J. Embryol. exp. Morph. Vol. 23, 2, pp. 463-71, 1970
Printed in Great Britain
463
Experiments on /3-mercaptoethanol as an inhibitor
of neurulation movements in amphibian larvae
By C A R L - O L O F J A C O B S O N 1
From the Wistar Institute of Anatomy and Biology
Philadelphia, Pennsylvania 19104
and
The Institute of Zoology, Uppsala, Sweden
The morphogenetic movements of the ectoderm during neurulation include:
(1) the movements taking place within the neural plate, which becomes longer
and more concentrated in a medio-lateral direction (Jacobson, 1962); and (2)
those found in the lateral epidermis layer which, in an epibolic way, moves in a
dorsal direction, thus exerting a pushing effect on the lateral edges of the neural
plate (Lewis, 1947). The former is, to a great extent, realized by a change of
form of the neuroepithelium cells, from cuboidal in early neurulae to the high
columnar cells observed during later phases of neural-tube closure. In the
epidermis, on the other hand, the case is the reverse. The dorsal spreading of the
layer is made possible by a flattening of the cells.
In a series of papers, Brächet and his group have show that /?-mercaptoethanol (HSCH 2 CH 2 OH; in this article, called ME) inhibits neurulation (for
review, see Brächet, 1964).
The aim of this investigation was to determine if this thiol acts on the whole
morphogenetic complex or if only parts of the processes involved are blocked.
Therefore, we extirpated the neural plates of the amphibian Amblystoma and
observed the differences in development between operated and unoperated
animals, both in ME solutions and in normal media. It is known that if the
neural-plate epithelium is extirpated in early neurulae, the epidermal part of the
ectoderm has the power to move up over the exposed chorda-mesoderm and
fuse in the dorsal midline (Jacobson, 1962).
MATERIALS AND
METHODS
Experiments were performed on two different species of the genus Amblystoma: A. opacum and A. mexicanum. Operations were made on stage-14
neurulae (open neural plate, ridges appearing), since experiments had shown
that larvae of later stages continue their change of form for a time even after
1
Author's address: The Institute of Zoology, Uppsala, Sweden.
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C.-O. JACOBSON
exposure to ME. The reason for this is obviously that the tube-shaping events
are taking place at an accelerated tempo after the narrowing of the neural plate
is initiated. Twenty-one extirpations of the neural plate were performed; in
most cases the parts within the neural ridge were excised (Fig. 1 A), but in six
larvae the ridges were included (Fig. IB). In order to loosen the ectoderm
without damaging the underlying mesoderm, the operations were made in a
hypertonic medium (4 x Steinberg's solution) using operational techniques
previously described (Jacobson, 1964). Sixteen of the larvae operated on were
transferred to a medium consisting of 001 M-/?-mercaptoethanol (Eastman
Organic Chemicals) in Steinberg's solution (pH 7-4); five larvae were transferred to a 0 03 M-ME solution.
A
B
Fig. l. Schematic drawing demonstrating the extirpation experiments. The neuralplate ectoderm was excised at stage 14 (early neurula stage) inside the broken lines.
Either (A) only material situated inside the neural ridges was excised, or (B) the extirpation included the ridges also.
Control. Five larvae with excised neural plates were cultivated in Steinberg's
solution. Thirty-one unoperated larvae were held in 001 M-ME in Steinberg's
solution; sixteen of these were decapsulated in advance. Fifteen larvae were
held in a 003 M-ME solution. In all experiments untreated controls were cultivated in Steinberg's solution. These animals gave data concerning the normal
rate of neurulation.
RESULTS
Unoperated larvae
When stage-14 larvae were transferred to ME, the neural plates had not
narrowed after 15 h of exposure to the substance; however, the ridges were well
developed (Fig. 2). The control larvae, cultivated in Steinberg's solution, had
finished the tube-shaping process after the same period of time (stage 21-22).
Twenty-four hours later the neural plates of the ME-exposed larvae remained
unchanged in form, but aggregations in the mesoderm, interpreted as somites,
were observed through the pigment-poor ectoderm. These results are in full
agreement with those reported by Brächet & Delange-Cornil (1959).
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neurulation
465
Larvae with excised neural plates
In the control animals, transferred to Steinberg's solution after the operations,
the epidermis fused over the chorda-mesoderm (Fig. 3C), confirming earlier
observations (Jacobson, 1962). The process is somewhat retarded compared to
the fusion of the ridges in normal larvae. In the operated larvae exposed to ME,
Fig. 2. A larva treated with 0 01 M-ME from stage 14 (early neurula stage). The
photograph was taken after 23 h of treatment and shows that the neurulation was
arrested at stage 15. Note the well-developed neural folds and the hint of mesodermal
differentiation sculpturing the neural plate, x 50.
the free ectoderm margins of the operation area had almost reached each other
after 6 h (Figs. 3B,4). No difference in the speed of the dorsad-moving epidermis
sheet could be seen either in larvae with, or larvae without, the neural ridges
included in the extirpate. It is remarkable that the ectoderm never fused in the
dorsal midline, in contrast to the case in the control experiments (Fig. 3). On the
contrary, in some of the experiments a slight widening of the furrow was
observed after 40 h of exposure to ME.
In one larva (stage 16) only the spinal portion of the plate including the ridges
30
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C.-O. JACOBSON
was extirpated, whereas the brain portion was left fully intact. After 6 h of
exposure to 001 M-ME, the anterior region remained unchanged; in the
operation area, however, the epidermal edges had almost reached each other
dorsally (Fig. 5).
Fig. 3. Schematic drawing illustrating the main experiment. The neural plate, here
including the ridges, was excised, leaving the archenteron roof intact (A). If the larva
after the operation was cultivated in an isosmotic salt solution containing
0 01 M-ME, the lateral epidermis moved up over the mesoderm and its margins came
quite close to each other, even if they never fused (B). In the control larvae, cultivated in a solution out of the thiol, complete dorsal fusion took place (C).
Some efforts were made to excise the neural plate of larvae treated with ME
for more than 20 h. As this was very difficult to do without damaging the
archenteron roof (the neuroectoderm had new, sticky qualities) the results were
generally unsuccessful. In one case, however, it was possible to extirpate all but
Inhibition of neurulation
467
the suprachordal parts of the plate. It was interesting to note that the epidermis,
in a couple of hours, had pushed the ridges into a position where they could
fuse to the intact median parts of the neural epithelium (Fig. 6).
No differences in movements or in the rate of development were observed
between experiments performed on A. opacum and those performed on A.
mexicanum, nor were any differences observed after treatment with 0 03 M-ME
compared to treatment with the 001 M-solution.
Fig. 4. (A) Photograph of a larva in which the neural plate inside the ridges has
just been extirpated. Medially in the archenteron roof the notochord material is seen;
laterally the first signs of segmentation of the mesoderm can be observed, x 40.
(B) Detail from the dorsal part of a larva photographed 6 h after the neural plate
including the ridges was extirpated. The larva was cultivated in ME solution continuously from the time of operation. Though the epidermis margins have not
fused over the mesoderm, the photograph clearly shows how a mass movement of
the lateral epidermis has taken place, x 75.
The jelly capsule
Amblystoma eggs are protected by a two-layered jelly capsule, the inner layer
being hard and elastic, the outer layer more soft and sticky. The latter layer was
totally dissolved after a short period of ME treatment of non-decapsulated
larvae.
30-2
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C.-O. JACOBSON
DISCUSSION
The inhibition of neurulation movements after treatment with /?-mercaptoethanol, which was pointed out by Brächet and his group (1964), was shown
in the experiments reported here to include changes in the form of the neural
plate, but not in the dorsal spreading of the epidermis. These two morphogenetic forces, claimed to be highly involved in the tube-shaping process, are in
turn the result of changes in cell form. The epidermal cells are becoming
flattened; the neuroectodermal cells, on the contrary, are rising from cuboidal
A
B
Fig. 5. (A) Schematic drawing demonstrating the ectodermal area extirpated in a
larva that was transferred to a ME solution after grafting. (B) 6 h after the operation the epidermis outside the extirpated area had moved up over the mesoderm;
in the intact area all movements were inhibited.
A
B
Fig. 6. (A) Schematic drawing demonstrating ectodermal areas extirpated in a larva,
that had been treated with ME for 20 h prior to grafting. Dotted area represents
intact neuroectoderm, the hatched area was excised. (B) 6 h after the operation the
lateral epidermis had moved up over the naked mesoderm to fuse with the intact
neuroectoderm.
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469
to high columnar shape. At the same time the neural plate is stretched in a
complicated way (Jacobson, 1962). These events in combination result in a
narrowing plate and a diminishing distance between the neural ridges.
The increased height of the neuroepithelial cells has been associated with an
increased intercellular affinity (Brown, Hamburger & Schmitt, 1941 ; Jacobson,
1962; cf. Gustafson & Wolpert, 1961) and with the appearance of bundles of
vertically arranged tubular filaments (Waddington & Perry, 1966).
The neural-plate epithelium of the ME-treated neurulae is not made up of
columnar, pseudo-stratified cells, as is clear also from the figures of Brächet &
Delange-Cornil (1959). Whether this elongation of normal cells is caused by
increased affinity or by microtubular formation (the truth may well be a combination of these two events), ME seems to interfere with the synthesis or with
the maintenance of stable proteins. (Moscona & Moscona (1963) claimed that
intercellular affinities are dependent on protein synthesis.) However, overall
protein synthesis is not disturbed. Neither changes of affinity within the epidermis (causing epibolic movements) nor the further differentiation of the
mesoderm (both events occur in the presence of ME) are likely to take place
without protein synthesis. Brächet (1964) has suggested that ME acts as an
inhibitor of neural induction by interfering with basic proteins, which may act
as inducers. This hypothesis is interesting, since it might explain why just neural
structures are affected.
However, a direct action by ME on proteins with sulphydryl and disulphide
groups or bonds can be anticipated under all circumstances. The negative
influence of ME on polymerizing of fibrillar subunits has recently been reported
(Burton, 1967; Wade & Satir, 1968).
It was observed that the epidermal free edges and neural ridges, respectively,
never fused over the uncovered mesoderm of ME-treated embryos, although
fusion occurred in the control larvae raised in normal medium, with a more or
less narrow cleft left between the ridges (Fig. 3) ; instead, after 40 h of treatment
with ME a widening of the fissure often took place. Most likely this is not only
a result of an incapacity to fuse, but also a general, but delayed, reaction to the
thiol substance within the epidermal sheet, perhaps as a result of inhibited
RNA synthesis or RNA transport. It is known that RNase treatment of amphibian embryos results in abnormal development of the ectoderm, but welldifferentiated chorda and somites (Brächet & Ledoux, 1955).
The possible interference of ME with the epidermis did not occur, however,
even after 20 h of treatment, as revealed by the experiment in which the neural
ridges moved inwards when the lateral parts of the plate had been excised.
It is obvious that the pushing forces of the epidermis are not strong enough to
overcome the resistance of a passive neural plate. The same phenomenon was
seen when the neural plate cells were poisoned by heavy doses of the vital dye
Nile-blue sulphate, bringing to a halt the movements of the plate (Jacobson,
1962). The neuroepithelium thus must take an active part in the bending
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C.-O. JACOBSON
process or, at least, after the medio-lateral concentration movements are finished,
must possess a plasticity that enables it to give in to pressure from the sides.
SUMMARY
1. A concentration of 0-01 M-/?-mercaptoethanol inhibits the neural-tube
shaping process in amphibian larvae.
2. Two forces instrumental in this process are the narrowing of the neural
plate and the dorsad movement of the lateral epidermis.
3. Experiments show that mercaptoethanol inhibits the morphogenetic
movements within the plate, but not, at least initially, the epibolic tendency of
the epidermis.
4. Possible backgrounds of this difference are discussed.
RÉSUMÉ
Expériences sur le ß-mercaptoethanol en tant qu'inhibiteur des
mouvements de neurulation chez les larves d''Amphibien
1. Une concentration de 0,01 M de /^-mercaptoethanol inhibe le processus de
formation du tube neural chex les larves d'Amphibien.
2. Les deux contraintes contribuant à ce processus sont le rétrécissement de
la plaque neurale et le mouvement dorsal de l'épiderme latéral.
3. Des expériences montrent que le mercaptoéthanol inhibe les mouvements
morphogénétiques à l'intérieur de la plaque, mais non, au moins au début, la
tendance épibolique de l'épiderme.
4. On discute des causes possible de cette différence.
I wish to express my gratitude to Dr H. Koprowski for placing all the facilities of the
Wistar Institute at my disposal during my stay there as a visiting scientist. The study was
supported by a grant from the Swedish Natural Science Research Council, from the Faculty
of Science, University of Uppsala, and Public Health Service Research grant 5-Sol-FR
05540-05 from the General Research Support Branch.
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(Manuscript received 23 April 1969)