/ . Embryol. exp. Morph. Vol. 19, 3, pp. 387-96, May 1968
Printed in Great Britain
387
The role of sodium chloride
in the process of induction by lithium chloride in
cells of the Rana pipiens gastrula
By LESTER G. BARTH 1 & LUCENA J. BARTH 1
Marine Biological Laboratory, Woods Hole, Massachusetts
A study of the effects of a series of monovalent cations, Li + , Na + and K + ,
and a series of divalent cations, Mn 2+ , Ca 2+ and Mg 2+ , upon small aggregates
of cells taken from the presumptive epidermis of Rana pipiens gastrulae revealed that these ions induce nerve and pigment cells (Barth, 1965). The effectiveness of both series of ions as inductors was similar to their effects on
decreasing the electrophoretic mobility of DNA as determined by Ross &
Scruggs (1964).
When it was found that sucrose in glass-distilled water also would induce
nerve and pigment cells the role of ions as inductors came under closer scrutiny.
A study of the nature of the induction by sucrose revealed that a relatively
high concentration of sodium ions was necessary in the culture medium used
after sucrose treatment (Barth, 1966). If after sucrose treatment the cells were
cultured in a medium containing a relatively low concentration of NaCl no
induction occurred, and the cells differentiated into ciliated and mucus cells, as
did untreated controls. One suggestion offered was that sucrose altered the cell
surface and sodium ions penetrated and brought about the actual induction.
In any case the dependence of the induction by sucrose upon sodium ions
brought into question the nature of the induction by other substances such as
LiCl. Does LiCl induce by itself or does it merely change the cell surface so that
sodium ions can enter and effect the actual induction? In the experiments recorded below small aggregates of presumptive epidermis cells were treated for
varying lengths of time with LiCl and then returned to relatively high and low
concentrations of NaCl in our standard culture medium.
A second question arises regarding the period of competence for induction
of nerve and pigment cells. If the induction actually occurs after the LiCl is
removed, then the competent period must be extended farther than revealed by
the LiCl treatment.
1
Authors' address: Marine Biological Laboratory, Woods Hole, Mass. 02543, U.S.A.
388
L G. BARTH & L. J. BARTH
METHODS
The methods of preparing, treating and culturing the aggregates of cells from
the presumptive epidermis of stage 11 (Shumway, 1940) Rana pipiens gastrulae
are given in earlier papers (Barth & Barth, \961a, for references).
RESULTS
The dependence of the inductive effect of lithium chloride
upon the concentration of sodium chloride
Although LiCl at a concentration of 3-0 mg/ml of our standard solution
usually induces nerve and pigment cells when the aggregates of stage 11 presumptive epidermis are exposed for 2 h and then returned to our standard
solution, little or no induction is obtained when the aggregates are returned to
a lower concentration of NaCl, 2-57 mg/ml (Table 1, Exp. 1). The concentration
of NaCl in our standard solution is 5-15 mg/ml. Thus the induction by LiCl is
dependent upon the concentration of NaCl into which the aggregates are
transferred.
A more extensive analysis of the effects of NaCl is recorded in Table 1.
Experiment 2 demonstrates that the LiCl-treated aggregates fail to form nerve
and pigment cells if held in low NaCl (2-0 mg/ml) for 18 h and then transferred
to a solution containing 5-15 mg/ml of NaCl. If, however, the LiCl-treated
aggregates are held in a solution containing 5-15 mg/ml of NaCl for 18 h and
transferred to a solution containing the same concentration, normal induction
of nerve and pigment cells occurs.
Experiment 3 shows that higher concentrations of LiCl do not induce if the
aggregates are held in 2-0 mg/ml NaCl for 18 h before transferring to 5-15 mg
NaCl/ml.
Experiments 4-6 were performed to test whether the low NaCl content was
incompatible with differentiation of the cells or with induction of the cells.
These experiments show that nerve and pigment cells will differentiate in low
NaCl if they have been induced by LiCl followed by high NaCl. The differentiation of the various cell types is the same as in high NaCl but the time required
for differentiation is longer in low NaCl. An interesting variation of the experiment is shown in Exp. 4, where a short exposure to low NaCl does not repress
induction of nerve. We conclude from Exps. 4-6 that high NaCl is necessary to
complete the induction process initiated by LiCl and that then differentiation
will proceed in low NaCl.
The time course of induction by high NaCl after LiCl treatment is shown in
Table 1, Exps. 7-9. Experiments 7 and 8 show that the minimal time in high
NaCl for induction is about 2-5 h. Shorter exposures of 0-1 h result in the
differentiation of ciliated cells. Longer exposures, 3-5 to 18 h, result in sequential induction, i.e. radial nerve, spreading nerve, pigment cells. Finally, in Exp. 10
Ions and induction
389
Table 1. The role of NaCl in induction by LiCl
The concentration of the sodium chloride in our standard solution is varied,
keeping the concentrations of all other ions constant. Stage no.: Shumway (1940);
treatment cone: mg/ml LiCl standard solution containing 5-15 mg/ml NaCl;
h: time in hours of treatment or post-treatment; post-treatment cone: mg/ml of
NaCl in solution; culture cone: mg/ml of NaCl in solution; UPS: unipolar
spongioblasts.
Treatment
Exp. Stage
h
no. no. Cone
No. of
aggregates
Post-treatment
Cone
h
Culture
cone
Types of cellular
differentiation
2-57
Ciliated epithelium, ciliated masses, rare nerve
Ciliated masses, mucus
Nerve, pigment cells,
slate-grey epithelium
Ciliated masses, mucus
Ciliated masses, mucus
Ciliated masses, mucus
Pigment cells, nerve,
mesenchyme
Spreading nerve, UPS
Pigment cells, nerve,
slate-grey epithelium
Ciliated masses
Pigment cells, nerve
Pigment cells, nerve
Pigment cells, nerve,
mesenchyme
Ciliated masses, mucus
Ciliated masses, mucus,
nerve
Radial nerve, spreading
nerve, ciliated masses,
mucus
Spreading nerve, radial
nerve, pigment cells
Ciliated masses
Ciliated masses, mucus,
rare nerve
Spreading nerve
Spreading nerve, rare cilia
Spreading nerve, no cilia
Spreading nerve, pigment
cells
Ciliated masses, ciliated
epithelium, rare nerve
Nerve, pigment cells
Nerve, pigment cells
Nerve, slate-grey epithelium, UPS
Nerve, slate-grey epithelium, UPS
1
11
30
20
75
2
11
11
30
30
20
20
60
60
200
5-15
18
18
515
515
3
11
11
11
11
30
30
30
30
20
2-5
30
20
40
200
200
200
515
18
18
18
18
5-15
515
515
200
11
11
30
30
20
20
30
10
200
515
30
18
515
5-15
11
11
11
11
30
30
30
30
20
20
20
20
40
30
30
75
200
515
515
515
18
18
18
18
200
200
515
200
11
11
30
30
20
20
35
40
515
5-15
01
2-5
2-57
2-57
11
30
20
35
5-15
3-5
2-57
11
30
20
40
515
180
2-57
8
,
11
11
30
30
20
20
40
35
2-57
515
10
10
2-57
2-57
9
11
11
11
11
30
30
30
30
20
20
20
20
40
35
40
50
515
515
515
515
4-5
2-5
50
18
2-57
2-57
2-57
2-57
11
30
20
20
2-57
18
2-57
11
11
11
30
30
30
2-3
2-3
2-3
35
35
35
3-40
5-15
3-40
18
18
18
3-40
3-40
5-15
11
30
2-3
35
515
18
515
4
5
6
7
10
390
L. G. BARTH & L. J. BARTH
the effect of 3-4 mg/ml of NaCl is compared with 5-15 mg/ml. Holtfreter's solution and Niu-Twitty solution, commonly used for urodele gastrulae cells, contain 3-4 mg/ml. It is clear that both concentrations induce after treatment with
LiCl.
Table 2. Induction with combinations of LiCl and NaCl
Cone: mg/ml; h: time of treatment; Na + : NaCl; Li + : LiCl. Other ions are present in the
normal concentrations used in our standard solution.
Treatment
Exp. Stage
no. no.
A
(
Cone.
h
No. of
aggre- Culture
gates cone.
Types of cellular
differentiation
Na+
1
2
3
.
4
5
111111
40 Li+
40 Li+
60 Li+
40
40
10
40
40
25
2-57
11
60 Li+
20
25
515
11
11
11
11
11
11
11
11
11
11
60 Li+
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na +
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na+
3-0 Li+, 2-57 Na+
4-3
40
40
70
70
20
20
20
20
50
25
30
30
30
30
30
30
25
25
25
515
515
11
11
11
11
3-0 Li+, 2-57 Na+
1-0 Li+, 5-15 Na+
30 Li+
30 Li+
19
18
40
18
25
40
40
40
2-57
11
40 Li+
40
40
4-50
11
40 Li+
40
4-50
18
515
515
2-57
5-15
2-57
515
2-57
515
2-57
2-57
200
4-50
4-50
Few pigment cells
Ciliated masses, mucus
Spreading nerve, radial nerve,
slate-grey epithelium
Pigment cells, spreading
nerve
Pigment cells
Pigment cells, nerve
Ciliated epithelium
Pigment cells, nerve
Pigment cells, nerve
Pigment cells
Pigment cells, nerve
Nerve, ciliated epithelium
Ciliated masses, mucus
Ciliated epithelium, pigment
cells
Pigment cells
Pigment cells
Ciliated masses
Pigment cells, ciliated
epithelium
Pigment cells, ciliated
epithelium
Many loose cells, large
pigment ring cells
The synergetic action of LiCl and NaCl
The foregoing section shows clearly that induction does not occur during a
2 h exposure to LiCl unless the treatment is followed by several hours exposure
to high NaCl. What part does LiCl play in the induction process? Table 2,
Exps. 1 and 2, shows that 6-0 mg/ml of LiCl is necessary to produce the usual
induction in the absence of NaCl. However, if LiCl and 2-57 mg/ml NaCl are
applied together, only 3-0 mg/ml of LiCl are needed to induce nerve and pigment
cells (Exps. 3 and 4). Therefore LiCl and NaCl act synergetically to produce the
induction. If the Li + and Na + chlorides act only from 2-4 h, the induction must
be completed by high NaCl (5-15 mg/ml). However, if the treatment is extended
Ions and induction
391
to from 5 to 20 h, then pigment cells are induced even when the treatment is
followed by low NaCl (2-57 mg/ml). Finally a very low concentration of LiCl
combined with a high concentration of NaCl (Exp. 5) will induce pigment cells
after 18 h even when followed by a low concentration of NaCl (2-0 mg/ml).
In summary, Tables 1 and 2 show that LiCl does not induce by itself in short
periods (2-4 h) and any induction obtained is dependent upon the concentration
of NaCl into which the aggregates are transferred. Thus a two-step process is
suggested. LiCl alters the cell surface and NaCl penetrates and induces.
Longer periods of exposure to Li + in the presence of NaCl (7-20 h) result in
induction regardless of the concentration of NaCl to which the aggregates are
transferred. Thus we suggest that LiCl not only alters the cell surfaces but also
may penetrate with NaCl to effect the induction.
Table 3. Competence for pigment cell induction in neunda stages
Stage: Shumway, 1940; cone: mg/ml of LiCl in standard solution (LiCl used in both
stages in all experiments); h: time of exposure.
First treatment
Exp. Stage
no. no. Cone.
Second treatment
r
Stage
no. Conc.
h
Types of cellular differentiation
11
11
11
11
11
11
11
30
30
30
10
10
10
10
2-25
2-25
2-25
200
700
1-50
300
15
15
30
30
10
3-5
15
15
1414-
30
30
30
30
2-5
2-5
30
30
11
10
1-50
14
30
2-5
11
10
1-50
5
11
10
1-50
15
10
1-5
6
11
10
10
10
10
10
10
10
3-50
1-50
1-50
1-50
15
1414-
10
30
30
30
1-5
30
Mesenchyme, pigment cells
Mesenchyme, pigment cells
Nerve
Rare nerve and pigment cells
Pigment cells, rare nerve
Ciliated masses, muscle, epithelium
Epithelium, ciliated masses, pigment
cells
Pigment cells, ciliated masses, rare
nerve, rare muscle, epithelium
Ciliated masses, rare nerve, muscle
and pigment cells
Ciliated masses, rare epithelium and
nerve
Rare pigment cells and nerve
Ciliated masses, rare nerve
Ciliated masses, nerve
Radial nerve, ciliated masses.
1
2
3
4
h
Competence in relation to the time of induction
The foregoing experiments show that induction does not necessarily occur
during the treatment with lithium chloride, and this fact requires a reinvestigation of competence for pigment cell induction. For example, the loss of so-called
competence for induction of pigment cells by the lithium ion is total by the end
of gastrulation whether the test cells be left in situ within the embryo (Lasher,
392
L. G. BARTH & L. J. BARTH
1965) or whether they are cultured in vitro and subjected to the lithium ion at
different times (Barth & Barth, 1967a). However, since pigment cells are at the
end of a sequence of induction which begins with nerve induction, it is clear
that when the competence to nerve is lost the whole sequence fails. Thus the
presumptive epidermis at late gastrula has lost the competence to be induced to
form nerve and one would not expect any of the rest of the sequence to be
induced. If, however, such late gastrula cells had received at the early gastrula
stage an inductive stimulus to induce nerve, would they then be competent for
further induction in the sequence, i.e. pigment cells?
To answer this question small aggregates of the presumptive epidermis from
mid-gastrula stages were prepared in the usual way. After treatment with lithium
chloride at a concentration known to induce nerve but not pigment cells, they
were returned to the standard solution for an overnight period. Whole egg controls showed that the cells were then stage 1 4 - to stage 15. At this time the
cells were transferred to lithium chloride for a second treatment which constituted a test for competence to respond to pigment cell induction.
Table 3, Exp. 1, records the results of a single and two double treatments
with lithium chloride. The single treatment at stage 11 resulted in the differentiation of nerve, while the second treatment, at stage 15, carried the induction
to pigment cells and mesenchyme. Clearly the induced nerve cells are competent
to be induced to pigment cells as late as stage 15.
When gastrula cells were treated with a lower concentration of lithium chloride as in Exps. 2-4, subsequent treatment at the equivalent of neurula stages
also gave pigment cell induction. The conversion from epidermal types of differentiation was, however, less complete so that pigment cells formed a lower
proportion of cell types after the second treatment at the neurula stage. The
same kind of subminimal induction is also shown in Exp. 5, where both first
and second treatments are made with low lithium chloride. But here too a few
pigment cells were induced by the longer exposures. Experiment 6 serves as a
partial control since the first lithium treatment was made at early gastrula stage,
a time of low competence to respond fully to a low concentration of the neural
inductor. The subsequent treatment at the neurula stage had no effect, and no
pigment cells were induced. The first treatment induced some radial nerve but
probably this is not far enough along in the sequence to be further induced to
pigment cells. Nerve induction must proceed to the spreading nerve step in the
sequence before pigment cells can be induced.
DISCUSSION
In a previous paper (Barth, 1966) the experiments reported suggested that
the induction process in the case of sucrose was at least a two-step process.
First, the sucrose altered the cells in some manner, possibly increasing their
permeability to NaCl; and then the NaCl in the culture medium actually effected the induction. At any rate, it was clear that induction by sucrose did not
Ions and induction
393
occur when transfer was made to a low concentration of NaCl, while excellent
induction was obtained in relatively high concentrations of NaCl.
Some of the data in this paper where LiCl is used as an inductor may be
interpreted in the same way. Relatively high concentrations of LiCl applied for
a short time do not induce unless followed by a high concentration of NaCl.
This fact suggests that LiCl changes the cell so that NaCl can penetrate and
carry out the process of induction.
Other data show that Li + may actually also participate with NaCl as an
inductor. For example, LiCl acting together with a low concentration of NaCl
for a relatively long time will induce the cells so that they will differentiate even
in a low concentration of NaCl. In these cases the induction by LiCl is independent of the concentration of the NaCl to which the cells are transferred.
The foregoing results with LiCl are diametrically opposite to those obtained
with sucrose. If sucrose is combined with a low concentration of NaCl there is
no inductive effect regardless of the concentration of NaCl in the medium to
which the cells are transferred. In other words, while sucrose and NaCl are
antagonistic as regards induction, LiCl and NaCl are synergetic. This latter
fact could be explained if LiCl in addition to altering the cell surface also
entered with NaCl to effect the induction.
A comparison of the effects of NaCl and LiCl on the presumptive epidermis
of Triturus pyrrhogaster gastrula has been made by Masui (1966). He comes to
the conclusion that while NaCl is effective in inducing neuralization, LiCl will
induce mostly mesodermal structures and little neural tissue. Treatment by
NaCl first followed by LiCl treatment results in an increased neuralization with
some mesodermalization. Thus there is some evidence for an antagonism between LiCl and NaCl as regards the type of induction. We have found that in
Rana pipiens NaCl and LiCl both will induce neural cells but that a higher
concentration of NaCl is required to produce the same induction as LiCl. In
addition, LiCl will induce pigment cells but NaCl will not. It would appear that
while Li + and Na + may add their effects as in the experiments reported in this
paper, LiCl alone can induce pigment cells, while NaCl alone cannot.
The reinvestigation of competence for pigment cell induction by LiCl shows
that competence lasts until stage 15 if the presumptive epidermis is first induced
to nerve at stage 11. The previous finding (Lasher, 1965; Barth & Barth, 1967 a)
that competence for pigment cell induction was lost at stage 12 is now open to a
different interpretation. In all probability what is lost at stage 12 was the competence for nerve induction, and since nerve induction must precede pigmentcell induction, the test was not an adequate one for competence for pigment cell
induction. The present test appears to be a more incisive one, i.e. first induce
nerve at stage 11 and then later test this induced nerve for pigment-cell
competence.
The origin of the concept of two primary inductive principles (activating or
neuralizing; and transforming or mesodermalizing) has been thoroughly docu-
394
L. G. BARTH & L. J. BARTH
mented by Saxen & Toivonen (1962). When Yamada (1958) reported that progressive denaturation of a mesodermalizing agent (bone-marrow factor) led to a
gradual decrease of mesodermal inductions but a gradual increase in the activating or neuralizing principle, he also suggested that the concept of two factors
be reconsidered in terms of a single factor whose effect could be modified by
the presence of different active groups of the same principle, or to a different
spatial configuration of one and the same molecule. This unifying idea was
reinforced by Nieuwkoop & Van der Grinten (1961) when their transplantation
experiments gave results which indicated that the chemical processes which
characterize activation and transformation are rather closely related. Transformation is even partially reversible under the influence of a renewed confrontation of competent tissue with a strong activating influence.
Meanwhile a divergent conceptual pathway had been proposed (for example,
Barth & Barth, 1959, 1962; Masui, 1961, 1966), which appears now to be
merging with the above-mentioned experiments with heterogeneous inductors.
This second approach was based upon the discovery that an ion (lithium)
applied for different durations or in different concentrations could evoke stepwise a series of different cell types in amphibian embryos.
When Masui (1966) reported that the mesodermalizing actions of the lithium
ion depended upon pH and also could be modified by the presence of other
ions known to be either weak or strong neuralizing agents, the need for the
concept of two specific heterogeneous, high-molecular-weight compounds
appeared to be removed. Qualified by differences among species with respect to
susceptibility or permeability, a single inorganic ion may act as an inductor
whose effect may be modified by the micro-environment of the responding cells.
The relation between the actions of heterogeneous and ionic inductors may,
as suggested by Barth (1966), consist in the former so altering the membrane
properties of responding cells as to make available the ions to interior cell
compartments. If, as suggested by Barth (1965), the ions enter into complexes
with DNA molecules, specific synthetic pathways might be set in motion. There
is also the possibility that ions can trigger release of energy sources stored within
mitochondria, or within yolk platelets (Flickinger, 1962; Karasaki, in Yamada,
1962; Tseng, Mo & Chang, 1965).
We can only speculate upon the mechanism of these primary inductive effects.
We find it interesting to point out, however, that Toivonen (1953) proposed the
idea that regional specificity in the living archenteron roof is based upon regional
differences in permeability of the cells from which active substances must diffuse.
Thus the composition of the immediate environment may control release of
inductors from inside the cells, or the entrance of an inductor from the medium.
Our own results (Barth & Barth, 1967 b) tend to suggest that primary induction
may consist in altering cell membranes so that ions normally present in the
environment of the cells may enter.
This idea of activation of pre-set sequences in differentiation may be traced
Ions and induction
395
to its origin in Holtfreter's (1938) studies, which first established the autonomous
character of the change with age in competence of cells to respond to natural
inductors within the embryo. Subsequent studies too numerous to mention in
detail (for example, recently, Nieuwkoop & Van der Grinten, 1961; Leikola,
1965) have been concerned with sequential changes in competence to respond to
neuralizing and mesodermalizing inductors. The present study confirms an
earlier suggestion of one of us (L.G.B.) that competence for later steps in a
necessary sequence of inductions is extended in time provided that during the
initial period an activating or neuralizing stimulus has been applied.
SUMMARY
1. The induction of Rana pipiens stage 11 presumptive epidermis by high
concentrations of LiCl applied for short periods of time is dependent upon the
concentration of NaCl in the solution in which the cells are cultured.
2. When low concentrations of LiCl are used for long periods of time the
induction becomes independent of the concentration of NaCl.
3. The period of competence for pigment-cell induction by LiCl extends to
stage 15 if the presumptive epidermal cells are first induced as far as nerve by
LiCl at stage 11.
RESUME
Le role du chlorure de sodium dans le processus de ^induction par le chlorure
de lithium, sur des cellules de la gastrula chez Rana pipiens
1. L'induction d'epiderme presomptif du stade 11 de Rana pipiens par de
fortes concentrations de chlorure de lithium appliquees pendant de courtes
durees, depend de la concentration en NaCl de la solution dans laquelle sont
cultivees les cellules.
2. Quand on utilise de faibles concentrations de LiCl pendant de longues
durees, l'induction devient independante de la concentration en NaCl.
3. La periode de competence pour l'induction de cellules pigmentaires par le
LiCl s'etend jusqu'au stade 15 si les cellules epidermiques presomptives sont
d'abord induites au stade 11 par le LiCl, jusqu'a pouvoir donner des cellules
nerveuses.
This work was supported by U.S. Public Health Service Grant no. HD00701-3 to the
Marine Biological Laboratory.
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