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/. Embryol. exp. Morph. Vol. 36, 2, pp. 343-354, 1976
Printed in Great Britain
343
On the determination of the
dorso-ventral polarity in the amphibian embryo:
suppression by lactate of the formation
of Ruffini's flask-cells
ByULF LANDSTROM1, HUGUETTE L0VTRUP-REIN1
AND S0REN L0VTRUP1
From the Department of Zoophysiology, University of Umed
SUMMARY
Cells isolated from the vegetal hemisphere of the blastula of Ambystoma mexicanum
differentiate spontaneously into fibroblast-like cells. Similar cells may be formed from
animal cells, provided they are induced either by vegetal cells or by Li+.
We have found that lactate and various inhibitors of RNA synthesis suppress the spontaneous cell differentiation. The effect of lactate differs from that of the other agents in so far
as lactate must be present before the second day of culture to suppress the outgrowth of
flbroblasts on the third day; the other inhibitors are active also when added on the second
day. An explanation of this difference may possibly be found in the fact that lactate suppresses incorporation of RNA but only by 40 %. The effect on the differentiation of various substances supposed to interfere with the metabolism of lactate was established. The results
obtained were suggestive, but not conclusive.
It is concluded that the effect of anaerobiosis may be explained as a lactate inhibition.
The amounts of lactate under aerobic conditions are so slight that it is unlikely, but not
impossible, that lactate is directly involved in the control of differentiation.
INTRODUCTION
Gastrulation in the amphibian embryo is initiated by the formation of the
dorsal blastoporal lip. The cells responsible for this event are the so-called
'Ruffini's flask-cells', large filopodium-possessing cells (Holtfreter, 1943). The
longest filopodia, attached to the embryonic surface, contain microtubules
(Perry & Waddington, 1966), suggesting that we are dealing with a kind of
fibroblast-like cell. In fact, it seems likely that these cells are engaged in pulling
in the surface in a way similar to that in which the secondary mesenchyme cells
carry out the imagination in the echinoid embryo as described by Dan and
Okazaki (1956) and Gustafson & Kinnander (1956). The activity of Ruffini's
cells leads to a condensation of surface pigment, and the appearance of black
1
Author's address: Department of Zoophysiology, University of Umea, S-901 87 Umea,
Sweden.
344
U. LANDSTROM, H. L0VTRUP-REIN AND S. L0VTRUP
spots or lines is therefore a diagnostic of their presence. In the normal embryo,
the blastopore gradually spreads around the egg until the two ends meet at the
ventral side. This time differential in the formation of Ruffini's cells imposes a
dorso-ventral polarity on the embryo.
It has long been known that gastrulation is completely suppressed by
anaerobiosis (Crawford & Wilde, 1966 a). In fact, by unilateral restriction of the
oxygen supply it is possible to invert the polarity (L0vtrup & Pigon, 1958;
Landstrom & Lovtrup, 1975). The oxygen dependency may be a question of
energy supply (Crawford & Wilde, 19666). Yet this explanation is contradicted
by the fact that when stage-10 embryos (early gastrula) are placed under anaerobic conditions, the process of gastrulation continues, although at a reduced
rate (Gregg & Kahlbrock, 1957). We therefore find it necessary to consider
another alternative, namely, that the formation of Ruffini's cells is dependent
upon the occurrence of certain oxidative processes. The oxidative metabolism
in question might concern either or both of two phenomena, namely, the synthesis of some component essential to the differentiation of Ruffini's cells or the
oxidative removal of some inhibitor. Since carbohydrate metabolism is quite
extensive during the early development, we have investigated the effect of various
glucose metabolities on the spontaneous formation of Ruffini's cells in cultures
of isolated vegetative cells. We here report that lactate completely suppresses
this differentiation.
MATERIAL AND METHODS
Isolation and cultivation of cells. The material for the present investigation was
obtained from embryos of the Mexican axolotl Ambystoma mexicanum. Spontaneously laid eggs were collected and allowed to develop in 7-5 % amphibian
Ringer. Appropriate stages (midblastula to late blastula) were decapsulated by
means of a pair of watch-maker's forceps. The embryos were rinsed in several
changes of sterile 7-5 % Ringer containing streptomycin (25 /^g/ml) and benzylpenicillin (25 i.u./ml). The subsequent experimental steps were made under
aseptic conditions.
Cell explants, containing about 15 cells, were dissected out by means of a glass
FIGURES 1-6
Fig. 1. Two-day culture of a vegetal explant.
Fig. 2. Three-day culture of a vegetal explant showing large yolk-laden fibroblastlike cells.
Fig. 3. Four-day culture of an animal-vegetal explant showing small pigmented
fibroblast-like cells.
Fig. 4. Six-day culture of a vegetal explant showing elongated fibroblast-like cells.
Fig. 5. Six-day culture of an animal-vegetal explants showing nerve cells.
Fig. 6. Three-day culture of a vegetal explant showing the effect of lactate inhibition.
Dorso-ventral polarity in the amphibian embryo
50 ^m
50/zra
n
50 ^m,
50//m
346
U. LANDSTROM, H. L0VTRUP-REIN AND S. L0VTRUP
needle and a hair loop. The explants were isolated either from the region around
the animal pole or from the vegetal surface, not too far from the pigmented
border. The latter explants are significantly larger than the former, owing to the
difference in size between the respective cells.
The explants were rinsed in several changes of a standard growth medium
(Barth & Barth, 1959) and finally transferred to a glass culture chamber (similar
to the one used by Jones & Elsdale (1963)) by means of a Speman pipette (one
to three explants per chamber). Each chamber was filled with 1 ml growth
medium, with or without additional substrates. A certain measure of
disaggregation occurred in the explants, but after 24 h the cells had reaggregated
(Fig. 1). Subsequently, the aggregates attached to and flattened out on the glass
surface of the culture chamber. Each explant was regularly observed and, when
required, photographed in a microscope with camera attachment. All experiments were carried out at room temperature (23 °C).
Our experimental conditions are similar to those employed by Niu & Twitty
(1953), Barth & Barth (1959), Becker, Tiedemann & Tiedemann (1959), Jones
& Elsdale (1963) and Deuchar (1971), except that our explants are much smaller.
As appears, we have not been able to corroborate Deuchar's finding that no
differentiation occurs in small explants.
Autoradiography. Vegetal explants were isolated and placed on slides in Petri
dishes (5 cm$). To the standard growth medium was added 5-[3H]uridine (1 fiCij
ml) and, except for the controls, Na-lactate (10 HIM) or Na-selenate (10 HIM).
The explants were incubated until fibroblast-like cells appeared in the controls,
i.e. for a period of 3 days. At this time the cultures were fixed in Carnoy for
1 h and post-fixed in glutaraldehye (2 % in 0-1 M phosphate buffer, pH 7-8) for
10 h. The slides, rinsed in distilled water and dried, were covered with a stripping
film, Kodak AR-10, and exposed for 3 weeks (Appleton, 1972). The number of
grains per nucleus was counted in selected samples of cells. Background counts
were determined on similarly incubated cells,but in absence of [3H]uridine.
RESULTS
Differentiation of Ruffini's cells. Before the induction of the neural plate has
occurred, the pigmented spots indicating the formation of Ruffini's cells are
always confined to the vegetal hemisphere. This differentiation thus apparently
occurs spontaneously only in vegetal cells. In agreement with this inference we
have observed that in cultures of vegetal cells an outgrowth of typical 'fibroblasts' occurs on the third day at room temperature (Fig. 2). From the appearance of these it may be inferred that they are Ruffini's cells.
The course of events in the normal embryo suggests that only a restricted
number of cells spontaneously undergo differentiation, and that these cells in
turn may induce the differentiation pattern in adjacent cells. This point was
confirmed by the following observations: (1) when the explanted cells were per-
Dorso-ventral polarity in the amphibian embryo
347
Table 1. The spontaneous and induced differentiation o
fibroblast-like cells in explants
Addition
Nature of explant
Vegetal
Vegetal
Animal
Animal
Animal + vegetal
(concentration ITIM)
LiCl 10
—
LiCl 10
—
Differentiated
explants (%)
85
95
0
67
62
No. of explants
150
21
46
49
39
mitted to remain in contact, a substantial fraction of all cells differentiated and
(2) when the original cells were scattered, differentiation was suppressed in most
of their descendants. In further support we may refer to Jones & Elsdale (1963).
who found that 'mesodermal' cells, i.e. cells located not too far above the pigment border, would differentiate in 10-30 % of the explants, provided that the
latter were about 10 times as large as ours, but never in explants of less than 30
cells. Evidently, the likelihood of spontaneous differentiation decreases with the
distance from the vegetal pole, as shown by the fact that we got differentiation
in a high percentage in our small explants. The number of cells is also important, the larger the explant, the greater the chance of differentiation.
Explanted animal cells never spontaneously gave rise to 'fibroblasts'. Two
kinds of treatment are known to provoke their differentiation, sublethal cytolysis (Barth 1941; Holtfreter, 1945) and treatment with Li + (Barth & Barth,
1968). By adding the latter agent to the medium we have found that vegetal
cells transform after 2, instead of 3 days, and that animal cells do so after 4 days.
The animal 'fibroblasts' are distinctly different from the vegetal ones, being
smaller (about 3-4 x ) and containing pigment granules rather than yolk platelets (Fig. 3). Exactly the same cell type appears if animal cells are mixed with a
few vegetal cells (Table 1). It thus appears that the latter through homotypic
induction can impress their differentiation pattern on the animal cells. We
suggest that the phenomenon here described is identical with the 'neural induction' occurring in the normal embryo when the presumptive neural plate during
gastrulation is brought in contact with the invaginating chordocytes.
When the cultures were continued, the cells underwent morphological
changes. The vegetal cells assumed the appearance shown in Fig. 4, but did
not display any distinguishable differentiation pattern. In contrast, we have
observed melanocytes, nerve cells (Fig. 5) and muscle cells in the cultures of
animal cells. In some cases the presence of the muscle cells was recorded through
spontaneous, rhythmical contractions in small cell aggregates. Nerve cells
appear after 6, muscle cells after 8, days of culture. The various results reported
here corroborate the interpretation of the mechanism of the dorsal 'organizer'
previously suggested (L0vtrup, 1974).
348
U. LANDSTROM, H. L0VTRUP-REIN AND S. L0VTRUP
Table 2. Suppression of the spontaneous differentiation of RuffinVs cells
by lactate
Addition
(concentration mM)
Differentiated
explants (%)
No. of explants
Lactate
10
5
2-5
0
7
76
48
27
25
Inhibition by lactate of the formation of RuffinVs cells. A number of substances
involved in the anaerobic and aerobic metabolism of D-glucose, or related to the
latter (sucrose, D-glucose, L-glucose, glucose-1-phosphate, glucose-6-phosphate,
pyruvate, lactate, NAD, NADH, NADP and NADPH) were tested for their
influence on the differentiation of Ruffini's cells. Only one of these, lactate, was
found to be active, inhibiting the transformation completely at 10 mM, and
slightly at 2-5 mM (Table 2 and Fig. 6).
Lactate might act merely by affecting cell morphology. We have dismissed
this possibility by showing that addition of lactate to already differentiated cells
is without any effect. In fact, addition of lactate after 2 days of culture does not
prevent the appearance of differentiated cells on the third day. Thus, the event
crucial for differentiation, which is inhibited by lactate, occurs within the first 2
days of culture. Once this process has taken place, lactate is ineffective.
Lactate inhibition and RNA synthesis. Cyanide, as well as anaerobiosis, suppress completely gastrulation in the amphibian embryo, and under either set
of conditions substantial amounts of lactate are produced (Cohen, 1954; 1955;
Gregg, 1962; Crawford & Wilde, 1966a). Judging from these observations one
might envisage that the inhibition is a direct consequence of the lactate accumulation.
Several authors have found that synthesis of RNA of the messenger type
begins in the late amphibian blastula and this activity is accelerated in the
gastrula (Brown, 1964; Brown & Littna, 1966; Denis, 1968). It is reasonable to
presume that some, at least, of this RNA is involved in the differentiation of
Ruffini's cells. In agreement with this inference it was found by Crawford &
Wilde (1966) that the RNA synthesis is completely suppressed under anaerobic
conditions. It is therefore possible that lactate acts by inhibiting the synthesis of
RNA.
In order to check this point we first studied the effect of various inhibitors on
the formation of Ruffini's cells. Thus, we verified the anticipated suppression of
this process by cyanide. From Table 3 it is seen that a concentration of 100 fM.
is required for complete suppression.
Next, we tried actinomycin D and cordycepin. In Table 3 it is shown that 10
/iu of either of these inhibitors of RNA synthesis suffice to inhibit completely
Dorso-ventral polarity in the amphibian embryo
349
Table 3. Suppression of the spontaneous differentiation of RujfinVs cells
by inhibitors of RNA synthesis
Addition
(concentration /iu)
Differentiated
explants(%)
No. of explants
KCN
100
10
1
0
7
76
48
27
25
0
30
60
20
20
20
0
20
50
20
20
20
0
9
40
20
33
20
Actinomycin
10
1
01
Cordycepin
10
1
01
Deoxynucleosides
2500
500
100
the appearance of Ruffini's cells. It is believed that the effect of cordycepin is
less extensive than that of actinomycin. A possible confirmation of this point
was observed in our cultures; in the presence of actinomycin the cells were completely scattered, while with cordycepin the appearance was similar to that of the
lactate cultures (Fig. 6).
We have previously found that deoxynucleotides suppress the synthesis of
RNA in the amphibian embryo (Landstrom, L0vtrup-Rein & Lovtrup, 1975).
As shown in Table 3, a solution of the four deoxyribosides in a total concentration of 2-5 mM prevents the formation of Ruffini's cells.
These various observations may suggest that even lactate works through
inhibition of RNA synthesis. Yet, there is one fundamental difference between
lactate and the inhibitors of RNA synthesis investigated by us. It was mentioned
above that if lactate is added on the second day, it does not prevent the appearance of Ruffini's cells on the following day. In contrast, actinomycin, cordycepin
and deoxynucleosides are active even when added on the second day. It thus
appears that synthesis of some RNA necessary for the differentiation of Ruffini's
cells occurs between the second and third day, and that this activity is not
inhibited by lactate.
In order to test the effect of lactate on the synthesis of RNA we followed the
incorporation of [3H]uridine as described above. The morphology of the differentiated cells being quite different from that of the undifferentiated ones, we
included a control of selenate-inhibited cells. Selenate inhibits the sulphatation of
mucopolysaccharides (Wilson & Bandurski, 1958), as well as the gastrulation in
23
EMB 36
350
U. LANDSTROM. H. L0VTRUP-REIN AND S. L0VTRUP
Table 4. Incorporation of 5-[3H]uridine in the nuclei of Ruffini's cells
and in cells inhibited by lactate and selenate (n = 10 in each group
Grains per nucleus
(mean ± standard error)
Controls
217 ± 6-6
Lactate (10 ITIM)
130±
Selenate (10 HIM)
Background
204+10-5
7± 1-0
5-1
the echinoid embryo (Sugiyama, 1972). We have convinced ourselves that at 10
mM it suppresses the differentiation of Ruffini's cells, giving rise to cultures indistinguishable from those with lactate (Fig. 6).
Since selenate acts by interference with the sulphate-transferring system, the
effect of selenate must be at a very late stage in the process of differentiation, and
no effect on RNA synthesis should therefore obtain. As shown in Table 4 this
expectation was corroborated, the difference between the incorporation of uridine in the nuclei of Ruffini's cells and of the selenate-inhibited ones is not
significant (P > 0-25). The difference between these and the lactate-inhibited
cells is evidently significant (Fig. 7 and 8), but, remarkably enough, lactate does
not suppress the incorporation completely, only by 40 %.
Kosher & Searle (1973) have shown that a major fraction (> 80 %) of the
sulphated glycosaminoglycans synthesized during pregastrula development is
heparan sulphate. Kraemer (1971) found that heparan sulphate always occurs
in various lines of cultivated fibroblasts, and this has been confirmed and extended to embryonic cells by Conrad & Hart (1975). These observations, in
conjunction with the observed suppression of the differentiation of Ruffini's
cells by selenate, suggest that the presence in the cytoplasm of heparan sulphate is
a necessary condition for the appearance of the typical fibroblast morphology,
with the formation offilopods, etc.
Lactate inhibition and oxidative metabolism. We have tested the effect of
various substances which in one way or another may affect oxidative metabolism
and thus elimination of lactate (Table 5).
The uncoupler dinitrophenol inhibits the formation of Ruffini's cells, in
agreement with the suppression of gastrulation by DNP reported by Wilde &
FIGURES 7 AND 8
Fig. 7. Incorporation of tritium-labelled uridine into the nucleus (N) of a differentiated vegetal cell (Ruffini's cell), x 4500.
Fig. 8. Incorporation of tritium-labelled uridine into the nucleus of a vegetal cell,
inhibited from undergoing differentiation by lactate. Figures 7 and 8 are reproduced
to the same scale, x 4500.
Dorso-ventral polarity in the amphibian embryo
351
.y*
23-2
352
U. LANDSTROM, H. L0VTRUP-REIN AND S. L0VTRUP
Table 5. Effect of the spontaneous differentiation of RuffinVs cells
various metabolic inhibitors
Addition
(concentration ITIM)
Differentiated
explants(%)
No. of explants
0
50
80
35
20
20
48
57
61
23
23
23
39
57
61
23
23
23
0
48
70
23
23
23
Dinitrophenol
01
001
0001
Sodium fluoride
10
1
01
Malonate
10
1
01
Chloramphenicol
1
01
001
Crawford (1963). Sodium fluoride, an inhibitor of enolase and thus of lactate
formation, does not further the formation of Ruffini's cells, rather a slight inhibition was observed. Malonate, an inhibitor of succinate dehydrogenase, has
very little effect on the differentiation. Chloramphenicol inhibits mitochondrial
protein synthesis. As seen, it also inhibits the formation of Rumni's cells.
The experiments reported here are for two reasons not very conclusive. First,
they interfere with the supply of energy and in some cases the inhibition may be
explained by this effect. Second, there are various observations in the literature
suggesting that the oxidative metabolism in the early embryo is substantially
different from that in adult organisms (cf. Lovtrup, 1974), and before we know
more on this point it is difficult to evaluate these result.
DISCUSSION
Our studies have shown, rather unexpectedly we believe, that lactate is an
inhibitor of RNA synthesis in amphibian embryonic cells. Since lactate also suppresses the formation of Ruffini's cells, it may be presumed that some of the
RNA in question is involved in the differentiation of these cells. It is of interest
that lactate is a rather peculiar inhibitor, directed only towards the early stages
of RNA synthesis, as contrasted with actinomycin D and cordycepin.
Is lactate an inhibitor of the differentiation of Ruffini's cells in the course of
normal embryogenesis ? Two observations support this contention. First,
except under very special conditions, the spontaneous formation of this cell
type always occurs in vegetal cells, in which the concentration of carbohydrate is
Dorso-ventral polarity in the amphibian embryo
353
much lower than in the animal ones (Gregg & Lovtrup, 1950), Furthermore, in
hybrids, in which the rate of oxygen consumption is much lower than in normal
embryos (Barth, 1946) blastopore formation begins further towards the vegetal
pole (Moore, 1946), i.e. towards regions of lower carbohydrate content.
Against this effect of lactate speaks the fact that the amount of lactate is very
low under aerobic conditions: less than 0-1 /*gper embryo (Cohen, 1954; Gregg,
1962). We found complete inhibition at 10 mM, and if this concentration were to
obtain in an embryo of 2 /A, the corresponding amount of lactic acid would be
2 jug. Admittedly, if an uneven distribution of lactate is assumed, it is still possible to ascribe an inhibitory effect to lactate under normal conditions.
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(Received 2 April revised 3 June 1976)