The Effect of Sodium Thiocyanate on Isolates of the
Presumptive Ectoderm and Medio-ventral Marginal
Zone of Triturus Gastrulae
by KEN-ICHI
6GI1
From the Department of General Education, Nagoya University
A s shown by Holtfreter (1944, 1945, 1947), the isolated presumptive ectoderm
of Triturus torosus develops into a mass of epidermal cells when cultured in a
physiological medium. However, if it is treated with injurious media such as
acid, alkaline, or Ca-free solutions, or alcohol, and then cultured in a physiological medium, it differentiates neural structures and mesenchyme. These
observations were confirmed in experiments made with the ectoderm of T.
pyrrhogaster (Yamada, 1950; Karasaki, 1957a) except for the following points:
treatment with Ca-free media or with alcohol does not cause neural or mesenchymal differentiation in the ectoderm of this species, although these treatments
lead to a disaggregation of the ectoderm just as with acid and alkali (Karasaki,
\9Sla). For the ectoderm of T. pyrrhogaster ammonia was found most effective
in producing neural structures. Even differentiation of the mesoderm is altered
by treating with ammonia: an isolate of the ventral marginal zone of T. pyrrhogaster, with an inherent tendency to form blood-island and mesothelium, differentiate notochord and muscle after a treatment with ammonia (Yamada, 1950).
It was emphasized by Yamada that ammonia shifts the mode of differentiation
of the isolated ectoderm and mesoderm from ventral towards dorsal. Such
effects were designated as dorsalization. Similarly it was demonstrated by
Kawakami & Okano (1955) that lactic acid is capable of dorsalizing the presumptive ectoderm and ventral marginal zone.
Ranzi & Tamini (1940) studied the morphogenetic effects of thiocyanate on
the ventral part of the early gastrula of Amblystoma tigrinum and found enhanced frequency of neural structures. However, no clear effect was observed
on the differentiation of mesodermal tissues. According to Ranzi & Tamini
(1939) and Ranzi (1945) treatment of embryos of Rana esculenta with thiocyanate leads to an increase of the notochordal rudiment. The present writer
observed a twisting of the notochord after a treatment of the embryos of T.
pyrrhogaster with thiocyanate but could not confirm any increase in the total
1
Author's address: Department of General Education, Nagoya University, Mizuho-ku, Nagoya,
Japan.
[J. Embryol. exp. Morph. Vol. 6, Part 3, pp. 412-417, September 1958]
KEN-ICHI OGI—DORSALIZAT1ON BY THIOCYANATE TREATMENT 413
cell number and total volume of the notochord. In spite of these negative results
it might be possible to obtain a dorsalizing effect of the agent on the mesoderm
by the method employed by Yamada for demonstrating the morphogenetic
effects of ammonia, because it enables a better penetration of the agent into the
cell interior. Hence in the present experiments thiocyanate was tested for its
morphogenetic effects on the isolated presumptive ectoderm and medio-ventral
sector of the marginal zone of the early gastrulae of T. pyrrhogaster.
MATERIAL AND METHOD
The solution of sodium thiocyanate was prepared immediately before use by
adding 0-3 c.c. of 1 M sodium thiocyanate to 9 c.c. of Holtfreter's solution. Its
pH was adjusted to 7-2 with sodium bicarbonate. A piece of the presumptive
ectoderm or of the medio-ventral marginal zone with the narrow strip of the
ectoderm directly overlying it was isolated with a glass needle, carefully avoiding the inclusion of the cells of the adjacent territories, and subjected to the
thiocyanate-Holtfreter's solution for 5 to 8 minutes. The isolates were then
transferred to Holtfreter's solution and cultured at 20°-23°C. for 9 to 10 days.
The operation and cultivation were carried out under sterile conditions.
EXPERIMENTS
Experiments on the presumptive ectoderm
(a) Untreated isolates. All 29 explants developed into atypical epidermis.
(b) Thiocyanate-treated isolates. A piece of the presumptive ectoderm was
isolated in Holtfreter's solution, and transferred to thiocyanate-Holtfreter's solution. Many of the isolates curled so as to expose their originally internal surface
TABLE 1
Structures differentiated in thiocyanate-treated isolates of
presumptive ectoderm
Available explants
.
.
.
.
.
Explants with structures other than epidermis
Explants with neural structures .
Explants with: Eye .
Fore-brain .
Nose .
Lens .
Pigment vesicle
Neural vesicle
Neural mass
Mesenchyme
Melanophores
85
61
51
12(14)
13(13)
7(11)
8(10)
6 (6)
13(13)
37
10
11
72%
60%
14%
15%
8%
9%
7%
15%
44%
12%
26%
In parentheses: Actual number of differentiated structures.
outward. After several minutes, mutual adhesiveness of cells apparently lessened and a number of peripheral cells were released from the isolates. Then the
latter were returned to Holtfreter's solution, in which the cells regained their
414 KEN-ICHI OGI—DORSALIZATION BY THIOCYANATE TREATMENT
adhesiveness and solid aggregates were formed. These aggregates were transferred to a further dish of Holtfreter's solution and cultured. The surface of the
aggregate seemed to be occupied by the originally internal surface of the
ectoderm. The results of histological observation, shown in Table 1, indicate a
considerable number of dorsalized isolates. In 22 of 61 dorsalized isolates
archencephalic structures could be identified. The pigment vesicle occurred
either isolated in the epidermis or attached to the brain-type structure. Mesenchyme and melanophores differentiated in the direct vicinity of the neural tissue.
Experiments on the medio-ventral marginal zone
(a) Untreated isolates. The medio-ventral marginal zone was isolated and
cultured in Holtfreter's solution. As summarized in Table 2, blood-island, bloodvessel, and mesothelium, which roughly correspond to the presumptive significance of the isolates, were obtained as the main differentiation. The nephric
tubules formed within an endodermal mass or in the mesenchyme were short
and poorly differentiated. A group of myoblast-like cells without myofibrils was
obtained in two cases. In no case was notochordal tissue observed.
TABLE 2
Differentiation of the isolated medio-ventral marginal zone
treated or non-treated with thiocyanate
Control
A v a i l a b l ee x p l a n t s .
.
.
. 39
.
E x p l a n t sw i t h :B l o o d - i s l a n d .
.
. 27 .(69%)
M e s o t h e l i u m .
. 13
. (33%)
.
B l o o d - v e s s e l .
. 27. (69%)
.
M e s e n c h y m e .
.
.24 (62%)
.
0
M e l a n o p h o r e s . . . .
10 (26%)
Nephric tubule
M y o b l a s t
.
.
.
. 2 (5%)
M u s c l e .
.
.
.
. 0
N o t o c h o r d
.
.
. 0.
N e u r a lt i s s u e.
.
.
. 0
0
E a rv e s i c l e .
.
.
.
Treated explants
89
36 (40%)
21 (24%)
39 (44%)
56 (66%)
17 (19%)
39 (44%)
13(15%)
25 (28%)
37 (42%)
10(11%)
2 (2%)
(b) Thiocyanate-treated isolates. The isolated medio-ventral marginal zone
was treated with thiocyanate-Holtfreter's solution until disintegration of the
isolate was indicated, and then transferred to Holtfreter's solution, where most
of the cells reaggregated. Peripheral cells of the isolates were lost during and
after treatment. In this case curling of the isolate so as to turn its originally
internal surface outwards was insignificant, probably because of the thickness
of the isolate. The results obtained in this series are summarized in Table 2.
As compared with the control series the occurrence of notochord and muscle
was noticeable. A well-formed notochord provided with a sheath was found in
only 3 explants out of 37. The remaining notochordal tissue was represented by
KEN-ICHI OGI—DORSALIZATION BY THIOCYANATE TREATMENT 415
an unorganized mass of vacuolated cells scattered among the myoblasts. Very
often the notochordal tissue was accompanied by muscle-cells with myofibrils,
or by neural tissue situated on the outer surface of the explant. The structure
like an ear vesicle was formed in the vicinity of an elongated notochord. No
archencephalic structure was observed. The nephric tubules were much more
advanced in differentiation than those of the control series. Although the frequency of blood-islands was only moderately reduced (Table 2), a decisive
reduction in the number of blood-cells in each blood-island was apparent, when
the present series was compared with its control.
DISCUSSION
The results obtained in the series of experiments on presumptive ectoderm
clearly indicate that thiocyanate affects development of the isolated ectoderm in
such a way that unorganized neural tissues and archencephalic structures, including mesenchyme and melanophores, are differentiated beside epidermal
cells which alone were formed in the control series. The series of experiments on
the medio-ventral marginal zone demonstrates that differentiation of notochord
and muscle is induced by thiocyanate in the ventral mesoderm, which has the
inherent tendency to form ventral structures of the trunk mesoderm such as
blood-island, blood-vessels, and mesothelium. Thus for both the ectoderm and
ventral mesoderm the effects of thiocyanate-treatment can be designated as
dorsalization. The results are closely comparable to those obtained with ammonia (Yamada, 1950), lactic acid (Kawakami & Okano, 1955), and urea (Leone,
1952; Karasaki, 1957a).
The neural tissue formed in the thiocyanate-treated mesoderm has probably
been derived from the ectodermal component of the isolate. As this component
must also have been exposed to thiocyanate, it would have been dorsalized as in
the pure ectodermal isolates. However, the fact that ear vesicles, which were
never formed in the ectoderm series, appeared in the mesoderm series, needs
a special interpretation. In view of their occurrence in the vicinity of the well-
formed notochord, it is probable that they were induced from the ectodermal
component by the mesoderm dorsalized in its turn by thiocyanate. It may be
added that induction of the ear vesicle by the presumptive notochord has been
well established (Borghese, 1943), while the capacity to induce is apparently
lacking in the ventral mesoderm. That the ventral mesoderm dorsalized by a
chemical stimulus is able to induce neural structures has been demonstrated by
putting the presumptive ectoderm on an ammonia-treated piece of the ventral
mesoderm (Yamada, unpublished). Thus subcytolytic treatment not only
changes the mode of differentiation of the ventral marginal zone, but evokes in
the latter an inductive capacity comparable to that of the normal organizer.
As stated in the introduction, treatment of the whole embryo of T. pyrrhogaster with thiocyanate causes a severe twisting of the notochord, which, however, is not accompanied by an increase in the volume or cell number of the tissue
416 KEN-ICHI OGI—DORSALIZATION BY THIOCYANATE TREATMENT
(Ogi, 1955,1957). The difference between those results and the present ones may
be due to a difference in the extent of penetration of the substance into the cell
interior.
Since there is an antagonistic effect of thiocyanate and lithium on development of the sea-urchin, it is interesting to note that in amphibian embryos the
two agents also have antagonistic influences: it is well known that lithium
suppresses the formation of the notochord of amphibian embryos, which is
enhanced by thiocyanate. Furthermore, the dorsalizing effect of thiocyanate as
demonstrated in the present experiments is more or less suppressed by lithium
(Okano, 1956; Karasaki, 1957a). According to Ranzi (1952) the antagonism is
reflected in the effect of both agents on viscosity of various protein solutions.
Data of Karasaki (19576) suggest a structural change of the cell as the basis of
dorsalization caused by subcytolytic stimuli. This change may be reduced by
lithium.
SUMMARY
1. Isolates from the presumptive ectoderm and the medio-ventral marginal
zone of the early gastrula of T. pyrrhogaster were briefly treated with sodium
thiocyanate, and cultured in Holtfreter's solution.
2. The treated isolates of the presumptive ectoderm differentiated archencephalic structures, unorganized neural structures, mesenchyme, and melanophores; whereas its control isolates only formed atypical epidermis.
3. The non-treated isolates of the medio-ventral marginal zone differentiated
blood-island, blood-vessel, mesothelium, mesenchyme, and nephric tubule; while
the treated isolates of this zone gave rise to notochord and muscle.
ACKNOWLEDGEMENT
It is a pleasure to acknowledge my gratitude to Professor Tuneo Yamada for
his valuable advice and critical reading of the manuscript.
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