J. Embryo!, exp. Morph., Vol. 15, 3, pp. 371-386, June 1966
With 1 plate
Printed in Great Britain
371
pH-dependence of the inducing activity
of lithium ion
ByYOSHIO MASUI
From the Department of Biology, Konan University
INTRODUCTION
Previous investigations of primary induction in amphibian embryos have
shown that there are two kinds of heterogenic inductors, one of which induces
the presumptive ectoderm towards neural differentiation alone, the other
towards mesodermal or endodermal differentiation, and that they are characteristically distinct from one another in chemical nature (Yamada, 1961; Saxen
& Toivonen, 1962). Various substances which are chemically dissimilar to each
other have been found to be effective in neural induction, though their inducing
activity was always thermostable. On the contrary, mesodermal and endodermal
inductions were found to be caused only by certain proteins from vertebrate
tissues, and their inducing activity was always thermolabile.
The neural induction of gastrula ectoderm can also be brought about by
exposing the tissue to saline solution under appropriate conditions but in the
absence of specific inductor, whereas this is not the case in the mesodermal and
endodermal inductions (Barth, 1941; Holtfreter, 1944, 1945, 1947). Hence, it
appears that the mechanism of neural induction is not the same as that of
mesodermal and endodermal inductions. However, recent investigations of the
action of lithium ion on the differentiation of the gastrula ectoderm have
disclosed that the ion induces a broad spectrum of differentiation, not only of
neural but also of mesodermal and endodermal character (Barth & Barth, 1959,
1962,1963,1964; Gebhardt & Nieuwkoop, 1964; Masui, 1958,1959, I960,1961;
Ogi, 1961).
This raises a question of whether the specificity of induction of the differentiation of the ectoderm of amphibian gastrula resides in the inducing substance.
A single kind of molecule such as LiCl, which presumably does not carry specific
information because of the simplicity of its molecular structure, is capable of
inducing a variety of differentiations. Some authors have claimed that the
differentiations of gastrula ectoderm are conducted by two different inductors,
but that a mutual control system operates between them so that the differentiations are qualified by the co-operative action of these inductors (Yamada, 1950,
1958; Nieuwkoop et al 1952; Toivonen & Saxen, 1955; Takaya, 1957, 1959;
1
Author's address: Department of Biology, Konan University, Okamoto, Kobe, Japan.
372
YOSHIO MASUI
Saxen & Toivonen, 1961). For the purpose of examining this hypothesis I have
attempted to test the effect of LiCl on the differentiation of gastrula ectoderm
under the influence of varying pH and some neuralizing agents.
MATERIALS AND METHODS
From gastrulae of Trituruspyrrhogaster at St. 11 or 12 a of Okada & Ichikawa's
(1947) developmental table pieces of presumptive ectoderm were dissected with
tungsten needles by referring to the map of Nakamura (1942). Care was taken
to avoid contamination with the presumptive mesoderm and endoderm, but the
purity of material was checked in each experiment by explanting some of the
pieces, which were sampled randomly, without treatment. Such control explants
examined in fifty-five cases were all found to differentiate into atypical epidermis
alone, proving them to be free of contamination.
For the treatment twelve pieces of the ectoderm at a time were transferred to
a 6 cm wax-coated Petri dish containing 25 ml of the test solution, and each was
placed in a separate depression in the wax of 3 mm diameter and 1-5 mm
depth, and allowed to stand for a specified period of time and at a specified
temperature.
As test solutions, LiCl, KC1, NaCl, CaCl2, MgCl2, ZnCl2 and BeCl2 solutions
and LiCl solutions containing one of NaCl, KC1, CaCl2, MgCl2, ZnCl2, NH4C1
or urea were used. These solutions were prepared by dissolving each substance
in distilled water unless otherwise specified. The pH of the solutions containing
LiCl was adjusted with 0-1 M-LiOH or 0-012 M-HCI, while the pH of those not
containing LiCl was adjusted with 0-1 M-NaOH or 0-012 M-HCI. Such pH
adjustment had negligible effect on the molarity of the solution. All the solutions
were autoclaved for 30 min at 100 °C before use.
In order to minimize variation of the pH of the test solution during the treatment, the dishes containing the solution and the tissues to be examined were
enclosed in an air-tight glass chamber together with a beaker containing 50 ml
of 20% NaOH. pH determinations, which were made with a glass electrode
immediately before or after the treatment and with test paper during the
treatment, showed that the pH usually remained at the specified value ± 0-2 pH
units throughout the treatment.
At the end of the treatment the test solution was replaced with Holtfreter's
solution by repeated pipettings to rinse the treated tissues. The tissues treated
were cultured each in a vessel containing 1 ml of Holtfreter's solution at 18 °C
for 2 weeks. During culture daily observation of the explants was made with a
low-power microscope. A final histological examination was made by usual
methods.
pH-dependence of inducing activity
373
RESULTS
The effect of lithium and some other metallic ions
Solutions of LiCl, KC1, CaCl2, MgCl2, ZnCl2 and BeCl2 were prepared by
dissolving each salt at a concentration of 006 M in a bicarbonate-free Holtfreter's
solution of two-thirds the normal strength. The ectodermal explants were immersed in the solution for 3 h at 25 °C, and at the pH indicated in Table 1. No
dissociation of the ectoderm was brought about in the period of treatment
except in the case of treatment with ZnCl2 solution in which the tissues were
always dissociated completely. The zinc-treated ectoderms, however, recovered
from dissociation within 24 h to form round bodies with a brown surface. The
lithium-treated ectoderms frequently formed humps with a whitish surface
Table 1. Differentiation of ectodermal explants treated with salt solution
Percentages for dissociation and survivors are of the total number of experimental explants,
but for those for differentiation are of the number of survivors.
LiCl
PH
70
KC1
5-4
MgCl2
CaCl2
ZnCl2
BeCla
5-4
5-4
41
4-5
24
34
Total number of
31
29
34
31
explants
Dissociation
0(0%)
0(0%)
0(0%)
0(0%) 34(100%) 0(0%)
during treatment
Survivors
28(97%) 29(94%) 32(94%) 29(94%) 33(97%) 14(58%)
25
32
14
29
27
17
Epidermis
0
0
6
0
0
8
Mesenchyme
Neural
0(0%)
0(0%)
1(3%)
1(3%) 27(82%) 0(0%)
0 (0%)
0 (0%)
Mesodermal
0(0%)
0(0%)
3(11%) 0(0%)
(overall)
0
0
0
0
0
0
Notochord
0
3
0
0
0
0
Muscle
0
0
0
0
0
0
Pronephros
Yolky tissue
10(35%) 0(0%)
0(0%)
(0(0%)
0(0%)
0(0%)
(endodermal)
which were often segregated from the ectodermal vesicles and attached to the
bottom of the culture vessel, giving rise to the migration of spindle-shaped cells
over the glass surface. All the ectoderms treated with ions other than lithium
and zinc were found to form wrinkled bodies covered with atypical epidermis.
Generally the explants were found to develop well, except that berylliumtreated explants were liable to disintegrate, expelling necrotic cells during
culture.
Histological examination showed that in some of the explants treated with
lithium chloride solutions muscles and massive yolk-laden tissues were produced (Table 1). On the other hand, in the zinc-treated explants neural tissues
were produced frequently and formed archencephalon (Plate 1, fig. A). Neuroid
or weakly differentiated neural tissues were found in some of the explants
24
JEEM 15
3-5-3-7
4-0-4-2
4-4-4-6
4-9-5-1
6-0-6-2
3-7^-1
4-9-5-3
5-7-6-1
6-8-7-2
7-7-81
7-8-8-2
8-8-9-2
9-8-10-2
8-6-9-0
9-2-9-6
30(67%) 34 (68%) 33 (92%)
13
16
10
8
3
3
5(17%) 6 (18%) 10 (30%)
8(27%) 18 (53%) 21 (64%)
0
4
14
21
8
18
0
2
0
17(57%) 22 (65%) 14 (42%)
28 (39 %)
0
0
19 (68 %)
23 (82%)
14
23
0
14 (50%)
45
72
50
36
11(24%) 34 (68%) 24 (67%) 72 (100%)
6-8-7-2
39
30
54
31
40
34
28
Total explants
28(100%) 22 (65%) 37 (95 % ) 24 (80 % ) 54 (100 % ) 31 (100 % ) 40 (100 % )
Dissociation during
treatment
28(100%) 26 (71%) 33 (85 % ) 30 (100 % ) 36 (67 % ) 26 (84 % ) 29 (72 % )
Survivors
16
23
33
30
36
25
27
Epidermis
3
6
3
8
9
0
0
Mesenchyme
24(86%) 4 (15%) 12(36%) 8(27%) 15(42%) 14(54%) 21(72%)
Neural
0(0%)
0(0%) 0(0%) 0(0%) 0(0%)
0 (0%)
0(0%)
Mesodermal (overall)
0
0
0
0
0
0
0
Notochord
0
0
0
0
0
0
0
Muscle
0
0
0
0
0
0
0
Pronephros
0(0%) 0(0%) 0(0%) 0(0%) 0(0%)
0 (0%)
Yolky tissue (endodermal) 0(0%)
PH
NaCl treatment
Total explants
66
34
42
46
52
Dissociation during
66(100%) 30(88%) 30(71%) 14(30%) 10(19%)
. treatment
Survivors
30(45%) 26(76%) 30(71%) 30(65%) 28(54%)
Epidermis
5
8
14
16
12
Mesenchyme
5
2
3
2
8
Neural
25(83%) 10(39%) 11(37%) 4(13%) 4(14%)
Mesodermal (overall)
20(67%) 16(62%) 17(57%) 8(17%) 8(29%)
15
6
Notochord
5
2
0
20
16
Muscle
17
8
8
0
2
Pronephros
0
0
0
Yolky tissue (endodermal) 0(0%) 10(39%) 14(47%) 20(67%) 22(79%)
pH
LiCl treatment
Percentages as in Table 1.
Table 2. Differentiation of ectodermal explants treated with 0-06 M-LiCl or NaCl at different pH''s
o
o
4
/. Embryo/, exp. Morph., Vol. 15, Part 3
PLATE
YOSHIO MASUI
facing p. 375
pH-dependence of inducing activity
375
treated with calcium or magnesium ions, though true neural differentiation
occurred only rarely, to form a small neural vesicle of archencephalon type. All
the explants other than those mentioned above were found to form a mass of
atypical epidermis alone.
The influence ofpH upon the effect of lithium ion
Explants were exposed to 0-06 M-LiCl for 3 h at 26 °C at a pH ranging from
3-0 to 10-2. As a control the same treatment was carried out with 0-06 M-NaCl
solution. Results summarized in Table 2 showed that in both NaCl and LiCl
treatments the treated ectoderms were more or less dissociated, the frequency
with which complete dissociation was brought about within the period of
treatment depending on the pH at which the treatment was carried out. The
frequency of dissociation was usually lower in the LiCl treatment than in the
NaCl treatment, and it was lowest near pH 6-0 in the LiCl treatment and near
pH 5-0 in the NaCl treatment. Reaggregation of the dissociated explants was
easily brought about by transferring into Holtfreter's solution after treatment
with NaCl solution between pH 3-7 and 9-6 or with LiCl solution between pH
3-5 and 10-2. However, the dissociated tissues showed no sign of reaggregation
if they had been treated outside the pH ranges indicated above.
The NaCl-treated explants showed differentiation of ectodermal tissues alone,
mainly resulting in neuralization and sometimes in the formation of mesenchyme
and sensory organs (Plate 1, fig. B). On the other hand, the LiCl-treated explants
underwent not only neural but also mesodermal differentiations. Most of the
neural tissues produced were lacking regional specificity, forming neural masses.
Only in a few cases were neural tubes resembling spinal cord formed as a part
of axial structures (Plate 1, fig. C). Together with or without neural tissues,
PLATE 1
Fig. A. Neural differentiation brought about in an explant treated with ZnCl2 at pH 4 1 .
x90.
Fig. B. Differentiation of nasal placode and eye vesicle brought about by treatment with
NaCl at pH 90. x90.
Fig. C. Neural and medosermal differentiations brought about by treatment with LiCl at
pH 4-5. Photograph shows bilateral development of the explant in which neural tube and
notochord are located in the median part and somites are in the lateral part, x 90.
Fig. D. Mesodermal and endodermal differentiations brought about by treatment with LiCl
at pH 10-0. A gut-like structure was found beneath the somites, x 90.
Fig. E. Completely mesodermal differentiation brought about by treatment with calciumcontaining LiCl solution. The explant shows the differentiation of voluminous notochord
and underdeveloped somites, but no neuralization. x 90.
Fig. F. Epidermis formation brought about by treatment with calcium-containing LiCl
solution. The section photographed shows only the formation of epidermis and a mass of
undifferentiated muscle-like tissue which early segregated from the epidermis and attached
to the glass surface, but from other sections of the same explant, notochord and somite are
shown to be formed in connexion with the undifferentiated tissue in the photograph, x 100.
24-2
376
YOSHIO MASUI
notochord and somites were frequently differentiated, but pronephric tubules
were rarely found. No blood cells were ever seen. Besides these tissues masses
of yolk-laden cells with a structure resembling primitive gut were found (Plate
1, fig- D).
100% r
o
90
80
.csi
70
iffer
<D
60
50
°
o
c
40
30
1 20
£
10
9
10
pH
Text-fig. 1. The frequency of neuralization as a function of the pH at which ectoderms
were treated. Open circles: neuralization of LiCl-treated explants. Filled circles:
neuralization of NaCl-treated explants. Vertical bars: confidence interval of the
frequencies on a level of confidence coefficient 0-95; the broken and straight bars
corresponding to the open and filled circles respectively.
100%
ion
90
80
70
ffere
c
60
50
(4-1
o 40
o
p
30
20
10
10
Text-fig. 2. The frequency of mesodermalization and endodermalization of Li-treated
explants at varying pH. Filled circles: mesodermalization frequency. Open circles:
endodermalization frequency.
From Text-figs. 1 and 2 it is apparent that all the modes of differentiation are
affected by the pH at which the ectoderms were treated. Neuralization occurred
with minimum frequency near pH 5-0 after both LiCl and NaCl treatments,
but it was brought about with increasing frequency as the pH was raised or
pH-dependence of inducing activity
311
lowered. Similarly, the mesodermal differentiations of LiCl-treated explants
were brought about with minimum frequency near pH 5-0, but increased in
frequency with shift of pH in either direction. On the other hand, the production
of atypical epidermis and yolk-laden tissues was brought about with maximum
frequency near pH 6-0, and decreased at more acid or alkaline pH.
From the result it is evident that while NaCl is effective only in inducing
neuralization of ectoderm, LiCl is capable of inducing mesodermal differentiation in addition to neuralization under the same conditions. This seems to imply
that mesodermal induction of the ectoderm results from a specific action of the
lithium ion which is not shown by the sodium ion. At the same time, it is indicated that the effect of the lithium ion becomes manifest only outside the
physiological range of pH, and shows strong pH-dependence.
The effect of treatment with alkaline NaCl solution combined with
treatment with neutral LiCl solution
In order to clarify the significance of pH influence on the inducing effect of
lithium ion, treatment of the ectoderm with 006 M-NaCl was tried at pH 9-0
immediately before or after the treatment with 0-06 M-LiCl at pH 6-0. Both
treatments lasted for 3 h at 26 °C. The results indicated in Table 3 showed that
Table 3. Differentiation of ectodermal explants passed through alkaline
NaCl solution before or after treatment with LiCl solution
NaCl and LiCl solutions were used at 006M concentration, and the high and low pH were
respectively 90 and 60. Percentages as in Table 1.
LiCl
treatment
alone
(pH 61)
Total explants
Dissociation during
52
10(19%)
treatment
28(54%)
Survivors
12
Epidermis
8
Mesenchyme
4(14%)
Neural
8(29%)
Mesodermal (overall)
0
Notochord
8
Muscle
0
Pronephros
Yolky tissue (endodermal) 22(79%)
NaCl
treatment
alone
(pH 8-8)
LiCl
treatment
after
NaCl
treatment
LiCl
treatment
before
NaCl
treatment
31
31 (100%)
55
55(100%)
45
28(62%)
26(84%)
25
37(67%)
6
1
32(86%)
10(27%)
4
9
0
10(27%)
25(55%)
8
14(54%)
0(0%)
0
0
0
0(0%)
13
5
10(40%)
10(40%)
1
9
0
14(56%)
all the explants without previous treatment with LiCl underwent complete
dissociation after the treatment with alkaline NaCl solution, whereas the explants
treated with LiCl beforehand were unaffected by the Na-treatment in one-third
378
YOSHIO MASUI
of the cases. Moreover, the explants dissociated in NaCl solution after LiCl
treatment were found to reaggregate more quickly after being transferred into
Holtfreter's solution than those dissociated without the previous LiCl treatment.
On the other hand, when the treatment with LiCl was carried out after the treatment with NaCl, it was observed that a mucous substance released from the
cells during the NaCl treatment became very viscous and sticky upon transfer
to LiCl solution, and the cells were compactly reaggregated more quickly than
when they were transferred directly into Holtfreter's solution. This observation
may suggest that the action of the lithium ion is to make the surface substance
of ectoderm cells sticky and so causes the resistance of the tissue to dissociation
following alkaline treatment.
It was found that the neuralization following treatment with alkaline NaCl
solution occurred less often in the explants which had first been treated with
LiCl than in those without previous LiCl treatment (x2 = 0*51, 0-50 > P > 0-25).
On the other hand, the explants treated with LiCl after NaCl treatment were
more frequently neuralized than those treated with NaCl solution alone. The
increase in the frequency of neuralization was large enough to be significant
(X2 = 6-66,001 >P> 0005). Thus, the effect of the lithium ion on neuralization
is quite different according to whether the LiCl treatment occurs before or after
NaCl treatment. While the lithium ion has the effect of suppressing neuralization if it is applied prior to alkaline NaCl solution, the same ion has an enhancing
effect if applied after the NaCl treatment. It was, however, noted that no
difference was found in the type of the resulting neural tissues in these cases.
In both cases the majority of the neural formation lacked regional character
and only in a few cases were hindbrain-like structures found.
Mesodermal differentiation was increased in frequency by treatment with
alkaline NaCl solution carried out after LiCl treatment. Although the differentiation frequency was not very high in the explants treated with NaCl
solution after LiCl treatment as compared with the explants treated with LiCl
alone, it is probable that the treatment with alkaline NaCl solution is, if carried
out after LiCl treatment, effective in enhancing mesodermal differentiation
(X2 = 3-20,0-10 > P > 0-05). However, no increase in mesodermal differentiation
was found when the NaCl treatment was carried out prior to the LiCl treatment.
In this case the mesodermal differentiations took place with the same frequency
as in the treatment with LiCl alone. Regarding the quality of mesodermal
tissues produced, no difference was found between explants treated with alkaline
NaCl solution before or after LiCl treatment. In both cases notochord and
muscle were the predominant products. In view of the fact that no notochord
was produced in the explants treated with neutral LiCl solution alone, the
process of mesodermal differentiations seems to be modified by the additional
treatment with alkaline NaCl solution so as to result in notochord differentiation.
pH-dependence of inducing activity
379
The influence of some substances on the effect of lithium ion
The influences of NaCl, KC1, CaCl2, MgCl2, ZnCl2, NH4C1 and urea on the
effect of lithium ion were examined by treating the ectoderm with 006 M-LiCl
solution containing one of these substances at a concentration of 0-04 M. The
treatment lasted for 3 h at 26 °C at the pH indicated in Table 4. In the solutions
Table 4. Differentiation ofectodermal explants treated with LiCl solution
containing various substances
Percentages as in Table 1
Substance added to
KC1
NaCl
CaCl2
ZnCl2
A
K
PH
8-6-9-4
8-4-9-4
7-8-8-0
5-3-5-4
Total explants
Dissociation during
treatment
Survivors
Epidermis
Mesenchyme
Neural
Mesodermal (overall)
Notochord
Muscle
Pronephros
Yolky tissue (endodermal)
46
38(82%)
39
30(77%)
41
0(0%)
35(100%)
38(82°/
20
6
10(26°/
20(53°/
10
20
0
28(74°/
33(85%)
15
6
9(27%)
21 (64%)
6
21
3
9(27%)
33(80%)
9
6
0(0%)
24(73%)
6
24
0
12(36%)
Substance added to
O-Ofi M.T i n
pH
Total explants
Dissociation during
treatment
Survivors
Epidermis
Mesenchyme
Neural
Mesodermal (overall)
Notochord
Muscle
Pronephros
Yolky tissue
(endodermal)
5- 5-6-5
40
0 (0%)
35
30(86%)
13
3
27(90%)
1(3%)
0
1
0
3(10%)
MgCl2
NH4C1
A
A
Urea
A_
5-8
7-6
35
35(100%)
0(0%)
7-6-S10
40
0(0%)
32 (;80%) :24(60 %)
32
11
0
16
5(21 %)
6 09%)
0'(0%) 19(79 %)
0
0
19
0
0
0
5(21 %)
12 (38%)
8-4-8:-8
44
31(70°/o)
44
6(14%) 33(94%)
34(77%)
2
27
18
0
1
5
4 (67 %) 12(36%) 2(6%)
0(0"%)
0(0%)
0(0%)
0
0
0
0
0
0
0
0
0
0(0-%)
6(18%) 22(65%)
containing only monovalent cation the explants were usually dissociated in the
period of treatment, whereas no dissociation was brought about by the treatment with solutions containing a divalent cation with the exception of zinc.
Comparing the results shown in Table 4 with those in Table 2, it is seen that
the differentiation of the explants treated with LiCl solution containing sodium
380
YOSHIO MASUI
or potassium ion showed no marked difference from that of the explants treated
at the corresponding pH with the solution containing LiCl alone. Treatment
with LiCl solution containing calcium caused mesodermal differentiation more
frequently than the treatment with the solution containing LiCl alone at the
same pH. However, treatment with calcium-containing LiCl solution brought
about no neuralization, resulting in the production of completely mesodermalized explants in many cases as shown in Plate 1, fig. E. In the cases in which the
ectodermal structure was formed, only epidermis was found to be completely
segregated from the mesodermal tissue, as shown in Plate 1, fig. F. Treatment
with magnesium-containing LiCl solution was found to give rise to mesodermal
differentiation with increased frequency near pH 8-0 as compared with treatment
with the solution containing LiCl alone carried out at the corresponding pH.
However, the effect of magnesium ion was peculiar in that the treatment with
the LiCl solution containing this ion failed to induce mesoderm, but induced
neuralization, near pH 6-0 or above pH 8-4, and that the solution showed
increasing toxicity as the pH was raised so that none of the explants treated
at above pH 9-0 survived.
Treatment with LiCl solution containing ZnCl2 or NH4C1 brought about
neuralization with considerable frequency, but mesodermal differentiation and
the production of yolk-laden tissue were strongly reduced in frequency. The
resulting neural tissues were archencephalic, and were sometimes accompanied
by the formation of sensory organs. No mesodermal differentiation was found
after treatment with the LiCl solution containing urea. In this case, neuralization was brought about in a few cases. These results may indicate that the
mesodermalizing effect of lithium ion can be enhanced by calcium or magnesium
ion, but it is counteracted by zinc and ammonium ions and urea.
DISCUSSION
Explants of the gastrula ectoderm of Triturus treated with lithium ion
produced various kinds of ectodermal and mesodermal structures. In addition,
they formed a mass of yolk-laden cells which sometimes differentiated into a
gut-like structure. It has already been demonstrated that definitive endodermal
tissues appeared in lithium-treated explants when cultured for a prolonged time
(Masui, 1960,1961). Hence, the formation of the yolk-laden tissue in the present
explants must indicate an occurrence of endodermalization of the ectoderm by
lithium treatment. Since among the substances tested in the present experiment
only LiCl was effective in inducing mesodermal and endodermal tissues, the
meso- and endodermalizations of ectoderm are due to the 'specific' effect of the
lithium ion.
It is often observed that the ectodermal, mesodermal and endodermal tissues
appeared together within a single LiCl-treated explant. Since all the cells
uniformly gave rise to epidermis if the explant was untreated, a possible assump-
pH-dependence of inducing activity
381
tion is that a single test-piece of the ectoderm consists of a heterogeneous cell
population, the cells differing from each other in their competence. Under such
conditions lithium ions may induce the mesoderm from the cells withmesodermal
competence, while those with endodermal competence develop into the endoderm. The cells which are less competent for induction by lithium ion may
undergo the formation of ectodermal structures. An alternative assumption is
that the cells of the explant are homogeneous with respect to the competence,
but difference of the conditions under which the cells respond to lithium ion
may lead to different ways of differentiation. Besides, we should consider possible
secondary interactions between the structures developed. For instance, the
ectodermal structures in the LiCl-treated explant such as neural tube and ear
vesicle may be induced secondarily by the mesoderm. It is known that the
anterior neural plate does not develop into an archencephaHc structure, as
would be normally expected, when it is attached to the trunk mesoderm (Takaya,
1955, 1959). Thus, the mesoderm may exert the influence on the adjoining
neural tissues to inhibit archencephalic differentiation. This can be a reason why no
archencephaHc structure was formed in the LiCl-treated explants. It is interesting that in the treatment with calcium-containing LiCl-solution mesodermalization occurred in the absence of neuralization. The explants thus treated wholly
transformed into mesoderm, or formed epidermis in the absence of contact
with mesoderm. In the latter case (Plate 1, fig. F), it seems that early complete
segregation of mesodermal and ectodermal components takes place, as observed
by Gebhardt & Nieuwkoop (1964). Therefore, in this treatment there must be
no chance for the inductive interaction between the mesoderm and ectoderm.
Secondary interaction may take place between the mesodermal and endodermal
components, as it was found that the factors regulating the differentiation of
endoderm reside in the mesenchyme and mesodermal tissues (Okada, 1960;
Takata, 1960).
The effect of lithium ion is strongly pH-dependent. Apparently the treatment
at high or low pH enhanced the mesodermalizing effect of the ion. The result
seems to contradict the finding using the ectoderm of Ambystoma mexicanum.
In this material Gebhardt & Nieuwkoop (1964) showed that the mesodermalization occurred frequently by the treatment with LiCl near neutral pH. It has been
known that Ambystoma ectoderm is more sensitive to inductive stimuli from
the surrounding saline solution than Triturus ectoderm: in the former the
neuralization occurs at any pH, while in the latter it occurs only outside the
physiological range of pH (Barth, 1941; Holtfreter, 1944, 1945, 1947; Yamada,
1950; Karasaki, 1957). Recent data (Englander, 1962; Johnen, 1964) indicate
that Ambystoma ectoderm responds more quickly to both the neural and mesodermal inductors than Triturus ectoderm. Therefore, it may be assumed that
there is some difference in the competence between Ambystoma and Triturus
ectoderms for mesodermalization as well as for neuralization by the action of
ions. Probably in Ambystoma the ectoderm is already provided with the meso-
382
YOSHIO MASUI
dermal competence to respond to lithium ion at any pH, while in Triturus it
may become as competent as in Ambystoma only when exposed to extreme pH.
On the other hand, the endodermalization of LiCl-treated explants of the
ectoderm occurred mostly at neutral pH in Triturus. So, the competence of the
ectoderm for the induction by lithium ion may change from the endodermal
to the mesodermal with the shift from neutral pH.
Mesodermalization of the LiCl-treated explants was also enhanced when the
ectoderm was exposed to alkaline NaCl solution after pretreatment with LiCl
at neutral pH. Similar results were obtained by the application of killed organizer to the LiCl-pretreated ectoderm or by shock-treatment with ammonia of
the LiCl-pretreated ectoderm (Masui, 1960, 1961). Recent works of Barth &
Barth (1963, 1964) also suggest the enhancement of the lithium effect by adding
neuralizing agents. In their experiments ectoderm cells only underwent neuralization when lithium ion was applied for a short period or at a low dose, but
differentiated frequently into pigment cells and sporadically into mesoderm cells
when the ion was applied for a prolonged time or in combination with other
neuralizing agents. It seems likely that the neuralizing agents may sensitize the
mesodermal competence of the ectoderm so as to respond to the induction by
lithium ion more readily. It should be remembered that some neuralizing agents
such as ZnCl2, NH4C1 and urea failed to enhance the mesodermalization of the
lithium-treated explants, as shown in the present results. Probably the strong
neuralizing action of the agent may affect the explant so profoundly that it is
biased only towards neuralization without being sensitized to induction by
lithium ion.
It is important to note that in the cell culture of Barth & Barth (1963, 1964)
mesodermal differentiation occurred only sporadically after lithium treatment.
This may imply that in order for the ectoderm cells to accomplish the mesodermal differentiation after induction by lithium ion, they require some complex
factors from the environment which are lacking in their experimental system
of cell culture in dispersed form. A mass effect caused by aggregation of a large
number of cells, as found by Muchmore (1957) in the case of somite differentiation, may be critical for the mesodermal differentiation of the ectodermal cells
induced by lithium ion as well. From the foregoing discussion it emerges that
for the mesodermalization of ectoderm under influence of lithium ion it is
necessary not only to sensitize the cells to make them competent, but also to
maintain them in an aggregate of a sufficient size until the induction is stabilized.
According to Ranzi (1957, 1962) the effect of lithium ion is referable to its
action on proteins of embryonic cells. He found that lithium ion increased the
viscosity of the protein solutions. This makes, according to his interpretation,
the proteins resistant to the action of such agents as thiocyanate and urea.
However, it is still open to question how lithium ion interacts with the ions
normally present in the embryonic cell, though some investigations have been
done (Lallier, 1955; Ranzi, 1962; Vahs & Zenner, 1965). In so far as the present
pH-dependence of inducing activity
383
experiment was concerned, sodium and potassium ions showed neither marked
synergetism nor antagonism to lithium ion. Calcium and magnesium ions were
found to act on lithium ion synergistically near neutral pH. Both Ca and Mg
ions did not have mesodermalizing activity when applied singly, although they
showed weak neuralizing action (Barth & Barth, 1963, 1964; Flickinger, 1964).
These divalent cations, therefore, can only sensitize mesodermal competence so
as to render the ectoderm more reactive to lithium ion near neutral pH. On
the other hand, zinc and ammonium ions were strong antagonists of lithium ion,
suppressing the mesodermalization as well as endodermalization by lithium ion.
Thus, we may tentatively assume that ions having weak or moderate neuralizing
action, such as hydrogen, hydroxyl, calcium and magnesium, act on the mesodermalizing activity of lithium ion synergistically, whereas the ions having strong
neuralizing action, such as zinc and ammonium, appear to act antagonistically.
For detailed analysis of the mechanism of interaction between lithium and other
ions, however, new approaches are necessary.
SUMMARY
1. Using ectodermal explants isolated from early gastrulae of Triturus
pyrrhogaster, the inductive effect of lithium ion was examined at different pH's
and under the influence of neuralizing substances.
2. Treatment with 0-06 M-LiCl brought about the differentiation of axial
mesoderm together with neuralization and the production of yolk-laden tissues
which sometimes formed gut-like structures. The neuralization and mesodermalization were brought about with minimum frequency by treatment carried out
near pH 5-0, but they increased when treatment was carried out at alkaline or
acid pH. On the contrary, the production of yolk-laden tissue was brought about
with maximum frequency by the treatment near pH 6-0 and decreased as the
pH at which the treatment was done was raised or lowered.
3. Treatment with alkaline NaCl solution carried out after the treatment
with LiCl solution was found to be effective in enhancement of mesodermalization, but the same treatment carried out before the LiCl treatment had no effect
on incidence of mesodermalization.
4. Treatment with LiCl solution containing calcium or magnesium ion which
alone have a weak neuralizing action brought about considerable increase of the
mesodermalization near neutral pH. On the other hand, zinc and ammonium
ions and urea, which act as strong neuralizing agents, had an inhibitory effect
or mesodermalization by lithium ion.
5. The results are discussed in relation to the significance of the influence of
pH and neuralizing agents on the mesodermalizing effect of lithium ion, and it
is suggested that these agents sensitize the mesodermal competence of the ectoderm to the inductive action of the ion, resulting in enhancement of the mesodermalization.
384
YOSHIO MASUI
RESUME
Dependance de Vactivite inductrice de Vion lithium a regard de pH
1. L'action inductrice de l'ion Li+ a ete examinee a differents pH et sous
l'influence de substances neuralisantes, sur des explants ectodermiques isoles de
jeunes gastrulas de Triturus pyrrhogaster.
2. Un traitement au LiCl 0,06 M a provoque la differentiation du mesoderme
axial en meme temps qu'une neuralisation et la production de tissus charges de
vitellus qui ont quelquefois forme des structures de type intestinal. La frequence
la plus basse de neuralisation et de mesodermalisation a suivi un traitement
mene aux environs de pH 5, mais elle s'est accrue apres un traitement realise en
pH alcalin ou acide. Au contraire, la formation de tissu charge de vitellus a ete
la plus frequente apres un traitement aux environs de pH 6 et a diminue quand
le pH auquel s'etait fait le traitement etait plus eleve ou plus bas.
3. Un traitement par une solution alcaline de NaCl, realise apres le traitement par une solution de LiCl, a accru la mesodermalisation, mais si le meme
traitement etait realise avant le traitement au lithium, il n'accroissait pas le
mesodermalisation.
4. Un traitement par une solution de LiCl contenant des ions Ca + + ou Mg + +
qui, agissant isolement, ont une faible action neuralisante, a provoque un
accroissement considerable de la mesodermalisation aux environs de pH 7
(neutralite). D'autre part, les ions Zn ++ , NH 4 + et l'uree, qui sont des agents
fortement neuralisants, ont eu un effet inhibiteur sur la mesodermalisation par
l'ion Li+.
5. La discussion des resultats est faite sous le rapport de la signification de
l'influence du pH et des agents neuralisants sur l'effet mesodermalisant de l'ion
Li+, et on suppose que ces agents sensibilisent la competence mesodermique de
l'ectoderme a l'action inductrice de l'ion, provoquant un accroissement de la
mesodermalisation.
The author thanks Professor D. R. Newth for his kind criticisms, and is indebted to Dr
T. S. Okada for reading the manuscript and for his criticisms.
REFERENCES
L. G. (1941). Neural differentiation without organizer. /. exp. Zool. 87, 371-83.
L. G. & BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula in
unconditioned medium. /. Embryol. exp. Morph. 7, 210-22.
BARTH, L. G. & BARTH, L. J. (1962). Further investigations of the differentiation in vitro of
presumptive epidermis cells of the Rana pipiens gastrula. /. Morph. 110, 347-67.
BARTH, L. G. & BARTH, L. J. (1963). The relation between intensity of inductor and type of
cellular differentiation of Rana pipiens presumptive epidermis. Biol. Bull. 124, 125-40.
BARTH, L. G. & BARTH, L. J. (1964). Sequential induction of the presumptive epidermis of
the Rana pipiens gastrula. Biol. Bull. 125, 413-27.
ENGLANDER, H. (1962). Die Differenzierungsleistungen des Triturus- und AmbystomaEktoderms unter der Einwirkung von Knochenmark. Wilhelm Roux Arch. EntwMech. Org.
154, 143-59.
BARTH,
BARTH,
pH-dependence of inducing activity
385
R. A. (1964). Stimulation of dorsal axial differentiation in ventral explants of
Rana pipiens embryos by CaCl2 and guanidine HC1. Experientia, 20, 23.
GEBHARDT, D. O. E. & NIEUWKOOP, P. D. (1964). The influence of lithium on the competence
of the ectoderm in Ambystoma mexicanum. J. Embryo I. exp. Morph. 12, 317-31.
HOLTFRETER, J. (1944). Neural differentiation of ectoderm through exposure to saline
solution. J. exp. Zool. 95, 307-40.
HOLTFRETER, J. (1945). Neuralization and epidermization of gastrula ectoderm. / . exp. Zool.
98, 161-209.
HOLTFRETER, J. (1947). Neural induction in explants which have passed through a sublethal
cytolysis. /. exp. Zool. 106, 197-222.
JOHNEN, A. G. (1964). Heteroplastische Explantationen bei Ambystoma und Triturus zur
Analyse der primaren Schritte bei der neuralen Induktion. Wilhelm Roux Arch. EntwMech.
Org. 155, 314-41.
KARASAKI, S. (1957). On the mechanism of the dorsalization in the ectoderm of Triturus
gastrulae caused by precytolytic treatments. I. Cytological and morphogenetic effects of
various injurious agents. II. The suppression of dorsalization by treatments with sucrose,
glucose and 2,4-dinitrophenol. Embryologia, 3, 317-41.
LALLIER, R. (1955). Animalisation de l'oeuf d'oursin par les sels de zinc et de cadmium.
Exp. Cell Res. 8, 200-31.
MASUI, Y. (1958). Effect of lithium upon the development of head of amphibian embryo.
Jap. J. exp. Morph. 12, 23-53. (Japanese text.)
MASUI, Y. (1959). Induction of neural structures under the influence of lithium chloride.
Annot. Zool. Japon. 32, 23-30.
MASUI, Y. (1960). Alteration of the differentiation of gastrula ectoderm under influence of
lithium chloride. Mem. Konan Univ. Sci. Ser. 4, 103-14.
MASUI, Y. (1961). Mesodermal and endodermal differentiation of the presumptive ectoderm
of Triturus gastrula through influence of lithium ion. Experientia, 17, 458-9.
MUCHMORE, W. B. (1957). Differentiation of the trunk mesoderm in Ambystoma maculatum.
II. Relation of the size of presumptive somite explants to subsequent differentiation. /. exp.
Zool. 134, 293-310.
NAKAMURA, O. (1942). Die Entwicklung der hinteren Korperhalfte beiUrodelen. Annot. Zool.
Japon. 21, 169-235.
NIEUWKOOP, P. D. et. al (1952). Activation and organization of the central nervous
system in amphibians. I. Induction and activation. II. Differentiation and organization. III.
Synthesis of a new working hypothesis. /. exp. Zool. 120, 1-108.
OGI, K. (1961). Vegitalization of the presumptive ectoderm of the Triturus gastrula by exposure to lithium chloride solution. Embryologia, 5, 384-96.
OKADA, T. S. (1960). Epithelio-mesenchymal relationships in the regional differentiation of
the digestive tract in the amphibian embryo. Wilhelm Roux Arch. EntwMech. Org. 152,
1-21.
OKADA, Y. K. & ICHTKAWA, M. (1947). Normal table of developmental stages of Triturus
pyrrhogaster Boie. Jap. J. exp. Morph. 3, 1-6.
RANZI, S. (1957). Early determination in development under normal and experimental
conditions. In The Beginning of Embryonic Development, pp. 291-318. Ed. A. Tyler,
R. C. Borstel and C. B. Metz. Washington, D.C.: American Association for the Advancement of Science.
RANZI, S. (1962). The proteins in embryonic and larval development. Adv. Morph. 2, 211-57.
SAXEN, L. & TOIVONEN, S. (1961). The two-gradient hypothesis in primary induction. The
combined effect of two types of inductors mixed in different ratios. /. Embryol. exp. Morph.
9, 514-33.
SAXEN, L. & TOIVONEN, S. (1962). Primary Embryonic Induction. London: Logos Press
(Academic Press).
TAKATA, C. (1960). The differentiation in vitro of the isolated endoderm under the influence
of the mesoderm in Triturus pyrrhogaster. Embryologia, 5, 38-70.
TAKAYA, H. (1955). Formation of the brain from the prospective spinal cord of amphibian
embryos. Proc. Jap. Acad. 31, 360-5.
FLICKINGER,
386
YOSHIO MASUI
H. (1957). Regionalization of the central nervous system in amphibian embryo.
Jap. J. exD. Morph. 11, 1-24. (Japanese text.)
TAKAYA, H. (1959). Regional specificities in the differentiating neural plate ectoderm of
amphibian embryos. Jap. J. Zool. 12, 345-59.
TOIVONEN, S. & SAXEN, L. (1955). The simultaneous inducing action of liver and bonemarrow of the guinea pig in implantation and explantation experiments with embryos of
Triturus. Exp. Cell Res. (Suppl.), 3, 346-57.
VAHS, W. & ZENNER, H. (1965). Der Einfluss von Alkali- und Erdalkalisalzen auf die
Embryonalentwicklung der Regenbogenforelle. Wilhelm Roux Arch. EntwMech. Org. 156,
96-100.
YAMADA, T. (1950). Dorsalization of the ventral marginal zone of the Triturus gastrula. I.
Ammonia-treatment of the medioventral marginal zone. Bio I. Bull. 98, 98-121.
YAMADA, T. (1958). Embryonic induction. In A Symposium on Chemical Basis of Development,
pp. 217-38. Ed. W. McElroy and B. Glass. Baltimore: Johns Hopkins University Press.
YAMADA, T. (1961). A chemical approach to the problem of the organizer. Adv. Morph. 1,
1-53.
TAKAYA,
(Manuscript received 6 August 1965, revised 29 December 1965)