Lithium inhibits morphogenesis of the nervous system but not neuronal differentiation in Xenopus laevis
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Development 99, 353-370 (1987) 353
Printed in Great Britain © The Company of Biologists Limited 1987
Lithium inhibits morphogenesis of the nervous system but not neuronal
differentiation in Xenopus laevis
LORNA J. BRECKENRIDGE1*, R. L. WARREN2 and ANNE E. WARNER1
^Department of Anatomy and Embryology, University College London, Cower Street, London WC1N 6BT, UK
^Department of Nuclear Medicine, The Middlesex Hospital Medical School, London Wl, UK
* Present address: Department of Physiology and Biophysics, SJ-40, University of Washington, Seattle WA 98102, USA
Summary
Xenopus embryos treated with 100 mM-lithium from increased. Sodium, potassium and lithium were
the 2- to 4-cell stage to the early blastula stage (4h) equally able to increase the permeability of the
failed to neurulate and developed without a discernible embryo. However, sodium ions enhanced, while pot-
anteroposterior axis. The internal structure of assium ions interfered with, the uptake of lithium.
defective embryos was grossly disorganized, but Treatment with lithium at progressively later stages
immunohistochemical staining with cell-type-specific reduced the developmental defects and neural differ-
antibodies revealed differentiated nerve and muscle entiation returned to normal levels. The uptake of
cells. Quantitative assay in tissue cultures from con- lithium did not decline concomitantly. We conclude
trol and acutely abnormal lithium-treated embryos that lithium does not inhibit neural induction, but
showed that neural differentiation was enhanced and interferes with dorsal patterning. The sensitivity of
muscle differentiation unaffected. The embryos took the embryo to lithium is determined by developmental
up about 0 5 mM-lithium at threshold, maximal effects stage. The very low, effective intracellular concen-
resulted at 2-3 mM. Most of the lithium was extruded trations may be important in understanding the mech-
from the cells into the blastocoel fluid, where lithium anism of lithium-generated defects.
reached 17 mM. The threshold intracellular concen-
tration was about 150 uM. Lithium uptake rose steeply Key words: lithium, Xenopus laevis, neural development,
as the osmotic/ionic strength of the bathing medium neurulation.
Introduction Pasteels, 1945; Hall, 1942; Backstrom, 1954). By
gastrulation, exposure of the embryos to lithium
The teratogenic consequences of exposure of early generates less-marked defects and once gastrulation is
sea urchin and amphibian embryos to lithium ions complete lithium treatment has little effect. Attempts
have been recognized for many years. In the sea to clarify the consequences of lithium treatment by
urchin, lithium treatment of the early embryo leads to exposing isolated ectoderm stripped from the am-
the over production of structures derived from the phibian embryo to lithium have demonstrated a
vegetal pole, at the expense of structures derived confusingly wide range of effects. These include the
from the animal pole (Herbst, 1892). In the am- induction of derivatives characteristic of all three
phibian embryo, lithium treatment affects the devel- germ layers (Englander & Johnen, 1967), the forma-
opment of the notochord and causes a reduction in tion of mesodermal structures (Ogi, 1961; Masui,
neural structures such as the optic rudiments (Mor- 1961; Grunz, 1968; Johnen, 1970) and the induction
gan, 1903). Backstrom (1954) obtained similar results of neural differentiation (Barth & Barth, 1962,1974).
and further suggested that neural differentiation was In this paper we examine the consequences of
reduced. Consequently he proposed that lithium treatment of early amphibian embryos with lithium
'ventralized' the embryo. ions in order to test whether previous suggestions that
Both sea urchin and amphibian embryos are most the lithium ion might interfere with neural induction
sensitive to lithium treatment during the early cleav- are correct. We have used the appearance of differen-
age stages (sea urchin: Lallier, 1964; amphibian: tiated neurones as one criterion of successful neural354 L. J. Breckenridge, R. L. Warren and A. E. Warner
induction. Phenotypically differentiated neurones are Assessment of abnormalities
identified in intact embryos using cell-type-specific The embryos were assessed for salt-induced abnormalities
antibodies and quantitative assessment of the extent and batch viability when the control embryos had reached
of neural differentiation is made using an in vitro stage 20-22. For each group the number of dead and/or
assay (Messenger & Warner, 1979; Breckenridge & abnormal embryos was determined. The number of em-
Warner, 1982). We conclude that neural differen- bryos that died in the control group gave the spontaneous
tiation is not prevented by lithium treatment, despite death within the batch. The percentage of embryos that
the gross disturbance of morphogenesis and pattern- died spontaneously was subtracted from the percentage
that died after each experimental regime, to give the death
ing. This conclusion parallels that drawn in a very
rate for each condition tested. In individual experiments
recent paper by Kao, Masui & Elinson (1986), who the effects of the different treatments were compared on
examined pattern formation in Xenopus laevis after sibling embryos from the same batch. Salt-treated embryos
treatment of cleavage-stage embryos with lithium. were scored for abnormalities as described in the Results
These authors suggest that lithium enhances the section.
development of dorsoanterior structures and that
this respecification of the pattern-forming system is Histology
responsible for the morphological appearance of the Control embryos at stage 25-28 and lithium-treated siblings
treated embryos. of an equivalent age were fixed overnight in Bouin's fluid,
The mechanism by which lithium ions generate washed in buffer, dehydrated in alcohols and embedded in
butoxyethanol-glycol methacrylate (Dupont Ltd). 7 fim
these effects on development is not known. Surpris-
serial sections were cut on a Dupont-Sorval JB4 microtome
ingly, there are no estimates of the intracellular and stained with haematoxylin and eosin.
concentration of lithium that is necessary to generate
the developmental defects. We have therefore Immunohistochemistry
measured the concentration of lithium within am- Unfixed embryos at stage 25-28 were embedded in Tissue-
phibian embryos after treatment, for comparison Tek, frozen in isopentane and 5-7 /an sections cut on a
with the teratogenic effects. Bright 5030 cryostat. Sections were incubated for 1 h at
Some of these results were included in a thesis room temperature with: (i) a monoclonal antibody raised
submitted to the University of London in part against rat neurofilament protein (Wood & Anderton,
fulfilment of the requirements for the Ph.D 1981); (ii) a monoclonal antibody (12/101) raised against
adult newt muscle (Kintner & Brockes, 1984). The sections
(Breckenridge, 1985).
were then washed three times with Ringer solution and
incubated with FITC-labelled goat anti-mouse antibodies
(Nordic Immunological Laboratories, Maidenhead). After
Materials and methods further washing to remove unbound antibodies, the sec-
tions were mounted in 'anti-fade' mountant (Chemistry
Xenopus laevis embryos were obtained from adult frogs Department, City University, London) and viewed on a
induced to mate and lay by injection of chorionic gonado- Zeiss epifluorescence microscope equipped with appropri-
trophin (Pregnyl, Organon Ltd). Embryos were reared in ate filters. Photomicrographs were taken on Kodak Tri-X
tap water until they reached the required developmental Pan film.
stage; staging follows the normal table of Xenopus laevis
(Nieuwkoop & Faber, 1956). Tissue culture
Neural tube, notochord, somitic mesoderm and some
overlying ectoderm were dissected from embryos at stage
Treatment with lithium and other salts 19-20, when the neural tube had just closed. The dorsal
For each batch of eggs at least 10, more usually 20, embryos surface and immediately underlying mesoderm were dis-
of the required stage were soaked in a 100 HIM solution of sected from embryos of the same age, which had previously
the appropriate salt for the required length of time. When been treated with lithium. The dissected portions were
some embryos were to be removed for ion analysis (see soaked for 3-5 min in calcium-free Ringer solution
below) the number of embryos was increased. The jelly (120mM-NaCl, 2-5mM-KCl, 2 mM-Tris-hydroxymethyl-
coat was deliberately left intact, even though it could aminomethane, lmM-EGTA) at pH8-0 and then dis-
provide a variable barrier to the uptake of lithium, because sociated into single cells by mechanical trituration. Material
chemical removal of the jelly coat with cysteine can disturb from three embryos was dispensed into each 35 mm culture
the distribution of small ions within the embryo (A. Stotter, dish (Falcon Ltd) and cultured at 22 °C in modified
R. L. Warren & A. E. Warner, unpublished results). An Ringer solution (100mM-NaCl, 2-5mM-KCl, 20mM-CaCl2,
equivalent number of sibling embryos were placed into tap 2-0mM-MgCl2, 5-0mM-NaHCO3) with 10% fetal calf
water. At the end of exposure to salt solution, the exper- serum (Gibco-Biocult) and 300i.u. penicillin/streptomycin
imental embryos were washed, returned to tap water and (Gibco) at pH7-4. Three culture dishes were set up from
left for 20-24 h, by which time the control embryos had both control and lithium-treated embryos. The cultures
reached stage 20-22. All experiments were carried out at were scored 18-24h after plating, when a monolayer of
room temperature (18-22 °C). 60000 to 70000 differentiated cells had formed. Neurones,Lithium and neural development in Xenopus embryos 355
muscle cells and the total number of cells were counted in Results
20-30 microscope fields selected at random from the three
Petri dishes. Further details of the culture method can be Observations on the abnormalities generated by
found in Messenger & Warner (1979). lithium treatment
The majority of embryos treated with 100 ITIM-
lithium from the 2- to 4-cell stage to the early blastula
Ion analysis
stage (4h) developed normally until neurulation be-
At the end of the treatment period, when the embryos had gan. In a few, very severe cases the embryo exogas-
reached stage 7, four to six embryos were removed, washed trulated; this was followed by cytolysis and death.
thoroughly in three changes of double-distilled deionized Fig. 1 shows a normal tadpole at stage 26 that had
water and all the jelly removed with clean, flamed forceps.
been exposed to 100mM-NaCl (A) and examples of
The embryos were divided between two Eppendorf tubes
the range of abnormalities seen in lithium-treated
each containing 1 ml of double-distilled, deionized water
embryos (B-F). The defects ranged in severity from
and broken up by repeated freezing and thawing. The
slight inhibition of elongation (B), severe inhibition
lithium concentration was measured in 1 fA aliquots of each
of elongation with an apparent enlargement of the
sample and the final wash solution by flame emission
belly (C), complete absence of elongation, with the
spectroscopy (Warren, 1980). For calculation of lithium
concentration, the volume of the embryo at stage 7 was
neural folds failing to close (D) to complete absence
taken as 1-33^1 (Slack, Warner & Warren, 1973). Since the
of neural folds, which was sometimes accompanied by
diameter of Xenopus laevis embryos can vary from batch to a long proboscis (E). The most abnormal embryos
batch, this assumption introduces some error. The range of often died shortly after the controls had reached stage
volumes within all the measurements of Slack et al. (1973) 20-22. A similar range of defects was observed by
was 1-02-1-5^1. Backstrom (1954), using the same treatment sched-
ule. Kao et al. (1986) treated embryos with higher
concentrations of lithium (0-2 and 0-3 M in 20%
Microinjection Steinberg's solution) for a few minutes only and
The jelly coat was mechanically stripped from 2-cell em-
noted equivalent defects.
bryos and approximately 1 nl of 1 M-LiCl or 1 M-NaCl To determine the teratogenic effect of lithium
injected into each cell, giving an intracellular lithium under different experimental conditions, the embryos
concentration of about 2 mM. Injection pipettes were pulled were scored for the percentage that died within each
from Corning 7740 omega dot glass capillary, backfilled batch and the percentage showing any of the ab-
with the injection solution and connected to a Picospritzer normal features characteristic of lithium treatment,
(General Valve Corporation). The tip of the pipette was illustrated in Fig. 1. The relative severity of the
broken back to approx. 5/im. Each injection pipette was abnormalities was not determined systematically, but
calibrated by measuring the volume of a drop expelled from when the percentage of embryos showing abnor-
the tip for a given pulse pressure (138-276 kNm" 2 ) and malities was low, the abnormalities were less severe.
width. Conversely, a high proportion of dead embryos was
accompanied by more severely abnormal embryos. A
proportion of the untreated embryos in each batch
Samples of blastocoel fluid died also, reflecting differing degrees of viability. All
Micropipettes were pulled from lmm external diameter, experiments included in this paper were made on
0-33 mm internal diameter glass capillary and the tip broken batches of high viability (greater than 80 %, average
back to 2-5/im. The volume of the pipette tip was 90%). The consequences of lithium treatment
measured up to the shoulder. The pipette was inserted showed considerable variability from batch to batch,
between the cells of the animal pole into the blastocoel as seen by Stanistreet (1974). However, occasional
cavity of embryos at stage 7-8. Blastocoel fluid was rapidly batches showing extremely poor viability (20-40 %)
drawn into the tip by capillarity. The pipette was removed were no more sensitive to lithium than the rest. The
from the embryo and placed in a clean tube containing teratogenic consequence of lithium treatment was
double-distilled, deionized water. Each sample was defined as the sum of dead and abnormal embryos,
analysed for potassium and lithium by flame emission after correction for the viability of each batch.
spectroscopy (Warren, 1980). Cell damage during insertion
or withdrawal of the pipette produced yolky samples and The uptake of lithium
potassium levels well above that normally found in blasto- In order to resist the net loss of ions during develop-
coel fluid (l-4mM: Slack et al. 1973; Gillespie, 1983); ment the membranes of external facing cells of the
contaminated samples were discarded. As observed pre- amphibian embryo are relatively impermeable to ions
viously (Slack et al. 1973) cytoplasmic contamination was and water (Slack & Warner, 1973; Slack etal. 1973). It
rare. is, therefore, difficult to predict how much lithium356 L. J. Breckenridge, R. L. Warren and A. E. Warner enters the embryo even from high-salt solution. taken from 25 batches gave a mean of 2-54 ±0-18 ITIM- Measurements of lithium concentration made at the lithium (mean ± 1 standard error; 47 samples each end of a 4h exposure to lOOmM-LiCl in embryos containing two or three embryos), calculated using an Fig. 1. The external appearance of abnormal embryos generated by lithium treatment from the 2- to 4-cell stage to stage 7 (4h). (A) Normal embryo at stage 26 previously treated with NaCl. (B-F) The range of abnormalities seen in embryos of the same age that had been previously treated with LiCl. For detailed description see text.
Lithium and neural development in Xenopus embryos 357 embryo volume of 1-33 fA (see Methods section). The describe a linear regression curve fitted to the data large standard error reflects considerable variability with a correlation coefficient of 0-82 (P < 0-01, Mest). in lithium content from embryo to embryo (range: The results suggest that the threshold concentration 0-5-5 mid); part of this variability undoubtedly arises of lithium required to generate developmental abnor- because of differences in embryo volume. Thus very malities is close to 0-5 mM- and 2-5 mM-lithium is little lithium enters the embryos, despite the high sufficient to produce a maximal effect. Similar defects external concentration. Equivalent variability was were produced by the microinjection of lithium, into noted in the teratogenic consequences of lithium both cells of embryos at the 2-cell stage, to give a final treatment of embryos from these batches (abnormal: intracellular lithium concentration of about 2mM. 54-9 ± 7 % ; dead: 32-3 ±7-5%; range (abnormal Injection of sodium had no effect. + dead) 5-100%, 349 embryos; spontaneous death: 13-5 ±3-5%). The concentration of lithium in the blastocoel fluid Fig. 2A plots the relationship between extra- To determine the way in which lithium taken up by embryonic lithium and teratogenic effect (solid line), the embryo is distributed between the cells and the along with the concentration of lithium within some intercellular fluid, samples of intercellular fluid were embryos of each batch (dotted line) for four batches withdrawn from the blastocoel cavity of embryos that of eggs. The developmental effect of lithium is scored had been soaked in 100mM-LiCl for 4h. Seven as the sum of the abnormal and dead embryos from samples that were uncontaminated by cell damage each batch (60-65 embryos per point), after taking gave a lithium concentration of 17±6-3mM. In all account of spontaneous death in untreated controls cases the concentration of lithium in the blastocoel (8 ± 2 % ; four batches, 60 embryos). An external fluid was substantially greater than the average con- lithium concentration up to 50 HIM had no significant centration within the whole embryo after 4h in teratogenic effect and the embryo lithium concen- lOOmM-LiCl (average: 2-5mM). This suggests that a tration was less than 0-5 mM (average of 20-24 em- substantial proportion of the lithium taken up by the bryos). 100 mM-lithium generated the usual substan- embryo is extruded from the cells into the intercellu- tial teratogenic effect with an average lithium uptake lar fluid. A rough estimate of the intracellular lithium of 2-5 mM, close to the overall average given above. concentration can be made using the values for Lithium uptake and teratogenic effect were simi- blastocoel volume and protein content of the stage-7 larly related when the time of exposure to 100 mM- embryo given in Slack et al. (1973). This calculation lithium was varied (Fig. 2B). The spontaneous death suggests that the intracellular concentration of lith- in the batches used for these experiments was again ium that produces the maximum teratogenic effect close to 10%. A significant teratogenic effect was may be as low as O-Smmolesl"1 cell water. seen after l h in lOOmM-LiCl (seven batches, 100 embryos) by which time the embryos contained, on The sensitivity to lithium depends on developmental average, 0-5 mM-lithium (eight batches, 16 samples stage each containing two or three embryos). Both the Fig. 3A shows the developmental consequences of teratogenic effect and the lithium uptake increased exposure of Xenopus embryos to 100 mM-lithium for linearly with time of exposure, suggesting a high 4 h, initiated at progressively later times during devel- correlation between the intraembryonic concen- opment. At least three time points were tested on tration of lithium and the developmental defects. The every batch. The spontaneous death within the proportion of severely affected embryos and the batches used for this series of experiments was 19 %. proportion that died as a result of lithium treatment Each column gives the sum of the abnormal and dead increased together. Fig. 2C combines the results embryos; the hatched portion indicates the pro- given in A and B with data obtained in a number of portion that died as a result of treatment, taking into other experiments (see later) and plots the concen- account the batch viability. A 4 h exposure to lithium tration of lithium within the embryos against the became progressively less effective as the embryos percentage lithium effect, taken as the sum of the proceeded through development. Lithium treatment abnormal and dead embryos for each batch, after beginning at the 32- to 64-cell stage was significantly correction for spontaneous death (average 12%). less teratogenic than treatment starting at the 2- to 4- The figures at the foot of each column give the cell stage (2- to 4-cell versus 32- to 64-cell P
358 L. J. Breckenridge, R. L. Warren and A. E. Warner
stage onwards. At stage 10?, when lithium treatment completely lacked the notochord. Embryo C had no
no longer produced any developmental defects, the recognizable anterior-posterior axis and the internal
embryos nevertheless contained 1-5 mM-lithium at the structures were again disorganized. Patches of appar-
end of the 4h exposure. Since this concentration ently differentiated, but unidentifiable tissue, are
would have been sufficient to generate a 60 % lithium scattered throughout.
effect in embryos treated from the 2- to 4-cell stage, However, when tissue-specific antibodies were ap-
Xenopus embryos must become less susceptible to the plied to frozen sections of such disorganized embryos
teratogenic effects of lithium with increasing age. both differentiated nerve cells and muscle cells could
be recognized. Fig. 5 shows horizontal sections
Internal structure of lithium-treated embryos through the head of a control embryo (A) at stage 28
The most characteristic external feature of embryos and the dorsal region of an abnormal sibling of the
that have been treated with lithium is the inhibition of same age that had been treated with lithium (C), each
the elevation of the neural folds. The internal organ- labelled with an anti-neurofilament monoclonal anti-
ization of structures within such embryos also proved body (Wood & Anderton, 1971). In the control
to be grossly disorganized. Fig. 4 shows the external embryo, neurites emerging from the developing
appearance of a normal embryo (A) at stage 26 and brain, and particularly the otic nerve, are clearly
the dorsal view of two sibling embryos of the same labelled. The pattern of staining in the lithium-
age that had previously been treated with lithium treated embryo was quite different, with labelled cells
(B,C). Transverse sections through the head (Al, Bl, scattered throughout the internal structures. In both
Cl) and trunk (A2, B2, C2) of these embryos are control and lithium-treated embryos the antibody
shown below. By comparison with the control, both also stained epidermal cells. However, incubation of
lithium-treated embryos showed gross disorgan- similar sections with an antibody that is specific for
ization of normal patterning. No recognizable organ- epidermal cells (2f7.c7: Jones, 1985) showed staining
ized structures could be found in embryo B, which of the epithelium only, confirming that the scattered
100 -1 A 100 T B
- 3
60-
35.
40- 3
20
- 1
0-
20 40 60 80 100 1 2 3 4
Cone, of lithium (rain) Time (h)
3-1 C
Fig. 2. The effect of increasing lithium concentration (A)
and time of exposure to 100 mM-LiCl (B) on lithium
effect (solid line and solid circles) and lithium uptake
(dotted line and crosses). Left-hand ordinates: lithium
2-\ effect (%). Right-hand ordinates: lithium uptake (HIM).
Abscissae: lithium concentration in bathing medium (A)
and time of exposure (B). Each point gives the mean and
•5 1 -
i the bars ±1S.E. of results from four batches of embryos.
The lithium effect is taken as the sum of the percentage
abnormal and percentage dead embryos after taking
account of spontaneous death (8 ± 2 %) in controls.
(C) Histogram giving the relation between lithium uptake
30 6 21 10 12 10 4 12 13 68 and percentage lithium effect defined as in A and B. Bars
give ±1S.E.M. Regression line (broken line) calculated
20 40 60 80 100 from the means. The number of batches for each point is
Lithium effect (%) given at the bottom of each column.Lithium and neural development in Xenopus embryos 359
staining observed in the lithium-treated embryo with shows the pattern of muscle cell differentiation recog-
the anti-neurofilament antibody arose from neurones nized with an anti-muscle monoclonal antibody
(unpublished observations of S. Rowe). Fig. 5B,D (12/101: Kintner & Brockes, 1984) in the trunk
region of a control (B) and a lithium-treated embryo
100 n Lithium effect (D). In the control embryo, myotomal muscle cells
flank the notochord (see also Gurdon, Mohun,
Brennan & Cascio, 1985). By contrast, the lithium-
treated embryo contained groups of disorganized
(34) axial muscle cells. Six lithium- treated embryos,
showing varying degrees of internal disorganization,
tr 60- all gave positive staining with both antibodies. These
(4)
results show that both neurones and muscle cells
E differentiate in lithium-treated embryos, despite the
•3 40 H (24)
(V) gross disruption of pattern formation.
20-
L
(7)
Quantitative assessment of neuronal differentiation
Although differentiated neurones can still be recog-
V9 (3) nized in embryos treated with lithium, it is possible
_*#
that the amount of neural differentiation is reduced as
2-4
cells
m
8-16
cells
32-64
cells
128 cell
-st. 7
st. 8-9 st. lOi
compared with controls. If the hypothesis that lithium
inhibits neural induction is correct, then absolute
levels of neural differentiation ought to be reduced in
3-i B Lithium uptake embryos previously treated with lithium. To test this
possibility we compared neural differentiation in
monolayer cultures prepared from the neural tube
and somitic mesoderm of control embryos at stage 20,
2-
just before outgrowth from the neural tube begins,
(23) and from the dorsal surfaces of abnormal, lithium-
treated embryos of the same age. 18 h after plating,
control cultures have formed a monolayer of differen-
(6)
tiated cells in which neurones and muscle cells are
1- readily identified using morphological criteria (see
Messenger & Warner, 1979; Breckenridge & Warner,
1982). Cells identified morphologically as neurones
generate action potentials, drive contractile activity
in innervated muscle cells and are specifically recog-
2-4 cells 64-128 cells st. nized by the anti-neurofilament antibody. Morpho-
logically identified muscle cells generate end-plate
Fig. 3. The sensitivity to lithium declines with stage of potentials and contract when innervated, develop
development. (A) The teratogenic effect of 4 h exposure striations and stain with the anti-muscle antibody
to lithium initiated at progressively later stages of (Breckenridge, unpublished observations, see also
development. Ordinate: lithium effect (%). Abscissa:
Warner, 1985). The cultures also contain pigment
stage at which treatment was initiated. The hatched area
within each bar gives the proportion of embryos that died cells, fibroblastic and epithelial cells.
as a result of treatment, the clear area the proportion of The number of nerve and muscle cells, together
abnormal embryos. Bars give ± 1 S . E . , the numbers in with the total number of cells (100-500 cells/field),
parentheses the number of batches for each point (14 was counted in each of 20-30 microscope fields taken
embryos per batch). Treatment initiated at the 32- to 64- at random from three cultures prepared from control
cell state and later was significantly less effective than embryos or from sibling embryos previously treated
treatment beginning at the 2- to 4-cell stage. *PL. J. Breckenridge, R. L. Warren and A. E. Warner Fig. 4. Lithium treatment generates gross internal disorder. (A-C) Photographs of control embryo at stage 26 and two severely abnormal lithium-treated siblings of the same age. Bar, 1 mm. (Al, Bl, Cl) Sections taken through the head at the level of the optic cups in the control and the best estimate of the equivalent level in the lithium-treated embryos. (A2, B2, C2) Sections through the midtrunk region. Bar, 10;um. Note extreme internal disorder. differentiated from lithium-treated embryos was sig- field in eight experiments in which both total cell nificantly (F
Lithium and neural development in Xenopus embryos 361 Fig. 5. Cell-type-specific antibodies recognize nerve and muscle cells even in severely disorganized, lithium-treated embryos. Sagittal frozen sections through control embryo at stage 28 (A,B) and lithium-treated embryos of same age (C, D). A and C stained with antibody directed against neurofilament protein. Note labelling of white matter at periphery of developing brain and the otic nerve in the control. Neural staining is widely scattered and disorganized in the lithium-treated embryo. B and D stained with antibody that recognizes axial muscle. Note clear staining of myotomal muscle in the control and the scattered patches of labelled muscle cells in the lithium-treated embryo. (E) Section through control embryo treated with second layer antibody alone. There is no background staining. Bar, 100 fim.
362 L. J. Breckenridge, R. L. Warren and A. E. Warner
D Control P>0-01). There was no effect on either total cell
10
number or the proportion of differentiated muscle
cells. These results suggest that the increase in
6- neuronal differentiation observed in embryos treated
with lithium from the 2-cell stage is linked with the
severity of the abnormalities.
Factors determining the uptake of lithium
2 4 6 8 10 12 2 4 6 8 10 12 In the course of experiments to examine the mechan-
o
ism of lithium uptake by the amphibian embryo we
B 2-4 cells Li E 32 cells Li made a number of observations on the conditions that
determine the ease of entry of lithium ions. Although
these experiments were not able to shed any light on
the mechanisms underlying the teratogenic effects of
lithium, they nevertheless provide information that
may be useful for those who wish to design future
2-
experiments and are therefore described briefly be-
low.
2 4 6 8 10 12 2 4 6 8 10 12
% Neurones % Neurones
The effect of ionic strength
C % Neurones F % Neurones
The lithium uptake by treated embryos increased
2 8 fivefold for a doubling of the external concentration
6 of lithium from 50 to 100 mM. This implies that the
T *
§ 4- 4 j
CO
entry of lithium is not simply determined by the sum
in
of the electrical (the membrane potential) and chemi-
06
1 2- 2-
cal (the concentration gradient) inward driving forces
Control Lithium Control Lithium and that the permeability to lithium must have risen.
2 cells >32 cells To determine whether this increase in lithium per-
meability was induced by lithium itself or by the rise
Fig. 6. Neuronal differentiation after lithium treatment in ionic and osmptic strength of the bathing medium,
assayed quantitatively in tissue culture. (A) Frequency we examined the uptake of lithium from 50 mM-LiCl
distribution of the percentage of neurones in each field in when the ionic or osmotic strength of the external
cultures prepared from control embryos. (B) Similar
solution had been increased to that of 100 mM-salt by
distribution for neuronal differentiation from embryos
treated for 4 h with 100 mM-lithium from the 2- to 4-cell the addition of other compounds. Fig. 7 shows the
stage. Ordinates: number of observations. Abscissae: % teratogenic effect of 50mM-LiCl (A) and the corre-
neurones per field. (C) Pooled data from eight similar sponding lithium uptake (B) under these conditions.
experiments. Ordinate: % neurones/field. Arrows at the As in all previous figures the numbers in parentheses
median of each distribution. Bars give mean ± ls.E. give the number of batches used for each set of
Total number of fields counted given in parentheses. measurements and the values in panel A take account
(D-F) A similar set of plots for an experiment where of spontaneous death (19 % for this series). Columns
lithium treatment for 4h began at the 32-cell stage. 1 and 2 in each panel show the effect of 100 mM- and
*P0-l; lithium. The permeability of the embryo to lithium
Mann-Whitney test). The pooled data from eight must, therefore, depend on the osmotic strength of
experiments in which treatment with lithium began the medium bathing the embryo. The increase in
between the 32-cell stage and stage 11 is shown in permeability induced by raising osmotic strength may
Fig. 6F. The proportion of neurones differentiating extend to all ions and small molecules. Loeffler &
from lithium-treated embryos is slightly higher than Johnston (1964) found that the uptake of tritiated
found in controls but is barely significant (002> thymidine by urodele embryos could be increased byLithium and neural development in Xenopus embryos 363
100
link the apical edges of each cell and are of very high
resistance (Slack & Warner, 1973; see also Regen &
Steinhardt, 1986), so allowing direct access to the
(9) internal, more permeable and potassium-selective
80
cell membranes (Woodland, 1968; Slack & Warner,
(10) (11) 1973; de Laat, Buwalda & Habets, 1974). This would
r 60 explain the increased potassium sensitivity of the
(9) membrane potential of cells in embryos at high ionic
(10) strength (see below and Appendix). Electrophysio-
E
•2 40
r 11 I
logical tests to distinguish between these two possi-
bilities are described in the Appendix. They make it
most likely that high ionic or osmotic strength acts
I
20- directly on the properties of the cell membranes,
rather than the integrity of the tight junction. How-
ever, this conclusion may not be valid when the
100 Li 50 Li 50 Li 50 Li 50 Li 50 Li embryo is subjected to solutions of very high ionic
strength, such as those used by Kao etal. (1986). If we
50 Na 50 Tris 50 Ch 100 sucrose assume that about 2-5 mM intraembryonic lithium is
responsible for their finding that 6min exposure to
0-3 M-LiCl generates similar defects to those reported
here, then the permeability to lithium must have been
3- 40 times greater than that in 50mM-LiCl. Such a
massive increase in lithium uptake could well reflect
transient disruption of the properties of tight junc-
D.
3
2- tions, in addition to effects on cellular membranes.
E
3
The cation composition of the bathing solution
influences lithium uptake
T" "'
The properties of the channels opened by an increase
(8) (8) (8) (6) (6) (4) in osmotic or ionic strength may be quite complex
100 Li 50 Li 50 Li 50 Li 50 Li 50 Li since the increase in lithium uptake was strongly
influenced by the identity of the added ion. Fig. 8
50 Na 50 Tris 50 Ch 100 sucrose
shows the effect of adding the chlorides of potassium,
caesium, rubidium or magnesium to 50mM-LiCl
Fig. 7. Raising the osmotic or ionic strength increases the compared with that of NaCl. The presence of 50 mM-
defects generated by lithium (A) and the lithium uptake potassium had little influence on either the terato-
(B). (A) Each column gives the sum of the percentage
genic effect (A, column 4) or uptake of lithium (B,
abnormal embryos (clear) and the percentage of embryos
that died as a result of treatment (hatched). Bars ± 1 S . E .
column 4) from 50 mM-lithium^ Caesium, rubidium
The treatment schedule is given at the bottom of each and magnesium all increased the abnormalities and
column. Number of batches used given in parentheses. lithium uptake in 50mM-lithium, but were signifi-
Treatment with 50 mM-lithium alone is significantly less cantly less effective than sodium. Thus some cations
effective (**P< 0-001) than all solutions at double the may protect the embryo from the consequences of
ionic/osmotic strength. All the cations and sucrose increasing the ionic strength.
increase the effect of lithium equivalently. (B) The This protection could be the consequence of com-
lithium uptake in some embryos from a number of petition between lithium and other cations for the
batches (given in parentheses). Uptake from 50mM- sites that mediate the transfer of lithium across the
lithium alone significantly lower than in other conditions outermost membranes. Alternatively sodium and
tested.
lithium ions may bring about a larger increase in
permeability to lithium than the other salts used to
the addition of salt to the solution bathing the raise the ionic strength. Experiments designed to test
embryo. this point directly using electrophysiological tech-
The increase in overall permeability induced by niques are described in the Appendix. They show
100 mM-salt could reflect a change in the properties of clearly that the overall permeability of the amphibian
the outermost cell membranes. An equivalent effect embryo is closely similar in a 100 mM solution of all
would be produced if high osmotic strength solutions the monovalent cations. The increase in permeability
interfered with the integrity of the tight junctions that is, however, greatly reduced when the ionic strength364 L. J. Breckenridge, R. L. Warren and A. E. Warner
lithium achieves a small, but not significant, protec-
3 4 5 6 7 tion of the embryos against the teratogenic effects of
100
lithium. However, the most striking finding was that
soaking the embryo in NaCl from the 2- to 4-cell stage
80- to the 32- to 64- cell stage and then transferring the
(8)
embryos to lithium potentiated both the teratogenic
tT 60 effects (B) and the uptake of lithium (C).
These findings imply that the ionic composition of
the solution initially used to raise the ionic strength
.2 40 13) (6)
(13) 13) influences the properties of the channels which then
XI
20 - I become available for the movement of small mol-
ecules across the outer membranes of the embryo.
I
100 Li
(3)
50 Li 50 Li
+
w.
50 Li
+
50 Li 50 Li 50 Li
+ + +
We were unable to identify the nature of this influ-
ence. The only difference between the membrane
properties of embryos exposed to NaCl and KCl was
B 50 Na 50 K 50 Cs 50 Rb 33 M g in the membrane potential: cells of embryos in
4 -i
100 mM-lithium or in 50 mM-lithium together with
sodium had resting potentials that were about 10 mV
3- more negative than those of embryos in 100 mM-
potassium or in 50 mM-lithium together with potass-
2-
(4) (4) ium. The membrane potential was also depolarized in
the presence of rubidium and caesium, but to a lesser
extent. However, the difference in membrane poten-
(4)
.•§ 1 -
(4) tial did not persist beyond the period of exposure to
(2) (4) potassium ions. Embryos that had been exposed to
100 mM-potassium for 2 h promptly hyperpolarized on
100 Li 50 Li 50 Li 50 Li 50 Li 50 Li 50 Li transfer to 100 mM-lithium.
50 Na 50 K 50 Cs 50 Rb 33 Mg
Discussion
Fig. 8. Comparison of the ability of different cations to
increase the teratogenicity (A) and uptake (B) of lithium.
Lithium ions generate teratogenic effects in the
(A) Sodium and lithium added to 50 mM-lithium are
equivalent^ effective. K + , Cs + , Rb + and Mg2+ all
amphibian embryo at very low internal concen-
generate a lower lithium effect than Na + . *FLithium and neural development in Xenopus embryos 365
A Post-treatment B Pretreatment
1 2 lOO-i 1 2
100-1
4-
(7)
C Pretreatment
4 -i
80
(6)
T
60- 60- 3- 1
(5)
T
•g 40-
(9)
40- ** 2-
1
(9) (8)
(13)
(8)
20 20- 1- (8)
(9)
i
Li4h
1
Li2h
Water
Li 2h
Na 2h
Li2h
K2h
Li4h Water
Li2h
Na2h
Li2h
K2h
Li2h
Li4h Water
Li 2 h
Na2h
Li 2 h
K2h
Li2h
Fig. 9. The consequence of treating embryos with 100 mM-NaCl or 100 mM-KCl either after a 2 h exposure to 100 mM-
lithium beginning at the 2- to 4-cell stage (A) or before a 2h exposure to lithium (B). Treatment schedule given below
each column. (A) There is no difference between exposure to Na + , K+ or water after LiCl. (B) Sodium potentiates the
teratogenic effect of a subsequent exposure to lithium. (C). Lithium uptake in embryos treated with water, NaCl or KC1
for 2 h before treatment with lithium. Sodium significantly increases the subsequent uptake of lithium. **P < 0-001.
would be even higher. The free ion concentration, of the lithium permeability in 100 mM-lithium using
which may be most relevant for the teratogenic the Goldman equation (see Hodgkin & Horowicz,
effects, could be five times lower than the estimates 1959), with the influx taken from the net uptake
given above. There are no measurements of the of lithium and the membrane potential set at
lithium taken up by embryos in previously published -30 mV, suggests that even in 100 mM-lithium the
experiments although the treatment schedules of Hall permeability coefficient is probably not much greater
(1942) and Backstrom (1954) suggest very similar than 0-04xl0" 6 cms~ 1 . The sensitivity of the mem-
final lithium concentrations to those found here. brane permeability to increased ionic strength almost
The intracellular concentration of lithium that is certainly explains why Kao et al. (1986) were able to
sufficient to generate a substantial teratogenic effect generate substantial teratogenic effects by a few
is comparable to the therapeutic dose of lithium minutes' exposure to 0-2-0-3 M-LiCl. If the effects
used to treat patients with manic depressive illness observed in those experiments were achieved by
(0-7-1-5 meq I"1 plasma). There is full equilibration intraembryonic lithium concentrations similar to
of lithium across the placental membrane (Schou & those measured here, then their results imply a
Amdeson, 1975) and lithium may also enter the lithium uptake at a rate at least 40 times greater than
uterine fluid, an ultrafiltrate of plasma. Studies on the in lOOmM-LiCl, making the lithium permeability
amphibian embryo could therefore be more relevant
coefficient in the region of 2x 10~ 6 cms" 1 . This would
to possible teratogenic effects in human embryos than
suggest that the outer membrane of the Xenopus
has hitherto been realized.
embryo becomes at least as permeable to small ions
The absolute permeability to lithium of the outer as frog muscle under conditions of very high ionic
membranes of the amphibian embryo is normally strength.
very low, as for all other small ions (Slack et al. 1973;
de Laat etal. 1974). However, the lithium uptake, and The external defects generated by lithium treat-
therefore the permeability, was strongly dependent ment in the present experiments were closely similar
on both the osmotic/ionic strength of the bathing to those seen by others (e.g. Hall, 1942; Backstrom,
medium. A rise in osmotic strength to that equivalent 1954; Kao et al. 1986). They were accompanied by
to lOOmM-salt substantially increased the uptake of gross internal disorganization of all the structures of
lithium, probably because of a general increase in the the embryonic axis, showing that pattern formation
permeability of the outer membranes of the embryo had been severely disrupted. Nevertheless differen-
to all ions and small molecules. However, calculation tiated neurones and differentiated muscle cells could366 L. J. Breckenridge, R. L. Warren and A. E. Warner be identified with cell-type-specific antibodies, con- experiments would be in the region of 2mM, close to tradicting former suggestions that lithium ventralizes the concentration found by us to be maximally (e.g. Backstrom, 1954) or mesodermalizes (Ogi, 1961; effective. If lithium were rapidly redistributed Masui, 1961) the amphibian embryo. This conclusion throughout the embryo after injection into one cell is strongly supported by the observation that after then one would expect injections into the animal or lithium treatment, neuronal differentiation, assayed vegetal pole or into ventral or dorsal vegetal cells to in tissue culture, was substantial even from acutely be equally effective. This is not observed experimen- abnormal lithium-treated embryos. Muscle differen- tally, suggesting that movement of lithium within the tiation was unaffected. A further, important con- embryo is relatively slow. clusion is that lithium has separate effects on the An additional complication arises from the marked elements of neural induction that lead to the differen- stage dependence of the sensitivity of the Xenopus tiation of nerve cells and those that lead to the embryo to lithium treatment. In the present exper- patterning of neural and other axial structures. iments the teratogenic consequences of exposure of The finding that the differentiation of neurones was the embryos to lithium declined steadily as develop- significantly enhanced in lithium-treated embryos was ment proceeded. The lithium uptake did not decline unexpected. Although lithium has been shown to equivalently, suggesting that some relatively early induce the differentiation of neurones from com- event is sensitive to the intracellular lithium concen- petent Rana ectoderm (Barth & Barth, 1974), it does tration. The increase in neural differentiation seen in not appear to do so in Xenopus (Messenger, 1979). embryos exposed to lithium from the 2- to 4-cell stage One possibility is that the failure of the neural tube to also no longer took place, or was greatly reduced, close allows an inducing factor to spread further when lithium treatment began at times beyond the 32- within the ectoderm (Warner, 1979). Alternatively, cell stage. This observation provides further evidence the suggestion of Kao et al. (1986) that lithium that the quantitative increase in the number of enhances dorsoanterior development could mean neurones that differentiate is directly linked to the that the amount of neural tissue is increased. If this teratogenic effects of lithium. Kao et al. (1986) also enhancement were extensive then it might mechan- observed that the effects of lithium were stage depen- ically impede closure of the neural tube. In that case dent. Brief treatment with 0-2 or 0-3M-LiCl was most the inhibition of neurulation movements would be a successful in rescuing embryos from the consequence casual rather than a causal consequence of lithium of u.v. irradiation when applied at the 32- to 64-cell treatment. stages. If the teratogenic effect of lithium is mainly The finding that both lithium treatment and micro- determined by the intracellular concentration of lith- injection of lithium into vegetal cells can restore the ium at the 32- to 64-cell stage, then the effective development of dorsal structures in radially sym- concentration must be even lower than indicated metric, ventral embryos generated by u.v. irradiation above, since total lithium was little more than 1 mM prior to first cleavage (Kao et al. 1986) provides strong by the time embryos initially exposed to lithium support for the view that lithium enhances the devel- chloride at the 2- to 4-cell stage had progressed opment of dorsal. structures rather than inhibiting through the requisite number of cleavage cycles. neural induction. This is strengthened by Kao et a/.'s However, Kao et a/.'s (1986) finding that exposure to (1986) finding that microinjection of lithium into lithium up to the 32-cell stage was substantially less ventral vegetal cells of normal embryos at the 32-cell effective than at the 32-cell stage makes interpret- stage generated a high proportion of embryos lacking ation of the stage dependence difficult. Our exper- posterior development, but possessing two heads. To iments suggest that the outer membranes of the achieve substantial rescue, or duplication of dorsal amphibian embryo return to the low permeability structures, Kao et al. (1986) microinjected 4nl of state promptly (within less than lmin) on lowering O3M-lithium in 200% Steinberg's solution into a the ionic strength (see Appendix). Lithium that single vegetal pole cell. If the lithium remains within entered during earlier stages in Kao et a/.'s (1986) the injected cell then the intracellular concentration experiments ought therefore to be present at the 32- would be in the region of 40 mM, about 10 times to 64-cell stage and available to influence the puta- higher than a lethal dose. Some of the injected tive, stage- and lithium-dependent event. However, if lithium must be distributed throughout the embryo, the increase in permeability produced by a 0-2 or since our results show that most of it is destined to be 0-3 M bathing solution persists beyond the exposure, extruded into the blastocoel fluid, which has access to much of the lithium that enters the embryo would be all cells. The rate at which this redistribution of immediately lost. We found that microinjection of lithium takes place is not known and is difficult to lithium at the 2-cell stage was able to induce abnor- estimate. At complete redistribution, the intra- malities. Acute stage dependence would aid the embryonic lithium concentration in Kao et a/.'s (1986) identification of the sensitive cells by microinjection
Lithium and neural development in Xenopus embryos 367
because the distribution of lithium within the embryo and potassium in the putative lithium-sensitive, stage-
may be too slow for lithium ions in the appropriate dependent event. Until this event, which appears to
cell(s) to reach threshold within the sensitive period, control the fundamental process of patterning in the
ii .""ay be possible to resolve some of these issues with amphibian embryo, is identified, the effects of lithium
lithium-sensitive microelectrodes. are unlikely to be understood.
The uptake of lithium was markedly affected by the
identity of the cation used to raise the ionic strength. This work was supported by the Medical Research
Most notable was the interaction between lithium and Council. We thank Dr J. I. Gillespie for collaborating in
potassium, so that lithium uptake was substantially some preliminary experiments. B. Anderton, J. Brockes,
impeded when potassium ions, and to a lesser extent C. Kintner and E. Jones kindly donated samples of anti-
body.
rubidium and caesium, were also present in the
bathing medium. Lallier (1960) and Wolcott (1981)
found that the teratogenic effect of lithium on sea Appendix
urchin development was reduced in the presence of
potassium suggesting that potassium may impede The experiments comparing the uptake of lithium
lithium entry also in the sea urchin embryo. The when the ionic strength had been raised to 100 mM by
evidence suggests that potassium ions compete with the addition of various cations suggested that the
lithium at the sites made available by the rise in permeability of the embryo might be much less in the
osmotic strength. Whether interactions between lith- presence of potassium than in sodium-containing
ium and the other monovalent cations also take place solutions. Direct information on this problem can be
at intracellular sites is difficult to ascertain. The obtained by measuring the electrical resistance of the
finding that exposing the embryo to NaCl up to the embryo under the appropriate experimental con-
32-cell stage increased the subsequent uptake of ditions. This Appendix briefly describes electro-
lithium suggests that the intracellular environment physiological experiments which address this issue.
may be important. The sodium and potassium con- Some experiments that show that the alterations in
centrations in the embryo did not change significantly permeability probably occur in the outermost cell
in high external sodium (unpublished observations), membranes, rather than by disruption of the tight
although the naturally high concentrations and the junctions that link the apical edges of the cells, are
substantial variations- from embryo to embryo and also included. The major conclusions drawn from the
batch to batch (Slack et al. 1973; Gillespie, 1983) results are indicated at the appropriate positions in
could easily have obscured relatively small, yet im- the main text.
portant alterations in individuals. The internal levels Standard electrophysiological techniques were
of other monovalent cations may also have influenced used. Low resistance (5-10 megohm) microelec-
the experiments of Kao et al. (1986). If in their trodes filled with 3 M-KC1 with insignificant tip poten-
experiments the high permeability state persisted tials recorded the membrane potential or injected
much beyond the exposure period, then significant current pulses. After removing the jelly coat (either
net loss of other small ions, which experience a much manually or by brief exposure to 2 % cysteine in
greater outward driving force than lithium, may have Holtfreter's solution at pH8-0) the embryo was
occurred. An additional factor would be the shrink- transferred to a 3 ml volume recording bath. Solution
age on exposure to hypertonic solution and sub- changes were effected by injecting 10 ml of sol-
sequent swelling on return to low ionic strength ution into the bath and removing the excess with
media. constant suction. To measure the input resistance two
The mechanism underlying the profound pertur- electrodes were inserted into the same cell, one to
bations of development initiated by treatment of inject rectangular hyperpolarizing current pulses
early embryos with lithium is not known. In the (50-300 nA, 400-1200 ms in length, one every 2-9 s)
amphibian embryo the threshold concentration is while the other recorded the membrane potential and
very low, in the 100-500//M range, which restricts the the resultant electrotonic potentials. The ratio of the
range of possible mechanisms. In the appropriate electrotonic potential to the injected current gives the
concentration range, lithium has been reported to input resistance of the embryo. The vitelline mem-
inhibit adenyl cyclase (Thams & Geisler, 1981) and brane offers a negligible resistance to the flow of
microtubule-mediated movement of sperm flagellae current.
(Gibbons & Gibbons, 1984). Additionally it is known
that lithium interferes with a wide range of other The ionic permeability at different ionic strength
cellular processes. The complicated interactions be- The input resistance of the cleavage-stage embryo is
tween lithium and the other monovalent cations focus determined by both the properties of the low resist-
attention on a possible physiological role of sodium ance, relatively potassium-permeable membranes368 L. J. Breckenridge, R. L. Warren and A. E. Warner
generated at each cell division and the high resist- affect the overall conductance of cleavage-stage am-
ance, outermost membrane derived from the egg. phibian embryos equivalently. Comparison of the
Nevertheless, measurements of input resistance input resistances recorded in 100iriM-NaCl, 100 mM-
should reveal major differences in the ability of the LiCl and 100 mM-KCl showed the input resistance in
various cations used in experiments on lithium up-
(A) Intact embryo
take (Fig. 7) to increase the overall permeability.
100 Na 100 K 100 Na
Fig. 10A,B shows measurements of input resistance
of one cell of a 16-cell embryo in 50 and 100 mM-NaCl
(A) and KC1 (B). In both cases the measurement
begins in 100 mM salt solution and the input resistance mV
doubles within a minute of the reduction in ionic
strength to 50 mM. Similar results were obtained in 14 -J - 3 0
other experiments. Thus both sodium and potassium
100 Na 50 Na
(B) Internal membranes exposed
100 Na 100 K 100 Na
-20
mV
3-6 min -40
22 s 28 s
Fig. 11. The effect on the resting potential of changing
from 100 min-sodium (100 Na) to lOOmM-potassium
100 K 50 K (100 K). (A) Intact embryo. 100mM-potassium produces
an 8mV depolarization, which is reversed on return to
lOOmM-sodium. Electrode withdrawn from the cell at end
of trace and the trace returns to the OmV baseline. (B)
Vitelline membrane removed and two cells pulled apart
to reveal internal membranes. 100 mM-potassium
produces 30mV depolarization. Electrode withdrawn
before repolarization in 100 mM-sodium is complete.
Fig. 10. The input resistance of the embryo is the same
in NaCl and KC1. Pen records of the membrane potential
2-9 min (continuous trace) and the electrotonic potential
generated by injection of constant, hyperpolarizing
28s 36 s current pulses (vertical deflections) in (A) 100 inM-NaCl
(100 Na) and (B) 100 mM-KCl (100 K). For measurement
100 K 66 Mg 100K of the electrotonic potential the chart speed was
increased. The solution was changed to 50mM-salt (50Na,
-15 50 K) as indicated and a further set of electronic
potentials recorded. Right-hand calibration gives the
resting potential of the cell. The increase in input
-35 resistance seen on halving the ionic strength is fully
mV established within 1 min of the solution change. Na + and
II - -55
K+ are equivalently effective. (C) Continuous record of
membrane potential and the electrotonic potential in
100 mM-KCl. 66mM-MgCl2 (66 Mg) replaced KC1 during
part of the record. The input resistance doubles in
magnesium, even though the ionic strength of the two
solutions is the same.Lithium and neural development in Xenopus embryos 369
KC1 to be about 10 % less than in either NaCl or LiCl. References
The findings illustrated in Fig. 7 require the ionic
resistance of the embryo to be much higher (by at BACKSTROM, S. (1954). Morphogenetic effects of Li on the
least a factor of 2) in potassium than in sodium. embryonic development of Xenopus. Arkiv. fur.
Zoolog. 6, 527-536.
Fig. IOC shows how the input resistance altered in BARTH, L. G. & BARTH, L. J. (1962). Further
one of seven experiments in which the bathing investigations of the differentiation in vitro of
medium was switched from 100 HIM-KG to 66 mM- presumptive epidermal cells of the Rana pipiens
MgCl2 (the equivalent ionic strength). The input gastrula. /. Morph. 110, 347-367.
resistance doubles in the presence of divalent mag- BARTH, L. G. & BARTH, L. J. (1974). Ionic regulation of
nesium ions; the overall ionic permeability is there- embryonic induction and cell differentiation in Rana
pipiens. Devi Biol. 39, 1-22.
fore very similar to that found in 50 mM salts of the
BRECKENRIDGE, L. J. (1985). The role of small ions in
monovalent cations. In these circumstances the up- nerve and muscle cell differentiation in Xenopus laevis.
take of lithium from 50 mM-LiCl would be expected to Ph.D. Thesis. University of London.
be little different in the presence and absence of BRECKENRIDGE, L. J. & WARNER, A. E. (1982).
magnesium, as observed experimentally (Fig. 7). Intracellular sodium and the differentiation of
amphibian embryonic neurones. J. Physiol., Lond. 322,
393-491.
Is the increase in permeability the result of disruption DE LAAT, S. W., BUWALDA, R. J. A. & HABETS, A. M.
of tight junctions? M. C. (1974). Intracellular ionic distribution, cell
membrane permeability and membrane potential of the
Although there was little difference in the input Xenopus egg during first cleavage. Expl Cell Res. 89,
resistance of embryos in 100mM-NaCl, LiCl and KC1, 1-14.
the membrane potential always depolarized by about ENGLANDER, H. & JOHNEN, A. G. (1967). Die
10 mV when potassium ions were present. An morphogenetische wirkung von Li-ionen auf gastrula-
example is shown in Fig. 11A. The membrane poten- Ektoderm von Ambystoma und Triturus. Wilhelm Roux
tial recorded from intact embryos is not usually Arch. EntwMech. Org. 159, 346-356.
GIBBONS, B. H. & GIBBONS, I. R. (1984). Lithium
influenced by external potassium, except during
reversibly inhibits micro tubule based motility in sperm
cleavage, when new, potassium-sensitive membrane flagella. Nature, Lond. 309, 560-562.
is transiently exposed in the cleavage furrow (Wood- GILLESPIE, J. I. (1983). The distribution of small ions
land, 1968; Slack & Warner, 1973). Since the mem- during the early development of Xenopus laevis and
brane potential was only sensitive to alterations in Ambystoma mexicanum embryos. /. Physiol., Lond. 344,
external potassium concentration at high ionic 359-377.
GRUNZ, H. (1968). Experimental studies on competence
strength, it seemed possible that disruption of the
in early development of amphibian ectoderm. Wilhelm
tight junctions in 100 mM salt might lead to the Roux Arch. EntwMech. Org. 160, 344-374.
relatively potassium-permeable inner membranes be- GURDON, J. B., MOHUN, T. J., BRENNAN, S. & CASCIO, S.
ing continuously accessible to the bathing solution. (1985). Actin genes in Xenopus and their
However, this seems unlikely. Fig. 11B shows that developmental control. J. Embryol. exp. Morph. 89
when the internal membranes were deliberately ex- Supplement 125-136.
posed to the bathing solution (by removing the HALL, T. S. (1942). The mode of action of lithium salts in
embryo from the vitelline membrane and gently amphibian development. J. exp. Zool. 89, 1-36.
HERBST, C. (1892). Experimentelle Untersuchungen Uber
pulling two of the surface cells slightly apart) 100 mM- den Einfluss der veranderten chemischen
potassium generated a very substantial depolariz- Zusammensetzung des Umgebenden Mediums auf die
ation, with the membrane potential approaching the Entwicklung der Thiere. Z. wiss. Zool. 55, 446-578.
potassium equilibrium potential (OmV). Further- HODGKIN, A. L. & HOROWICZ, P. (1959). The influence of
more the input resistance of the fertilized egg was as potassium and chloride ions on the membrane potential
sensitive to alterations in ionic strength as multicellu- of single muscle fibres. J. Physiol., Lond. 148, 127-160.
JOHNEN, A. G. (1970). The influence of Li and SCN ions
lar embryos. Thefindingthat the membrane potential
on the differentiation capacities of Amblystoma
of cells becomes potassium sensitive at 100 mM salt ectoderm and their changes as a result of combined
suggests that the channels made available for ion treatment with both ions. Wilhelm Roux Arch.
transport by the increase in ionic strength may reflect EntwMech. Org. 165, 150-162.
the unmasking of channels originally present in the JONES, E. (1985). Epidermal development in Xenopus
oocyte membrane which normally cease to take part laevis: the definition of a monoclonal antibody to an
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