J. Embryol. exp. Morph. Vol. 24, 3, pp. 535-553, 1970
535
Printed in Great Britain
Some bio-electric parameters of early
Xenopus embryos
By J. F. PALMER1 AND CHRISTINE SLACK2
From the Departments of Physiology and Biology as Applied to Medicine,
The Middlesex Hospital Medical School
SUMMARY
Membrane potential and resistance were measured in eggs, cleavage stages and blastulae
of the South African toad Xenopus laevis, using intracellular microelectrodes.
The membrane potential increased from -6-5±2mV in eggs to - 5 7 ± 8 0 m V at the
mid-blastula stage.
The input resistance of fertile eggs ranged from 0-5 MCI to 50 Mfi corresponding to a
specific resistance of 20-200 k^cm2. During the first two or three division cycles the input
resistance usually decreased by a factor of 2-10 and then subsequently rose during the
blastula stages from a mean value of 600± 100kO at stage 5 to 20±0-5 MCI at stage 8.
At all developmental stages examined, point polarization of a surface cell in the embryo
by rectangular current pulses of 0-5-6 x 10~8 A produced voltage deflexions in other surface
cells. This was seen even when several (7-8) cell junctions intervened between the current
passing and voltage recording microelectrodes at distances of more than 1 mm. These
measurements suggest that the junctional resistance is low compared with that at the surface,
though the geometrical arrangement of cells is not favourable for calculation of absolute
values of membrane resistance.
Current spread between cells occurred apparently less easily during mid-blastula stages than
at earlier stages in development, perhaps indicating an increase in junctional resistance during
development.
A comparison has been drawn between the present measurements and similar ones made
in another amphibian, Triturus.
INTRODUCTION
A variety of events during early development have been correlated with
changes in bio-electric parameters in a number of embryos. Surface membrane
resistance and trans-membrane potential changes are known to be associated
with oocyte maturation in Xenopus (Kanno & Loewenstein, 1963), with the
activation of echinoderm, amphibian and Oryzias eggs (Tyler, Monroy, Kao &
Grundfest, 1956; Hiramoto, 1959; Maeno, 1959; Ito, 1962) and with cleavage
in Triturus and R. pipiens embryos (Ito & Hori, 1966; Woodward, 1968). Such
changes could reflect alterations in surface membrane permeability which may
be important in the initiation and control of developmental processes.
1
Author's address: Department of Physiology, Middlesex Hospital Medical School,
London, W.I., U.K.
2
Author's address: Department of Biology as applied to Medicine, Middlesex Hospital
Medical School, London, W.I, U.K.
34
E M B 24
536
J. F. PALMER AND C. SLACK
Electrophysiological techniques have in addition demonstrated that the
junctions between some embryonic cells are low resistance pathways to the
flow of ionic current. Measurements of this electrical coupling have been made
in squid embryos (Potter, Furshpan & Lennox, 1966), in chick (Sheridan, 1968),
the amphibian Triturus (Ito & Hori, 1966; Ito & Loewenstein, 1969) and in the
fish Fundulus (Bennett & Trinkaus, 1968). In this paper we report the results
of some electrical measurements in pregastrular stages of Xenopus laevis, the
clawed toad. The observations provide information on some of the membrane
properties of Xenopus embryos and indicate that the cells are electrotonically
coupled to each other.
100 MQ
Steinberg's
solution
Embryo
(stage 7)
Paraffin
To
current
amplifier
Fig. 1. Experimental set-up to measure input resistance and voltage decay across
cell junctions. Microelectrode A passed rectangular current pulses, B recorded
electrotonic voltage Vx in the same cell as A. Electrode C was positioned intracellularly at different points across the surface to measure current spread across the
cell junctions. Cal. = Calibrator.
MATERIALS AND METHODS
Xenopus laevis embryos stages 1-9 (Nieuwkoop & Faber, 1967), obtained by
injection of mature animals with chorionic gonadotrophin, were used. The
embryos were kept in culture medium until they had reached the required stage
and were then transferred to fresh solution for removal of jelly with fine forceps.
The culture medium employed throughout was Steinberg's solution which has
the composition: NaCl 60 mM; KC10-7 HIM; Ca(NO3)2.4H2O 0-3 HIM; MgSO4.
Bio-electric parameters o/Xenopus embryos
537
7 H2O 0-8 HIM; Tris buffer 1-4HIM; and was adjusted to pH 7-2-7-4 with HC1.
Measurements were made at room temperature (18-25 °C).
Fig. 1 illustrates the experimental set-up. Intracellular microelectrodes filled
with 3 M-KC1 with resistances in the range 16-60 MO and tip potentials of
- 1 0 raV or less, were used. The embryos were placed in paraffin wax depressions in a perspex bath. The bath was filled with Steinberg's solution and
earthed via a large Ag/AgCl electrode which made contact through a Steinberg/
agar bridge. Membrane potentials were measured with respect to the external
solution and were uncorrected for changes in electrode tip potential between
Steinberg's solution and cell cytoplasm. Input resistance was recorded in all
stages by two microelectrodes inserted into the same cell, one of which passed
rectangular current pulses, the other recording the resistive voltage produced
across the surface membrane. The voltage recorded close to (< 25/*) the
polarizing electrode, divided by the total current injected at that point gave a
measure of the input resistance of the embryo. In some experiments a third
microelectrode was positioned at different points in the embryo to record the
electrotonic spread of voltage across cell junctions. Current pulses were delivered from a stimulator through a 100 MO resistor in series with the polarizing
electrode, the absolute value of current being measured across a 100 kO resistor
in the earth return of the circuit. The currents used in these experiments were
in the range 0-5-6 x 10~8 A and the pulse width was usually 1 sec. Voltages were
recorded conventionally by d.c. amplifiers with cathode follower input stages,
and were displayed simultaneously on an oscilloscope and pen recorder.
A deflexion due to current flowing across the 100 kO resistor in the earth
return was recorded on all voltage traces. This was subtracted from the records
for the purposes of membrane resistance calculations.
RESULTS
Measurements were confined to surface cells of the animal pole and vegetal
margins. Those at the vegetal pole were not examined since measurements from
this region could be carried out only after removal of the vitelline membrane—
intact embryos always rotate within this membrane until the animal pole region is
uppermost. Whilst the membrane could be fairly easily removed from blastulae
using fine forceps, removal from early cleavage stages was difficult because
mechanical damage to the cells almost always occurred. In many instances also,
embryos which had their vitelline membranes removed at early cleavage divided
in an unoriented fashion. The cells tolerated prolonged impalement well as
judged by their rate of division, and experimental embryos subsequently gastrulated forming normal tadpoles.
Potential measurements
Fig. 2 shows a characteristic microelectrode penetration of a cell, this particular measurement was made from a stage 4 embryo. Passage of the electrode
34-2
538
J. F. PALMER AND C. SLACK
through the vitelline membrane was accompanied by a transient negative
deflexion, which returned to the base-line as the tip entered the perivitelline
space. The vitelline membrane had considerable elasticity and as a result proved
difficult to penetrate on some occasions. When the microelectrode tip pressed
against the plasma membrane a gradually developing positivity was observed,
which gave way to a sudden negative resting potential indicative of cell penetration. The size of the positive deflexion was governed by the magnitude of the
tip potential on the microelectrode, which was — 10 mV in this instance; when
the tip potential was very low, < - 3 mV, the positivity was absent. Within a
minute or two of initial impalement, the membrane potential often increased by
10
•HH
Fig. 2. Pen record showing penetration of animal pole cell in a stage 4 (8-cell)
embryo. At A, the microelectrode tip was in the external medium, B shows the
negativity associated with its passage through the vitelline membrane. At C the
electrode pressed against the cell surface and penetrated at D. At E, F and G the
record was stopped for 10 sec and the membrane potential increased during this
period from - 20 to - 28 mV. The electrode was withdrawn at H.
more than 10 mV (Fig. 2) and at the same time a region of pigment accumulation developed around the point of entry of the microelectrode. Recent experiments have provided evidence for the existence of a contractile system in the
cortex of Xenopus embryos (Gingell & Palmer, 1968; Gingell, Garrod &
Palmer, 1970; Gingell, 1970). It is probable that the accumulation of pigment
is a result of the response of this system to the presence of the electrode, thereby
sealing the membrane around the tip and preventing short-circuit of the intracellular potential. Spontaneous ejection of the microelectrode often occurred,
particularly during cleavage of the cells.
The effect of tip potentials on membrane potential measurements has been
discussed at some length by Adrian (1956) and the necessity for use of microelectrodes with low tip potentials for accurate measurements has been emphasized. The size of the tip potential is inversely related to the ionic strength of the
bathing solution (Adrian, 1956) and in Steinberg's medium, which is at 60 mM
osmotically equivalent to half strength Ringer's solution, microelectrodes tended
to have high tip potentials (> - 40 mV). This effect was minimized by selecting
Bio-electric parameters 0/Xenopus embryos
539
electrodes which had tip potentials of —10 mV or less for use in recording
membrane potentials.
Table 1 shows membrane potentials recorded from surface cells at different
stages of development. The membrane potential increased from - 6 - 5 ± 2 m V
(s.D.) in the uncleaved egg to a value of — 57 ± 8 mV (S.D.) at the mid-blastula
stage. The size of the standard deviations shows that there was considerable
variation in the potentials of different embryos at the same stage. The possibility
that cells from different parts of the surface may not have exactly the same
membrane potential has not been explored here.
Confirmation of the rise in membrane potential came from measurements of
potential in individual embryos at intervals during development. The results of
one experiment on three embryos from the same batch are plotted in Fig. 3.
Two of the embryos showed a fall in potential at stage 5 after an initial rise but
this had recovered in both by stage 7; the third rose smoothly between stages 2
and 8. In this particular experiment the rise in potential was very marked and
the membrane potentials of all three embryos at stage 8 were the highest ever
recorded in this investigation.
60
S 40
20
Nieuwkoop stage
H
6
7
H
h
3
4
Age (h)
Fig. 3. Increase in membrane potential of animal pole cells in three embryos
( # , O and ©) during cleavage from stage 3 (4-cell) to stage 8 (blastula).
540
J. F. PALMER AND C. SLACK
Table 1. Membrane potentials from pregastrulation stages o/Xenopus laevis
Cell diameter (/*)
—
—
—
—
—
300-350
175-200
125-150
50-75
Nieuwkoop stage
1
2
3
4
5
6
d\
7
8
(egg)
(2-cell)
(4-cell)
(8-cell)
(16-cell)
(32-cell)
(morula)
(early blastula)
(mid-blastula)
Membrane potential
in raVi s.D.
-6-5 ± 2 0
-190±100
-240 ±90
-27-0 ±6-3
-300 ±70
-31-0±4-5
-35-0 ±5-5
-45-0 ± 4 0
-57-0 ±8-0
No. of
embryos
15
21
20
21
14
14
20
22
15
Membrane potentials from surface cells of animal pole and vegetal margins have been
pooled in this Table.
Resistance measurements
Eggs and early cleavage stages. The vitelline membrane had no measurable
resistance to ionic current flow.
Fertile eggs, measured shortly before cleavage, had variable input resistances,
in the range 0-5-5 MQ. Assuming the egg to be a uniformly polarized sphere
with a diameter of 1-1 mm, this corresponds to a specific resistance of 20200 kQ cm2. The resistance of the egg surface before cleavage did not remain
constant but often fluctuated in an irregular manner: the reason for this is not
understood. The current/voltage relation of eggs was linear but this is not
unexpected from a cell in which the membrane potential is low. The egg capacitance, calculated by measuring the time taken for the electrotonic voltage to rise
to 67 % of its final value and dividing this figure by the steady-state resistance,
was in the range 0-02-0-05 JLOF yielding a specific capacitance of 0-6-1-6 /^F/cm2.
During first cleavage the membrane resistance of most eggs decreased by a
factor of between 2 and 10. Some eggs, however, showed little change in
resistance and in a few cases the resistance actually rose at the first cleavage,
falling only during the subsequent two or three division cycles. Fig. 4 illustrates
the changes in input resistance of four embryos during the first three division
cycles. Although the resistance of all four embryos fell between first and fourth
cleavage the size and pattern of change was variable. Embryo A had a high
(4 MQ) input resistance before cleavage and this fell sharply to 500 kiQ during
the first division, rising slightly before the appearance of the second constriction.
A rise in resistance before second cleavage after an initial fall was also shown by
two of the other embryos, C and D; the resistance of B, however, fluctuated
between first and second cleavage. The input capacitance did not in general
undergo large changes during first division, but again considerable variation was
observed. Some eggs showed a gradual increase, others an overall decrease, whilst
a third group remained, with slight fluctuations, at a more or less constant level.
Bio-electric parameters o/Xenopus embryos
541
Blastulae. Further development of the embryos was accompanied by a rise
in input resistance from an average value of 600 + 100 (s.D.) kQ at stage 5 to
2-0 ±0-6 (s.D.) MQ at mid-stage 8. Fig. 5 shows the input resistance of three
embryos measured at intervals during development from early to mid-blastula
stages. The rise in resistance of all three embryos was most marked between
40 -
0
1st cleavage
10
20
30
2nd cleavage
40
50
3rd cleavage
60
70 (min)
4th cleavage
Fig. 4. Input resistance changes of four embryos (A, B, C and D) during the first
four cleavages. Ordinate: total measured input resistance. Abscissa: time (min) after
appearance of first cleavage furrow. The appearance of this and successive furrows is
indicated by arrows. The slight variation in length of division cycles (range ± 3 min)
has been removed for simplicity by adjusting the time scales of individual embryos
to conform to a mean value.
stages 7 and 8. The current/voltage relation of blastulae was found to be almost
linear and there was little evidence of rectification (Fig. 6). It has been pointed
out, however (Noble, 1962), that in a situation where current spreads from a
point source in more than one dimension, non-linearity of the current/voltage
relationship is effectively masked by the geometry of the tissue. Thus, even if
542
J. F. PALMER AND C. SLACK
the surface membrane possessed rectification properties at the blastula stage, the
current/voltage relation of intact embryos would probably not show it. If the
membrane potential obeys the constant field equation (Goldman, 1943) some
non-linearity might be expected from mid-blastula stages since the potential
has increased to more than — 50 mV.
In some experiments a third microelectrode was introduced into cells adjacent
to that containing the other two electrodes in order to measure the ease with
which current spread from cell to cell in the embryo. This was expressed as the
20
r
15
2 10
0-5
Age(h)
Nieuwkoop stage
3
6
4
7
5
8
Fig. 5. Input resistance of three embryos ( # , O and A)
during blastula stages 6-8.
ratio V2jV1 of the voltages recorded in the two cells simultaneously (Fig. 1),
hereafter called the coupling ratio. Measurements of the ratio V2\VX in embryos
at various stages were as follows:
In a dividing egg, the voltages recorded across the cleavage plane were identical, in fact there was usually no measurable voltage decay across any junction
until the eight-cell stage. Thereafter the coupling ratio varied between 0-9 and
0-3, the lower values usually being recorded from mid-blastulae (stage 8). Fig. 7
shows the simultaneous voltage displacements recorded in adjacent cells of a
stage 7 embryo resulting from a hyperpolarizing current pulse (i) of 6 x 10~8 A.
The coupling ratio in this embryo was 0-85.
A further series of experiments investigated the decay of voltage as the third
microelectrode was inserted at different points across the surface of blastula
stages. It was found that measurable voltages were produced in all cells examined, even in those diametrically opposite the current source at a distance of
Bio-electric parameters o/Xenopus embryos
543
20
Currentx10"8(A)
l
40
30
20
10
10
20
40
60
s
J
80
Fig. 6. Current/voltage relation of a late stage 7 embryo measured in one of the
animal pole cells. Ordinate: displacement of membrane potential from the resting
value. Non-linearity of the relation is seen for hyperpolarization beyond — 30 mV.
Strong rectification of the current-passing electrode limited voltage displacements
in the depolarizing direction. Input resistance = 2 0 M£\
10 mV
. .
steps
Vl
—
. ,,
6x10" e A
-MOmV
Fig. 7. Electrotonic voltages (Vx and K2) recorded in adjacent animal pole cells of a
stage 7 embryo. Electrode set-up as in Fig. 1. Coupling ratio V2/V1 = 0-85. Input
resistance = 11 MO. Pulse width = 1 sec. Voltages corrected for 100 kQ resistor
in earth return.
544
J. F. PALMER AND C. SLACK
more than 1 mm. The voltage decay was found to be discontinuous, there being a
greater voltage drop across intercellular junctions than across the diameter of the
cells, even in early blastulae where the cells are large (200-300 JLC). When voltage
decay was measured as a function of the number of cell junctions intervening
between the electrodes, the shape of the decay usually depended on the age of the
embryo. In these experiments the number of junctions was measured across the
shortest distance between the current electrode and the third microelectrode.
Fig. 8 shows the result of an experiment where the decay was measured in an
embryo during cleavage from early to mid-blastula stages. It can be seen that the
10
Stage 7
S 0-5
10
20
30
40
50
60
70
80
Number of junctions
Fig. 8. Voltage decay across the surface of the same embryo measured at three
different blastula stages (7A, advanced 7 # and 8O)- Ordinate: ratio VX\VX where
Vx = voltage recorded in same cell as the current source, Vx = voltage measured
simultaneously in other cells at the surface. The point VJV-i = I, when all microelectrodes were in the same cell, is common to the three curves. Abscissa: number
of junctions intervening across the shortest distance between current electrode and
Vx. Input resistance at early stage 7 = 500 kQ, and 1-6 MQ at stage 8. Constant
current used throughout was 1 x 10"8 A.
voltage decayed more quickly in the later stages so that, for instance, when four
cell junctions separated the electrodes at stage 8, the voltage had fallen more
than when a similar measurement was made at early stage 7, even though the
electrodes were actually closer together during the later measurements. The input
resistance of the embryo was rising during the course of the experiment and
therefore the ordinate of Fig. 8 was plotted as the ratio VxjVx, where Vx = the
voltage recorded by the third electrode and Vx the voltage recorded simultaneously in the same cell as the current electrode. It is conventional to plot
Bio-electric parameters o/Xenopus embryos
545
voltage decay as the logarithmic function of distance from the current source in
cable-like tissues, such as muscle and nerve fibres. This has not been done in
Fig. 8 since the blastula does not approximate to a cable geometrically and an
exponential relationship between the two parameters probably does not exist
(see Discussion).
25
mv
30 sec
Fig. 9. Pen record showing fall in membrane potential of a stage 7 animal-pole cell
produced by insertion of another microelectrode into asecondcell separated from the
first by four junctions. Upper trace: voltage record from second cell; lower trace:
voltage record from first cell. Input resistance monitored by constant current pulses
of 2 x 10~8 A and 500 msec duration passed between inside and outside of the first
cell. At A first cell had a membrane potential of —40 mV and second cell had not
been penetrated. B—microelectrode entered the second cell recording an initial
potential of - 2 5 mV, at the same time potential in the first cell fell by 13mV. At C
the potentials in both cells had risen to - 3 5 mV. After 3 min both were - 4 0 mV;
this is not illustrated. Time scale = 30 sec.
These experiments show that current spreads easily from cell to cell across
the surface of the embryo and indicate that the junctions between the cells are of
low resistance relative to that at the surface.
Further evidence for the presence of low resistance junctions came from the
frequent observation that the membrane potential levels of different cells in the
blastula were interdependent. This was best illustrated by the fact that penetration with a third microelectrode often produced a fall in potential of the cell in
which input resistance was being recorded (Fig. 9). Subsequently, the potentials
in both cells recovered, at the same rate, to approximately the same final level.
If the cell is the generator of the membrane potential such a simultaneous fall
seems likely to result only if an absolute low resistance exists between the cells,
in other words, the embryo behaves as an electrical syncytium. A feature of
records from some blastulae (stages 7 and 8) was the appearance of apparent
time-dependent voltage changes where the input resistance rose during the
application of a constant current pulse: this is illustrated in Fig. 10. Whilst this
may be a true time-dependent permeability change in the membrane a similar
effect could be produced if the membrane actually had a steep non-linear
current/voltage relation. The exact conditions which give rise to this effect have
not been determined and it will not be discussed further at this point.
546
J. F. PALMER AND C. SLACK
Fig. 10. Time-dependent voltage change shown by stage 8 embryo during measurement of input resistance. Current (i) = 1 x ]0~8 A. Pulse width = 1 sec.
t
14
16
3 mM EDTA Steinberg's medium
Fig. 11. Effect of 3 mM EDTA at pH 8-2 on input resistance and coupling ratio in a
stage 7 blastula. Electrode set-up as in Fig. 1. Ordinate: electrotonic voltages
recorded in adjacent cells (O, • ) produced by constant current pulses of 2-3 x
10~8 A. Abscissa: time (min) after application of EDTA. At first arrow, Steinberg's
medium was rapidly replaced by EDTA solution, second arrow indicates replacement of EDTA by Steinberg's medium.
Bio-electric parameters of Xenopus embryos
547
The effect of ethylenediaminetetra-acetic acid (EDTA)
Interest in this chelating agent arose from the fact that it is used in alkaline
solution for the disaggregation of Xenopus embryos and also has been shown to
increase the resistance of intercellular junctions in electrotonically coupled
tissues (Loewenstein, 1966). For the purpose of these experiments, stage 7
blastulae were removed from their vitelline membranes using fine forceps—if
this was not done the embryos swelled against this membrane in EDTA and this
often led to cell damage. The coupling ratio V2/V1 was measured in Steinberg's
medium and the embryo then perfused with a 3 mM disodium EDTA in calciumfree magnesium-free medium at pH 8-2 for periods of time varying from 2 to
10 min. Fig. 11 shows the results of an experiment in which an embryo was
treated with EDTA for 3 min. The voltage deflexions in adjacent cells fell with
a similar time course during the application of EDTA, and at the same time the
membrane potential fell from —40 to —30 mV. On return to Steinberg's solution the voltages recovered simultaneously, although the reversal had a longer
time course. This suggests that the primary effect of EDTA is to lower the surface
resistance. An effect on the junctional resistance cannot be excluded but the
time course of any change must be the same as that at the surface. This result was
invariably obtained with EDTA. It must be pointed out, however, that 3 mM
EDTA is known to disaggregate stage 7 blastulae completely within 1 h, so the
coupling ratio must eventually change.
DISCUSSION
Membrane potentials. The measurements of membrane potential in Xenopus
laevis showed the same trend as that reported by Ito & Hori (1966) in Triturus
embryos, namely, that this rises progressively during the stages prior to gastrulation. Interpretation of the intracellular potential and thus of any changes which
may occur during development requires a knowledge of the relative permeability
of the membrane to ions and their intracellular concentration. At present we
have no such information for Xenopus embryos. Morrill and his co-workers,
have, however, shown that in R. pipiens embryos there is a high level of
intracellular sodium ions in early cleavage stages which falls as the blastocoel
develops (Morrill et al. 1966; Kostellow & Morrill, 1968). If the membrane
potential in these embryos can be described by the constant field equation
(Goldman, 1943),
RT
(K+)in + a ( N a + ) i n
m
~ F
(K+) out + a (Na+) out'
where R = the gas constant,
T = absolute temperature,
F = Faraday's constant,
a = permeability ratio P
548
J. F. PALMER AND C. SLACK
and if the membrane is permeable to both sodium and potassium ions, a
decrease in the intracellular sodium concentration would lead to a fall in the
membrane potential. The measured rise in potential suggests that if the intracellular sodium concentration falls during cleavage of Xenopus embryos there
must be a change in the relative permeability of the surface membrane to sodium
and potassium ions. It would be of considerable interest to know whether there
is an increase in intracellular potential of R. pipiens embryos during development and also whether loss of sodium ions from cells is a general feature of
amphibian development.
Resistance measurements. The range of specific resistance of Xenopus eggs
(20-200 kQ cm2) is comparable to the values quoted for R. pipiens eggs by
Woodward (1968) and is slightly lower than that of Bufo eggs (Maeno, 1959).
It is considerably lower than that of Triturus eggs before cleavage (Ito & Hori,
1966).
The variation in input resistance (500 kQ-5 MO.) of eggs was not correlated
in any way with viability since those eggs at the lower end of the resistance range
developed just as well as those with higher values. Nor was the variation confined to eggs from different batches since eggs from the same batch often had
different input resistances.
Several factors could affect the measured input resistance of the embryos
during development and these will be mentioned in turn. First, an increase in
external surface area resulting from cleavage would lead to a reduction in input
resistance. Secondly, changes in resistivity of the surface membrane, associated
with alterations in permeability could produce a rise or fall in the recorded
values. Thirdly, the magnitude of the junctional resistance might indirectly
determine the input resistance by limiting current spread in the embryo. The
problem is therefore to decide whether changes in measured input resistance
could be due to any or all of these factors.
During the first three cleavages it was not possible to record any voltage drop
across the intercellular junctions so it seems likely that the junctional resistance
does not affect measurements of input resistance at this stage and the embryo
may be considered electrically as a uniformly polarized sphere. The decrease in
input resistance which was measured during early cleavage could therefore be
due to an increase in area of the surface membrane in contact with the exterior.
Selmann & Waddington (1955) have, however, concluded that the increase in
external surface during cleavage of newt eggs is confined to a small area in the
region of the furrow. The absence of changes in input capacitance indicates that
large areas of new external surface membrane are not formed during cleavage
of Xenopus eggs. Woodward (1968) has shown electrically that the new membrane formed at first cleavage of R. pipiens eggs is located specifically within the
furrow, but he has suggested that this membrane has, initially, a low resistivity.
If in Xenopus even a small area of new membrane having a high conductivity
were formed in the cleavage furrow the fall in resistance might be explained in
Bio-electric parameters of Xenopus embryos
549
terms of a leakage current to the exterior through the furrow gap. In this case
the variation in resistance change may be a direct consequence of the magnitude
of this leak which would be controlled by the width of the furrow gap. There is,
however, no direct evidence that this explanation is correct and the existence of a
permeability increase over the whole surface during early cleavage cannot be
excluded. With further cleavage the total number of intercellular junctions
increases and it is clear from the voltage decay experiments that the presence of
more junctions has a considerable effect on the value of input resistance, which
begins to rise at the blastula stage. Developmentally, it is important to know
Fig. 12. (A) Schematic equivalent circuit of a portion of cable-like tissue. Rm and
Rc are the surface membrane and cytoplasmic resistances respectively. (B) Equivalent circuit of two adjacent blastomeres. Rm and Rc represent the surface and
intercellular resistances respectively. The intercellular cleft resistance is Rx, whilst
Ry is the resistance pathway to earth through all other cells.
whether there are relative changes in the absolute values of surface and junctional membrane conductances, but the essential difficulty in performing such a
calculation lies in the geometry of the blastula. In a cable-like tissue, such as
single nerve fibres, the equivalent electrical circuit may be represented by
Fig. 12 A, where jRm and Rc represent the surface membrane and cytoplasmic
resistances respectively. Current applied through an intracellular microelectrode
at A will spread longitudinally down the fibre and the ratio of the voltages
VB\VA will depend on the relative resistances of Rm and Rc. In such a situation
these relative resistances can be calculated from the one-dimensional cable
equations (Hodgkin & Rushton, 1946) which neglect the radial spread of
550
J. F. PALMER AND C. SLACK
current within the fibre and predict an exponential relationship between voltage
and distance from the current source. In the blastula, cells are arranged such
that, in principle at least, current can spread in two and possibly three dimensions away from the polarizing microelectrode and the equivalent circuit is
shown in Fig. 12 B. In this circuit Rm and Rc represent the surface and junctional
resistances of two adjacent blastomeres, Rx is the resistance from the intercellular cleft to the exterior and Rv represents the lumped series/parallel combination of all current pathways to earth through other cells of the embryo.
The relative resistances Rm and Rc cannot be easily related to the coupling ratio
VB\VA because the value of Rv at any particular instance is not known,
moreover the value of Rx, although likely to be high, cannot be directly determined. One of the difficulties in the interpretation of input resistance and voltage
decay experiments from blastulae lies in deciding whether the arrangement of
cells more nearly approaches a two or three dimensional pathway to current
spread during successive cleavages. Thus, the increase in input resistance between stages 7 and 8 when considered in conjunction with the steeper voltage
decay may indicate an increase in resistivity of cell junctions. However, it is
possible that the results reflect a change in the geometry of current spread arising
from the formation of more junctions in the embryo and the increase in number
of cell layers between the blastocoel and the exterior.
The present measurements of current spread in Xenopus blastulae show some
differences from similar ones made in Triturus embryos (Ito & Hori, 1966; Ito
& Loe wen stein, 1969). In the urodele blastula electrical coupling between surface
cells was tighter, little decay of voltage being detected over distances of 1000 /<•
(5-6 cell junctions in the mid-morula). Fig. 8 shows that at an equivalent stage,
the voltage decay in Xenopus is somewhat sharper. The apparent close coupling
in Triturus has been attributed to the presence of the surface coat described by
Holtfreter (1943), which in Triturus appears to have a high electrical resistance
and is a very strong permeability barrier (Ito & Loewenstein, 1969). The decay
experiments in Xenopus indicate that the two embryos are different, though
whether the difference lies in surface properties is not certain. The decrease in
input resistance and membrane potential of Xenopus blastulae in 3 HIM EDTA
suggests that under the present experimental conditions the surface permeability
barrier is not as high as that in Triturus where treatment with 10 mM EDTA for
30 min has no significant effect on the membrane potential (Ito & Loewenstein,
1969). This difference in surface resistance may be due to the use of different
culture media. Triturus embryos were cultured in Holtfreter's solution containing 1 mM CaCl2 whilst the present experiments utilized Steinberg's solution
where the level of free calcium is approximately 0-3 mM. Alternatively, the
junctional resistance may be higher in Xenopus embryos. Experiments on cells
isolated from intact embryos may help to resolve this problem.
The present measurements indicate that the junctions between these cells were
low resistance pathways to ionic current flow. Whilst there is no direct evidence
Bio-electric parameters o/Xenopus embryos
551
that low resistance junctions have any function in differentiation or regulation
of embryonic tissues it has been suggested that they may serve as channels for
information flow during development (Furshpan & Potter, 1968). Dye injection
experiments in Drosophila salivary gland (Kanno & Loewenstein, 1966) have
demonstrated the movement of large molecules—up to 69000 M—from cell to cell
across low resistance junctions. In crayfish and lobster giant axons, fluorescein
(M 332) has been shown to cross electrotonic junctions fairly rapidly (Pappas
& Bennett, 1966; E. J. Furshpan, unpublished observations) and to move easily
between coupled cells in tissue culture (Furshpan & Potter, 1968). In Xenopus
blastulae, the junctions between cells are not freely permeable to fluorescein
(Slack & Palmer, 1969), but in chick and squid embryos, the dye, Chicago blue
6B (M992), can diffuse from cell to cell under some circumstances (Potter,
Furshpan & Lennox, 1966; Sheridan, 1968). It is clear that until more measurements of cell to cell transfer in early embryonic tissues have been made, the
question as to whether differentiation could involve movements of macromolecules remains open. The use of uncoupling agents, such as halothane
(Palmer & Slack, 1969), which increase the junctional resistance between cells
may also provide information concerning the role of low resistance junctions
during development.
RESUME
Les parametres bio-electriques des embryos jeunes de Xenopus
Le potentiel de membrane et la resistance ont ete etudies dans des oeufs de crapaud Sud
Africain Xenopus laevis, aux cours du clivage et de la blastula, en utilisant des microelectrodes
intracellulaires.
Le potentiel de membrane s'accroit de — 6,5 ± 2 mV dans les oeufs pour atteindre — 57 ± 8,0
mV au cours de la blastula moyenne.
La resistance d'entree des oeufs fertiles presente des valeurs comprises entre 0,5 MCI et
5,0 M£2 ce qui correspond a une resistance specifique de 20-200 kQcm2. Au cours des deux et
trois premiers cycles de division, la resistance d'entree decroit habituellement d'un facteur de
2-10, et s'eleve ensuite, au cours de la blastula, d'une valeur moyenne de 600± 100kQ au
stade 5 pour atteindre 2,0 ±0,5 MQ. au stade 8.
A tous les stades de developpement, la polarisation ponctuelle d'une cellule de surface dans
1'embryon par des ondes rectangulaires de 0,5-6 x 10~8 A, produit des abaissements de voltage
dans les autres cellules de surface. Ceci s'observe meme lorsque plusieurs (7-8) jonctions
cellulaires sont interposees sur des distances de plus d'un mm entre le passage du courant et
les microelectrodes de voltage. Ces mesures suggerent que la resistance de jonction est faible
comparee a celle de la surface, bien que l'arrangement geometrique des cellules ne soit pas
favorable pour le calcul des valeurs absolues de la resistance membranaire.
La diffusion du courant entre les cellules se produit en apparence moins facilement pendant
les stades de la blastula moyenne que dans les stades plus precoces du developpement, ce qui
indique, peut-etre, une augmentation de la resistance de jonction au cours du developpement.
Une comparaison a ete effectuee entre les mesures ci-mentionnees et des mesures similaires,
realisees sur un autre amphibien, Triturus.
We are grateful to Professor E. Neil for providing facilities and to Professor L. Wolpert
for his encouragement. This work was supported by the Nuffield Foundation.
35
EMB 24
552
J. F. PALMER AND C. SLACK
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