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 ±2 mV in eggs to –57 ±8· 0 mV at the mid-blastula stage.

The input resistance of fertile eggs ranged from 0·5 MΩ to 5 ·0 MΩ corresponding to a specific resistance of 20–200 kΩ 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 ± 100 kΩ at stage 5 to 2·0 ±0·5 MΩ 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 × 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.

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.

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.

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 DIM; KC10·7 HIM; Ca(NO3)2.4H2O 0·3 mM; MgSO4. 7 H2O 0·8 MM; Tris buffer 1·4mM; 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-KCI with resistances in the range 16–60 MΩ and tip potentials of –10 mV 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 μ) 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 MΩ resistor in series with the polarizing electrode, the absolute value of current being measured across a 100 kΩ resistor in the earth return of the circuit. The currents used in these experiments were in the range 0·5–6 × 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.

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 V1 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.

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 V1 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.

A deflexion due to current flowing across the 100 kΩ resistor in the earth return was recorded on all voltage traces. This was subtracted from the records for the purposes of membrane resistance calculations.

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 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 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.

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.

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.

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 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 mV (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.

Table 1.

Membrane potentials from pregastrulation stages of Xenopus laevis

Membrane potentials from pregastrulation stages of Xenopus laevis
Membrane potentials from pregastrulation stages of Xenopus laevis

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.

Fig. 3.

Increase in membrane potential of animal pole cells in three embryos (•,○ and ◓) during cleavage from stage 3 (4-cell) to stage 8 (blastula).

Fig. 3.

Increase in membrane potential of animal pole cells in three embryos (•,○ and ◓) during cleavage from stage 3 (4-cell) to stage 8 (blastula).

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 MΩ . Assuming the egg to be a uniformly polarized sphere with a diameter of 1·1 mm, this corresponds to a specific resistance of 20–200 kΩ 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 μE yielding a specific capacitance of 0·6–1·6 μF/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 MΩ ) input resistance before cleavage and this fell sharply to 500 kΩ 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.

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.

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.

Blastulae. Further development of the embryos was accompanied by a rise in input resistance from an average value of 600 ± 100 (S.D.) kΩ at stage 5 to 2·0 ±0·6 (S.D.) MΩ 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 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 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.

Fig. 5.

Input resistance of three embryos (•, ○ and ▴) during blastula stages 6–8.

Fig. 5.

Input resistance of three embryos (•, ○ and ▴) during blastula stages 6–8.

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 MΩ .

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 MΩ .

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 ratio V2/V1 of the voltages recorded in the two cells simultaneously (Fig. 1), hereafter called the coupling ratio. Measurements of the ratio V2/V1 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 × 10−8 A. The coupling ratio in this embryo was 0-85.

Fig. 7.

Electrotonic voltages ( V1 and V2) 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 = 1·1 MΩ . Pulse width = 1 sec. Voltages corrected for 100 kΩ resistor in earth return.

Fig. 7.

Electrotonic voltages ( V1 and V2) 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 = 1·1 MΩ . Pulse width = 1 sec. Voltages corrected for 100 kΩ resistor in earth return.

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 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 μ). 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 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 Vx/V1, where Vx = the voltage recorded by the third electrode and V1 the voltage recorded simultaneously in the same cell as the current electrode. It is conventional to plot 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).

Fig. 8.

Voltage decay across the surface of the same embryo measured at three different blastula stages (7▴, advanced 7• and 8○)Ordinate: ratio Vx/V1 where V1= voltage recorded in same cell as the current source, Vx= voltage measured simultaneously in other cells at the surface. The point Vx/V1 = 1, 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 kΩ, and 1·6 MΩ at stage 8. Constant current used throughout was 1 × 10−8 A.

Fig. 8.

Voltage decay across the surface of the same embryo measured at three different blastula stages (7▴, advanced 7• and 8○)Ordinate: ratio Vx/V1 where V1= voltage recorded in same cell as the current source, Vx= voltage measured simultaneously in other cells at the surface. The point Vx/V1 = 1, 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 kΩ, and 1·6 MΩ at stage 8. Constant current used throughout was 1 × 10−8 A.

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.

Fig. 9.

Pen record showing fall in membrane potential of a stage 7 animal-pole cell produced byinsertionof 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× 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 –25 mV, at the same time potential in the first cell fell by 13mV. At C the potentials in both cells had risen to –35 mV. After 3 min both were –40 mV; this is not illustrated. Time scale = 30 sec.

Fig. 9.

Pen record showing fall in membrane potential of a stage 7 animal-pole cell produced byinsertionof 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× 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 –25 mV, at the same time potential in the first cell fell by 13mV. At C the potentials in both cells had risen to –35 mV. After 3 min both were –40 mV; this is not illustrated. Time scale = 30 sec.

Fig. 10.

Time-dependent voltage change shown by stage 8 embryo during measurement of input resistance. Current (i) = 1×10−8 A. Pulse width = 1 sec.

Fig. 10.

Time-dependent voltage change shown by stage 8 embryo during measurement of input resistance. Current (i) = 1×10−8 A. Pulse width = 1 sec.

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.

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 (○, •) produced by constant current pulses of 2·3 ×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.

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 (○, •) produced by constant current pulses of 2·3 ×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.

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),
formula

where R = the gas constant,

T = absolute temperature,

F = Faraday’s constant,

α = permeability ratio PNa/PR

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 kΩ 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 kΩ –5 MΩ ) 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 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 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 Rm and Rc represent the surface membrane and cytoplasmic resistances respectively. Current applied through an intracellular microelectrode at VB/VA will spread longitudinally down the fibre and the ratio of the voltages Vu/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 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 Ry 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.

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.

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.

The present measurements of current spread in Xenopus blastulae show some differences from similar ones made in Triturus embryos (Ito & Hori, 1966 ; Ito & Loewenstein, 1969). In the urodele blastula electrical coupling between surface cells was tighter, little decay of voltage being detected over distances of 1000μ (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 m.\i 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 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.

Les paramètres bio-électriques des embryos jeunes de Xenopus

Le potentiel de membrane et la résistance ont été étudiés dans des oeufs de crapaud Sud Africain Xenopus laevis, aux cours du clivage et de la blastula, en utilisant des microéléctrodes intracellulaires.

Le potentiel de membrane s’accroît de – 6,5 ± 2 mV dans les œufs pour atteindre – 57 ± 8,0 mV au cours de la blastula moyenne.

La résistance d’entrée des œufs fertiles présente des valeurs comprises entre 0,5 MΩ et 5,0 MΩ ce qui correspond à une résistance spécifique de 20–200 kΩ cm2. Au cours des deux et trois premiers cycles de division, la résistance d’entrée décroit habituellement d’un facteur de 2–10, et s’élève ensuite, au cours de la blastula, d’une valeur moyenne de 600± 100 kΩ au stade 5 pour atteindre 2,0 ±0,5 MΩ au stade 8.

A tous les stades de développement, la polarisation ponctuelle d’une cellule de surface dans l’embryon par des ondes rectangulaires de 0,5–6 × 10−8 A, produit des abaissements de voltage dans les autres cellules de surface. Ceci s’observe même lorsque plusieurs (7–8) jonctions cellulaires sont interposées sur des distances de plus d’un mm entre le passage du courant et les microélectrodes de voltage. Ces mesures suggèrent que la résistance de jonction est faible comparée à celle de la surface, bien que l’arrangement géométrique des cellules ne soit pas favorable pour le calcul des valeurs absolues de la résistance 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 précoces du développement, ce qui indique, peut-être, une augmentation de la résistance de jonction au cours du développement.

Une comparaison a été effectuée entre les mesures ci-mentionnées et des mesures similaires, réalisées 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.

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