofluorescence technique was used. Slides were exposed to
frog myosin (actin) antiserum for 10 mins in a moist chamber at room temperature. Afterwards they were washed twice in buffered saline for 10 mins and
covered with pig anti-rabbit gamma globulin conjugated with fluorescein
isothiocyanate (Sw AR/FITC, Sevac) for 20 mins. After being rinsed in buffered
saline, cells were counterstained with Evans blue, rinsed again in buffered
saline, mounted in phosphate buffered glycerine and examined immediately.
Controls. Cells (1) transferred to saline without additional procedure, (2)
treated with Sw AR/FITC only, (3) treated with anti-myosin (actin) antiserum
only; (4) cells where normal rabbit serum was used in place of anti-myosin (actin)
antiserum, followed by Sw AR/FITC were examined in parallel with the experimental ones.
Controls gave negative results in all cases.
The slides were examined under a ML-2 fluorescence microscope (OMO,
Amphibian myoblast differentiation
117
USSR) equipped with a high-pressure mercury lamp DRS-250 with exciter
filter FS 1-2 and barrier filters BS 8-2 and S 3S 7-2.
RESULTS
1. Development of cells in culture
Both cellular systems of Pleurodeles and Ambystoma, have an identical
development in culture at 18 °C (Duprat, 1970). Only a general outline of the
major steps of their differentiation will be given here since a preliminary report
has already been published (Duprat & Mathieu, 1973).
After introduction to the culture, the cells of different categories are deposited
on the bottom of the culture chamber where they remain rounded for approximately 48 h. Three to four days are necessary for their attachment on to the
coverslip. In the same chamber, several cell types can be observed: neuroblasts,
melanoblasts, epithelial cells, etc., which are control cells because they do not
present immunofluorescence with anti-serum (myosin or actin). The myoblasts
are either isolated or grouped in clusters. They remain generally fusiform and
do not show any other morphological differentiation. After 5 days in culture,
myofibrillar striation appears first around the nucleus and later, in parallel
with the increase in the volume of the cytoplasm, myofibrils are found also in the
periphery of the cells which, although isolated, are transformed into otherwise
typical muscle cells. Cytological studies at the light and electron microscope
level have confirmed that the differentiation and myofibrillar organization of
these cells is entirely normal (Duprat & Mathieu, 1973). Morphological differentiation of muscular cells is complete and functional, as frequent cell contractions are observed, after 10 days of culture.
II. Effect of actinomycin D, cycloheximide, puromycin and puromycin
aminonucleoside, on morphological differentiation of cells
The effects of these inhibitors has been published elsewhere (Duprat et al.
1966; Duprat, 1969, 1970).
(a) When undifferentiated myoblasts are submitted to the action of actinomycin D (2 /Ag/ml of the culture medium) their differentiation is not inhibited and
not even delayed as compared with control cells. Treated cells show however
nuclear and cytoplasmic alterations typical of actinomycin action. Furthermore, the incorporation of [3H]uridine is inhibited in treated cells while
that of [3H]-leucine or [3H]lysine is not affected (Duprat 1965; Duprat et al.
1966).
(b) When cycloheximide (10-15 /tg/ml) or puromycin (0-10/ig/ml) are added
to the medium, morphological differentiation of myoblasts does not take place;
not all cellular functions, however, are blocked as cells remain alive for approximately 8 days (Duprat, 1969,1970). On the other hand, a substance analogous to
puromycin, puromycin aminonucleoside, which is not an inhibitor of protein
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A.M. DUPRAT AND OTHERS
Amphibian myoblast differentiation
119
synthesis, does not show an inhibitory effect on morphological differentiation
of myoblasts.
III. Analysis of appearance and development of contractile proteins
{myosin and actin) by means of immunofluorescence
The experiments performed with inhibitors of protein synthesis are in agreement with Yaffe's hypothesis (see p. 121) However, the possibility still exists that
the contractile proteins of muscle cells are synthesized in undifferentiated
myoblasts but not organized in myofilaments (Croisille, 1965); and in that case,
puromycin would only prevent this organization by inhibiting the required
enzymes or proteins.
To eliminate this possibility a more accurate detection of actin and myosin
was necessary, and this was achieved by immunofluorescence. The cells were
fixed after attachment to the coverslip at 3, 4, 5, 6, 7, 8, 9, and 11 days.
(a) Appearance and development of myosin and actin
After 3 days of culture only a small number of myoblasts showed weak
fluorescence. The fluorescence of the myoblasts increased on the 4th day of
culture (Fig. 1). This reaction was generally diffuse, its intensity increased with
the progress of cytodifferentiation and it was more pronounced on the periphery
of cells (Figs. 1-5). Later an arrangement of contractile proteins could be observed
as fluorescing unstriated filaments could be detected (Figs. 2, 6) in the whole
cytoplasmic area. Finally, in differentiated cells, light and dark zones of myofibrils appeared, corresponding to the typical striation described by many
authors (Figs. 3, 7). As already mentioned in our preliminary report (Duprat,
Romanovsky, Hurychova & Macha, 1975) no differences were observed in the
appearance and further development of actin and myosin in both Pleurodeles
and Ambystoma.
FIGURES 1-4
m, Myoblasts; ex., epithelial cells; N, nucleus; Y, yolk platelets; C, cytoplasm.
Fig. 1. Myoblasts, 3-4 days of culture. Thick fluorescence.
Fig. 2. Myoblasts, 6-7 days of culture. Fluorescence increases and progresses in
cytoplasm.
Fig. 3. Myoblasts, 8 days of culture. Intensefluorescencein the whole cytoplasmic
area.
Fig. 4. Myoblasts treated with puromycin (010/*g) immediately after their spreading.
Although the cytoplasm is growing, morphological differentiation does not appear
(Duprat, 1970). No immunofluorescence (myosin or actin antiserum).
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A. M. D U P R A T AND OTHERS
Fig. 5. Myoblast 3-4 days of culture. Diffuse fluorescence.
Fig. 6. Myoblast 6-7 days of culture. ->, Fluorescence showing myofilaments.
Fig. 7 Myoblast 9 days of culture. Intense fluorescence. ->, Striated myofibrils.
Amphibian myoblast differentiation
121
(b) Appearance of contractile proteins after treatment of cells with puromycin
In cells treated with puromycin (0-10 /*g/ml) no fluorescence could be detected
in myoblasts. One can assume therefore that no detectable synthesis of contractile proteins takes place either before or after the treatment (Fig. 4).
DISCUSSION AND CONCLUSIONS
The problem of the existence of template m-RNAs which would direct the
differentiation of myoblasts made it necessary to know when the contractile
proteins appear in our biological system. The observations presented above,
obtained with the immunofluorescence technique, indicate that myosin and
actin are not present in myoblasts which are not yet morphologically differentiated, but are synthesized in the course of differentiation of these cells. These
results, together with those from experiments combining treatments with
actinomycin D, which does not prevent normal myoblast differentiation, and
with puromycin, which completely inhibits it, support the aforementioned
hypothesis: m-RNAs, which are necessary for the differentiation of myoblasts,
are already present in cells which are not yet morphologically differentiated.
Moreover, the life-time of these RNAs is prolonged when the manifestation of
cytodifferentiation of the myoblasts is delayed by means of combined treatment
with actinomycin D and puromycin (Duprat, 1970). These RNAs are still
functional after at least 5-6 days.
The experiments allow us also to state that myosin and actin appear simultaneously in the myoblasts of the Urodela used in the present study. This result
is in good agreement with the previous studies on embryos of Anura and Urodela
in toto (Romanovsky, Hurychova & Macha, 1973; Smidova, Hurychova &
Macha, 1974).
Noteworthy is the fact that myoblasts of Urodela have a peculiarity:
they differentiate normally even if they are completely isolated in the culture.
Fusion of mononucleated myoblasts into multinucleated myotubes is a major
step during the differentiation of muscle cells in mammals or chick. If such a
fusion is prevented, the differentiation processes are stopped (Yaffe et al. 1973;
Luzzati et al. 1973). But this phenomenon does not occur in Urodela. This
system can offer a valuable experimental model, for example to permit electrophysiological studies of certain problems of cell physiology in relation to
cyto-differentiation.
In conclusion, it seems possible to confirm that myoblast differentiation of
Urodela, like that of mammals, is directed by template m-RNAs localized in
cells which are morphologically undifferentiated. However, the basic problems
of the nature of the regulatory mechanisms involved in the biochemical and
morphological expression of specific characters, remains to be investigated.
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A. M. DUPRAT AND OTHERS
RESUME
La technique d'immunofluorescence et l'utilisation d'antibiotiques inhibiteurs de la biosynthese de RNA et de proteines, nous ont permis de constater que la differenciation de myoblastes d'Amphibiens, Urodeles, cultives in vitro est dirigee par des m-RNA a 'vie longue',
6 jours au moins, deja en place dans les cellules morphologiquement indifferenciees.
Contrairement aux autres cellules myogeniques cultivees in vitro, la fusion des myoblastes
d'Urodeles n'est pas necessaire pour qu'une differenciation normale et fonctionnelle (contractions) de ces cellules se manifeste; puisque des cellules completement isolees se differencient
tres bien.
Enfin, l'apparition et revolution ulterieure de la myosine et de l'actine se fait de facon
synchrone dans ce systeme biologique.
We wish to acknowledge the encouragement of Professor J. C. Beetschen.
This work was supported by research grants (E.R.A. no. 327) from the 'Centre National
de la Recherche Scientifique', France.
REFERENCES
BORN, J., TIEDEMANN, H.
& TIEDEMANN, H. (1972a). The mechanism of embryonic induction:
isolation of an inhibitor for the vegetalizing factor. Biochim. biophys. Acta 279, 175—183.
BORN, J., TIEDEMANN, H. & TIEDEMANN, H., (19726). Inhibitors in amphibian morphogenesis:
enzymic degradation of an inhibitor for the vegetalizing factor. /. Embryol. exp. Morph. 28,
77-86.
BUCKINGHAM, M., CAPUT, D., COHEN A. & GROS, F. (1973). Synthese d'ARN dans les
cultures primaires des myoblastes. Colloque INSERM, 'Synthese normale et pathologique
des proteines chez les animaux superieurs, pp. 457-474.
CROISILLE, Y. (1965). Synthese et distribution des proteines contractiles, myosine et actine,
pendant le developpement des muscles cardiaque et squelettique chez 1'embryon de poulet.
Meth. nouv. Embryologie (ed. Hermann), pp. 175-199.
DUPRAT, A. M. (1965). Comportement de cellules embryonnaires vivantes de Pleurodeles
waltliicultivees in vitro en presence d'actinomycine D. C. r. hebd. Seanc. Acad. Sci., Paris, 261,
5637-5640.
DUPRAT, A. M., ZALTA, J. P. & BEETSCHEN, J. C. (1966). Action de 1'actinomycineDsur la
differenciation de divers types de cellules embryonnaires del'Amphibien Pleurodeles waltlii
en culture in vitro. Expl Cell Res. 43, 358-366.
DUPRAT, A. M. (1969). Action de la cycloheximide sur des cellules embryonnaires d'Amphibien en cours de differenciation. Etude comparative avec les effets de la puromycine.
Annls Embryol. Morph. 2, 179-190.
DUPRAT, A. M., (1970). Action de la puromycine et d'une substance analogue sur la differenciation de cellules embryonnaires d'Amphibiens cultivees in vitro. J. Embryol. exp. Morph.
24, 119-138.
DUPRAT, A. M. & MATHIEU, C. (1973). Differenciation normale des myoblastes de Vertebres
maintenus isoles en culture in vitro. C. r. hebd. Seanc. Acad. Sci., Paris 277, 1705-1707.
DUPRAT, A. M., ROMANOVSKY, A., HURYCHOVA, D. & MACHA, J. (1975). Experiential,
488-490.
LUZZATI, D. & DRUGEON, G. (1972). The effect of actinomycin D on RNA and protein
synthesis during differentiation of myoblasts of line L 6 E : the half-life of functional
myosin messenger. Biochimie 54, 1157-1168.
LUZZATI, D., LOOMIS, F. W., DRUGEON, G. & WAHRMANN, J. P. (1973). Synthesis of characteristic muscle proteins during myoblast differentiation. Colloque INSERM, 'Synthese
normale et pathologique des proteines chez les animaux superieurs', pp. 475-485.
ROMANOVSKY, A., HURYCHOVA, D. & MACHA, J. (1973). Appearance of actin in embryos of
Rana temporaria studied by immunofluorescence. Folia boi, Praha 19, 43-46.
Amphibian myoblast differentiation
123
A., HURYCHOVA, D., ROMANOVSKY, A. & MACHA, J. (1974). Development of actin
and myosin in Urodela embryos detected by immunofiuorescence. Folia bioi, Praha 20,
177-181.
TIEDEMANN, H. (1968). Factors determining embryonic differentiation. /. cell. Physio I. 72,
suppl. 1, 129-144.
TIEDEMANN, H., BORN, J. & TIEDEMANN, H., (1972). Mechanisms of cell differentiation:
affinity of a morphogenetic factor to DNA. Wilhelm Roux Arch. EntwMech. Org. 171,
160-169.
YAFFE, D. & FELDMAN, M. (1965). The formation of hybrid multinucleated muscle fibres
from myoblasts of different genetic origin. Devi Biol. 11, 300-317.
3
YAFFE, D. & FUCHS, S. (1967). Autoradiographic study of the incorporation of uridine- H
during myogenesis in tissue culture. Devi Biol. 15, 33-50.
YAFFE, D., LA VIE, G., DYM, H. (1973). Changes in gene activity during the differentiation
at multinucleated muscle fibers. Colloque INSERM, 'Synthese normale et pathologique
des proteines chez les animaux superieurs', pp. 451-456.
YAMADA, T. (1961). Chemical approach to the problem of organizer. Adv. Morphogen. 1,1-53.
YAMADA, T. & KARASAKE, S. (1962). Roles of protein and RNA in determining the specific
cellular differentiation. FednProc. Fedn Am. Socs exp. Biol. 26,163.
SMIDOVA,
(Received 31 December 1974, revised 28 February 1975)