Using the whole-cell voltage clamp technique, we have studied junctional conductance (Gj), and Lucifer Yellow (LY) coupling in 2-cell and 32-cell ascidian embryos. Gj ranges from 17.5 to 35.3 nS in the 2-cell embryo where there is no passage of LY, and from 3.5 to 12.2 nS in the later embryo where LY dye spread is extensive. In both cases, Gj is independent of the transjunctional potential (Vj). Manually apposed 2-cell or 32-cell embryos established a junctional conductance of up to 10 nS within 30 min of contact. Furthermore, since we did not observe any significant number of cytoplasmic bridges at the EM and Gj is sensitive to octanol, it is probable that blastomeres in the 2-cell and 32-cell embryos are in communication by gap junctions. In order to compare Gj in the two stages and to circumvent problems of cell size, movement and spatial location, we used cytochalasin B to arrest cleavage. Gj in cleavage-arrested 2-cell embryos ranged from 25.0 to 38.0 nS and remained constant over a period of 2.5 h. LY injected into a blastomere of these arrested embryos did not spread to the neighbour cell until they attained the developmental age of a 32- to 64-cell control embryo. Our experiments indicate a change in selectivity of gap junctions at the 32cell stage that is not reflected by a macroscopic change in ionic permeability.

Gap junctions appear to play a role in the regulation of early developmental events. First, experimental perturbation of junctional conductance using antibodies or anti-sense RNA to connexons can lead to developmental defects (Warner et al. 1984; Warner, 1987; Bevilacqua et al. 1989). Second, junctional conductance may change both qualitatively and quantitively at developmentally significant times (see reviews, Finbow, 1982; Caveney, 1985; Revel, 1986; Warner, 1988) and, third, cell populations of different developmental fate often form distinct communication compartments (Warner and Lawrence, 1982; Weir and Lo, 1982; Kjmmel and Law, 1985; Serras and van den Biggelar, 1987).

In holoblastic regulative embryos, gap junctions appear consecutively with initial events of differentiation, usually at the 8- to 16-cell stage. For example in the mouse, dye and electrical coupling occurs at the 8-cell stage when compaction first starts (Lo and Gilula, 1979a,b; Goodall and Johnson, 1984). Early blastomeres in the sea urchin embryo are not electrically coupled (Dale et al. 1982), while electron microscopy shows gap junctions first appear at the 16-cell stage (Andreucetti et al. 1987). In the meroblastic cephalopod embryo, blastomeres are extensively coupled by gap junctions before differentiation of the germ layers (Marthy and Dale, 1989). Mosaic embryos also express gap junctions early in development. Morphological studies have identified gap junctions in the 2-cell molluscan embryo Patella (Dorresteijn et al. 1982), although Lucifer Yellow (LY) does not spread between blastomeres until the 32-cell stage (Dorresteijn et al. 1983).

Intracellular voltage measurements indicate coupling between blastomeres in the 2- to 8-cell ascidian embryo (Dale et al 1982), and it has been suggested that ionic permeability between cells increases at the 32-cell stage, coinciding with the appearance of LY dye coupling in this embryo (Serras et al. 1988). Complex multicellular interactions, together with cell size, movement and division, render it difficult to compare junctional conductance in the 2-cell and 32-cell ascidian embryo. To circumvent this problem, we have used cleavage-arrested embryos, since elsewhere it has been shown that cleavage is not necessary for the temporally programmed differentiation of several cytoplasmic and membrane-located gene products (Whittaker, 1973; Hirano and Takahashi, 1984; Okado and Takahashi, 1988; Crowther et al. 1990). Coupling ratios between cells, as estimated from voltage measurements, give little information about the cell-to-cell communication pathways. By applying the whole-cell voltage clamp technique to a cell pair, junctional conductance may be measured directly (Neyton and Trautmann, 1985; Veenstra and DeHaan, 1988; DeHaan and Chen, 1990). The purpose of the present investigation was to measure junctional conductance in early control and cleavage-arrested ascidian embryos, using the wholecell voltage clamp technique, and to determine whether early ascidian blastomeres are coupled by gap junctions.

Experiments were carried out on embryos of the ascidian Ciona intestinalis collected from the Bay of Naples. Sperm and eggs were collected from the gonoducts and the sperm kept dry until use. The chorions were removed manually using fine steel needles. Nude eggs were inseminated in agar-coated Petri dishes in natural sea water at 20 °C and, at the appropriate stage, the embryos were transferred to glass slides for electrical recordings. Cleavage-arrested embryos were prepared by exposing zygotes or 2-cell embryos to 2 μgml−1 of cytochalasin B (Sigma, St Louis).

Two standard patch micropipettes were used in the wholecell clamp configuration to voltage clamp the two blastomeres independently The electrodes of about 10 Mohm resistance and 1–2 μm in tip diameter were filled with an intracellularhke solution composed of: 200 HIM KC1, 20mM NaCl, 250 mM sucrose, 10 mM EGTA, 10 mM Hepes at pH7.4. By using standard techniques, we obtained gigaohm seals on the two cells, set the pipette voltage to −40 mV, and ruptured the patches Whole cell currents were measured on two List EPC-7 amplifiers. In some experiments, one of the electrodes was filled with a 5 % solution of Lucifer Yellow (Sigma, St Louis) in 0 2M LiCl2 and back-filled with 0.2M LiCl2. The low resistance electrodes favoured rapid diffusion of the dye, which usually filled the cytosol within 10s of patch rupture. Elsewhere using either two patch electrodes (Dale, 1988), or a patch electrode in conjunction with a conventional intracellular electrode (DeFelice et al. 1986), it has been shown that the membrane potential of ascidian eggs, with a diameter of 130 μm, is under adequate voltage control in these configurations. The changes in resting potential and membrane resistance from the unfertilized egg to the 8-cell stage have been reported previously (Dale et al. 1982) Preparations with holding currents greater than 100 pA were discarded

Junctional current (Ij), in response to an applied junctional voltage (V,), is inward for the more negative cell and outward for its depolarized neighbour. The capacitive currents recorded from single blastomeres in response to 10 mV depolarizing steps, from a holding potential of −80 mV, display a single exponential decay. Since the electrode resistance was low compared to the junctional and non-junctional membrane resistance the potential established across the membrane was considered to be that applied to the electrode Senes resistance was negligible and therefore compensation was not used in these expenments (see DeFehce et al. 1986). Voltages and currents were stored on tape and played back on a chart recorder for subsequent analysis. Junctional conductance (Gj) was calculated from Ij/Vj measured dunng a series of voltage clamp pulses across the intercellular junction. Cells were clamped at −40, −60 or −80 mV. One of the pair was then depolarized or hyperpolanzed in 10 mV steps of 400 ms duration to create the junctional voltage difference. Voltage protocols were applied manually The currents generated reached a stable state within the step period.

Control 2-cell, 32-cell and cleavage-arrested embryos were fixed in 2.5% glutaraldehyde containing 1% paraformaldehyde in a buffer composed of 0.2 M sodium cacodylate and 20% sea water, pH 7.2 for 1h and postfixed in 1% osmium tetroxide. The material was then dehydrated in ethanol, embedded in Epon, cut on a Reichert-Jung ultramicrotome and examined with a Philips 400 electron microscope.

In Ciona intestinalis, the plane of first cleavage lies along the A-V axis and divides the embryo symmetrically into left and right halves. The second cleavage plane is perpendicular to the first, forming 4 blastomeres of almost equal size (Conklin, 1905). The third cleavage is equatorial, and subsequent divisions give rise to the three-dimensional holoblastic embryo. At 22°C, cleavage occurs about every 20 min. Shortly after cell division, the blastomeres are round showing little contact, whereas after 10 min cells become semicircular with the apposing parallel membranes in apparent close contact (Fig. 1A).

Fig. 1.

Lucifer Yellow and electrical coupling in early embryos of the ascidian Ciona intestmalis. (A) Phase fluorescence photograph of a control 2-cell embryo 15 min after 1st cleavage. The blur to the right is the LY-containmg microelectrode. (B) Fluorescence photograph of a 32-cell ascidian embryo, 10 min following injection of LY into one of the blastomeres. Note the extensive dye spread. (C) Phase-fluorescence photograph of a cleavage-arrested ascidian embryo 2h following exposure to cytochalasin B showing dye spread between the two blastomeres. (D) Two cleavage-arrested ascidian embryos mechanically apposed Ih following exposure to CB. The two outer cells are in the whole-cell clamp configuration. Scale bar, 100 μm.

Fig. 1.

Lucifer Yellow and electrical coupling in early embryos of the ascidian Ciona intestmalis. (A) Phase fluorescence photograph of a control 2-cell embryo 15 min after 1st cleavage. The blur to the right is the LY-containmg microelectrode. (B) Fluorescence photograph of a 32-cell ascidian embryo, 10 min following injection of LY into one of the blastomeres. Note the extensive dye spread. (C) Phase-fluorescence photograph of a cleavage-arrested ascidian embryo 2h following exposure to cytochalasin B showing dye spread between the two blastomeres. (D) Two cleavage-arrested ascidian embryos mechanically apposed Ih following exposure to CB. The two outer cells are in the whole-cell clamp configuration. Scale bar, 100 μm.

Lucifer Yellow injected into a blastomere of a 2-cell embryo within 5 min of cleavage spreads slowly to the sister cell. However, as reported by Serras et al. (1988), we did not detect spread when the dye was injected at later stages (Fig. 1A, n=15). Using the dual whole-cell voltage clamp technique and by applying rectangular voltage pulses across the junction, we found the junctional conductance of 2-cell embryos at this stage to range from 17.5 to 35.3 nS, with a mean of 25.9±5.6nS (n=13). Hyperpolarizing one cell of the pair from −40 mV to −80 mV induced an inward steady state current and capacitive transients in the stimulated cell (Si, Su, Fig. 2A). The non-junctional resistance of the stimulated cell in this experiment is approximately 50Mohm. Since the partner cell was independently voltage clamped at −40 mV, these non-junctional currents were not detected by the second electrode. The currents recorded by the second electrode are essentially the consequence of current flowing through the cell-cell junction (R1; Ru, Fig. 2A). Note that the current pulses reached a steady state within the 400 ms voltage step excursion. Each blastomere was in turn depolarized or hyperpolanzed in 10 mV steps of 400 ms duration from holding potentials of −40, −60 or −80 mV to create a transjunctional potential difference (Vj). In seven experiments, it was shown that Gj was not dependent on the transjunctional voltage (V0, or the holding voltage (Vm). Similarly the l/V characteristics of the junction were not directional, i.e. did not depend on which cell of the pair was stimulated. A typical experiment showing that Gj is independent of Vj, where Vm was −60 mV, and one cell was depolarized in 10 mV steps is shown in Fig. 2B. In several experiments, we were able to inject Lucifer Yellow into a blastomere while simultaneously measuring GJ

Fig. 2.

Junctional currents and conductance in a control 2-cell ascidian embryo. (A) Both cells are whole-cell voltage clamped by two independent circuits at −40 mV. I1 is current from one cell, I2 is current from its neighbour. Each cell is hyperpolanzed alternatively to −80 mV for 400 ms (Si and Sn) The current generated in the neighbour, of opposite direction and lacking capacitive transients, is the current flowing through the cell-cell junction (Ri and Rn, Ij). Note that Ij reaches a steady state within the 400 ms period. (B) Junctional conductance (G,) vs junctional voltage (V.) in the same embryo, calculated from a series of 10 mV depolarizing steps applied to one cell from a holding potential of −60mV, while the second cell was held at −60 mV.

Fig. 2.

Junctional currents and conductance in a control 2-cell ascidian embryo. (A) Both cells are whole-cell voltage clamped by two independent circuits at −40 mV. I1 is current from one cell, I2 is current from its neighbour. Each cell is hyperpolanzed alternatively to −80 mV for 400 ms (Si and Sn) The current generated in the neighbour, of opposite direction and lacking capacitive transients, is the current flowing through the cell-cell junction (Ri and Rn, Ij). Note that Ij reaches a steady state within the 400 ms period. (B) Junctional conductance (G,) vs junctional voltage (V.) in the same embryo, calculated from a series of 10 mV depolarizing steps applied to one cell from a holding potential of −60mV, while the second cell was held at −60 mV.

Fig. IB shows a 32-cell control embryo 10 min after Lucifer Yellow had been injected into one of the animal blastomeres. It can be seen that the dye has spread to many of the other blastomeres, including non-sister cells. By voltage clamping two non-adjacent cells in the 32-cell embryo, we measured the junctional conductance of the composite communication pathway. In 8 embryos, the composite junctional conductance ranged from 3.5 to 12.2 nS with a mean of 7.7±3.2 nS. Current flow in these stages is multi-directional and appears to be modified by the characteristics of the non-junctional membranes (Fig. 3A). The input resistance of the stimulated cell is approximately 200wohm (Fig. 3A). A typical experiment in which Vm was held at −40 mV in both cells, and one cell was hyperpolarized and depolarized in 10 mV steps is shown in Fig. 3C. Again it can be seen that Gj is essentially voltage independent.

Fig. 3.

Junctional currents in a 32-cell control embryo (A) and a cleavage-arrested 2-cell embryo of equivalent developmental age (B) Two non-adjacent blastomeres were picked at random in the 32-cell embryo Note the smaller currents in A. The voltage steps are indicated on the current traces. (C) Junctional conductance (Gj) vs junctional voltage (Vj) calculated from a series of 10 mV hyperpolarizing and depolarizing steps for A (* * * * * *) and B(…..). Cells were voltage clamped at −40 mV.

Fig. 3.

Junctional currents in a 32-cell control embryo (A) and a cleavage-arrested 2-cell embryo of equivalent developmental age (B) Two non-adjacent blastomeres were picked at random in the 32-cell embryo Note the smaller currents in A. The voltage steps are indicated on the current traces. (C) Junctional conductance (Gj) vs junctional voltage (Vj) calculated from a series of 10 mV hyperpolarizing and depolarizing steps for A (* * * * * *) and B(…..). Cells were voltage clamped at −40 mV.

Owing to the small size of the cells, cellular movements and the rapid mitotic interval, it is not easy to trace systematically the communication network in these multicellular stages. In addition, since it is probable that the various cell lines of the 32-cell embryo display graded degrees of coupling and other segregational programs have led to a heterogenous cell population, we exposed 2-cell embryos to cytochalasin B to arrest cleavage and studied junctional conductance and dye-coupling in these cleavage-arrested embryos. Shortly after cleavage arrest, LY was injected into one of the blastomeres and the embryo periodically observed at the fluorescence microscope for dyespread. In 14 experiments, spread occurred at 1.5 to 2.5 h following arrest, equivalent to the developmental age of 32- to 64-cell embryos (Fig. 1C). Junctional conductance in these cleavage-arrested embryos at this developmental time was comparable to that of the control 2-cell embryos ranging from 25.0 to 38.0 nS with a mean of 28.7±4.2nS (n=8, Fig. 3B and C).

In electron micrographs of the 2-cell, 32-cell or 2-cell cleavage-arrested embryos cytoplasmic bridges were virtually absent (Fig. 4). The intercellular spaces ranged from 5 to 50 nm and were similar in all embryonic stages studied. Gap junctions were not observed; however, desmosome-like structures were frequent. Cytochalasin B had no obvious deleterious effect on cellular morphology.

Fig. 4.

Electron micrographs of the intercellular junctions of a control 2-cell embryo (A and B), a control 32-cell embryo (C and D) and a cleavage-arrested 2-cell embryo, 2h after continuous exposure to 2μgml−1 of cytochalasin B (E and F) Note the similar morphology in all and the desmosome-like structures seen at high magnification. Cytoplasmic bridges are rare and CB does not appear to affect gross morphology. The scale in A is for A,C and E, while the scale in B is for B,D and F.

Fig. 4.

Electron micrographs of the intercellular junctions of a control 2-cell embryo (A and B), a control 32-cell embryo (C and D) and a cleavage-arrested 2-cell embryo, 2h after continuous exposure to 2μgml−1 of cytochalasin B (E and F) Note the similar morphology in all and the desmosome-like structures seen at high magnification. Cytoplasmic bridges are rare and CB does not appear to affect gross morphology. The scale in A is for A,C and E, while the scale in B is for B,D and F.

In seven experiments, embryos at various stages were mechanically apposed and one cell in each embryo was voltage clamped Within 30min, junctional coupling of 3.0 to 10 0 nS was established between the embryos. In one experiment, we manually apposed a cleavage-arrested zygote and a 2-cell embryo. When the embryo reached the 8-cell stage junctional conductance was 5.4nS. Lucifer Yellow pre-injected into the zygote did not spread to the blastomeres until the embryo reached the 32-cell stage, whereafter it spread to all the blastomeres. Similar results were observed when cleavage-arrested zygotes were apposed and also with two apposed 32-cell embryos. Fig. 5 shows Gj measured across two apposed cleavage-arrested 2-cell embryos (shown in Fig. 1D), 1 h after 1st cleavage. By depolarizing one of the blastomeres in 10 mV steps from a holding potential of -60 mV, it can be seen that junctional conductance is independent of the transjunctional voltage (between embryos in this case, Fig. 5B).

Fig. 5.

(A) Junctional currents established between to mechanically apposed cleavage-arrested embryos (shown in Fig. ID) 1 h following cleavage arrest and 30 min following contact. (B) Junctional conductance (G,) vs junctional voltage (Vj) in embryo A, when the membrane potential of one embryo was shifted in a series of 10 mV pulses from its holding value of −40mV to +60 mV.

Fig. 5.

(A) Junctional currents established between to mechanically apposed cleavage-arrested embryos (shown in Fig. ID) 1 h following cleavage arrest and 30 min following contact. (B) Junctional conductance (G,) vs junctional voltage (Vj) in embryo A, when the membrane potential of one embryo was shifted in a series of 10 mV pulses from its holding value of −40mV to +60 mV.

Finally, exposing 2-cell, 32-cell and cleavage-arrested embryos to 1 mM 1-octanol resulted in a gradual decrease in Gr starting about 5 min after bath application. In 5 experiments using the standard intracellular pipette solution, which is buffered with EGTA to maintain intracellular calcium to below 10−7 M Gj was reduced by about 50 %. For example, in one experiment using a 2-cell control embryo, Gj was reduced from 29.0nS to 19.7nS, while in a 32-cell embryo junctional conductance was reduced from 7 6 to 5.3 nS. To permit fluctuations in intracellular Ca2+ levels, we earned out a further 3 experiments using an intracellular-like pipette solution without EGTA. An example is shown in Fig. 6, in which Gj in a 2-cell embryo was annulled by octanol 10 min after exposure. Since current amplitudes increased in the stimulated cell, it appears that octanol also has an effect on the non-junctional membrane. Owing to the fragility of the nude embryos, it was not feasible to wash out the octanol and therefore we have no information on reversibility in this system.

Fig. 6.

Junctional currents in a control 2-cell embryo where one cell is depolarized by a senes of 400ms steps (A), and 10mm after exposure to 1mM octanol showing the loss of coupling (B). (C) Gjvs Vj. before (…..) and after (* * * * *) exposure to octanol. Holding currents in both cells increased from less than 100pA to about 300pA following exposure to octanol.

Fig. 6.

Junctional currents in a control 2-cell embryo where one cell is depolarized by a senes of 400ms steps (A), and 10mm after exposure to 1mM octanol showing the loss of coupling (B). (C) Gjvs Vj. before (…..) and after (* * * * *) exposure to octanol. Holding currents in both cells increased from less than 100pA to about 300pA following exposure to octanol.

The whole-cell voltage clamp technique has been used to measure junctional conductance in a variety of primary cell cultures, known to express extensive gap junctional communication, including pairs of rat lacrimal gland cells (Neyton and Trautmann, 1985, 1986) and chick embryonic ventricle cells (Veenstra and De Haan, 1988). In the latter case, cells of about 5μm diameter have a junctional conductance (Gj) ranging from 0.15 to 35.0nS (Veenstra and DeHaan, 1988). Similar values of Gj have been measured in isolated pairs of adult rat ventricular myocytes (White et al. 1985). A major difference in physiology of adult and embryonic heart gap junctions is that the former are reported to be voltage insensitive, whereas embryonic gap junctions are voltage sensitive (see DeHaan and Chen, 1990 for references).

In the 2-cell ascidian embryo, measurement of Gj using the same technique gave values of 17.5 to 35.3 nS, which, despite the difference in size, is comparable to the Gj in embryonic ventricular cells in culture (Veenstra and DeHaan, 1988). To our knowledge, Gj has not been previously measured in an intact early embryo using this technique. Gj in an embryonic cell line of Drosophila, Kc, was shown to be about 7 nS, and coupling in this system was suggested to be due to cytoplasmic bridges (Spray et al. 1989). Since, in the present study, we did not observe at the electron microscope any significant number of bridges connecting cells, and considering that Gj was both sensitive to Octanol (Spray et al. 1985) and reached values of 10 nS between manually apposed embryos, it is probable that early ascidian embryos express functional gap junctions We have not been able to successfully fix gap junctions in ascidian embryos, a problem common to other manne invertebrates.

Serras et al. (1988) have shown previously that dye coupling with LY starts at the 16- to 32-cell stage in the ascidian embryo, and we have confirmed this result. By studying coupling ratios as calculated from voltage excursions, these authors suggested that the change in communication pattern from the 2- to the 32-cell stage reflects an increase in ionic permeability. Direct measurements show that Gj in the 32-cell embryo is smaller than that in the 2-cell embryo, possibly as a result of cell size, although position and movement of these smaller blastomeres precludes such a conclusion. In addition, Gj in cleavage-arrested 2-cell embryos at 2 h was not significantly different from that of control 2-cell embryos, supporting the idea that there is no significant change in ionic permeability. Finally, in contrast to the suggestion of Serras et al. (1988), Gj appears to be independent of Vj in all stages up to the 32-cell stage.

Since cytoplasmic bridges are absent in cleavage-arrested embryos, and Gj does not change in time, what is the molecular basis for the change in dye coupling properties at the 32-cell stage? The experiments with cytochalasin B, which functionally blocks cell movement and cleavage, rules out spatial organization of the apposing junctional membranes as an important factor. In the early mouse embryo, it has been shown using cytochalasin B that gap junction assembly is independent of cell flattening and cytokinesis (Kidder et al 1987). One possibility is that the gap junctions in the 32cell stage are products of the embryonic genome, while the gap junctions in the 2-cell stage are the result of maternal gene expression. Although macroscopic Gj does not change in time, the embryonic junctions may contain fewer functional channels of larger single channel conductance. An alternative possibility is that junctional properties are modulated by removal or synthesis of an accessory protein that controls permeability. Studies with antibodies to gap junction proteins and single channel measurements may resolve this problem.

This work was supported by Nato grant No (30)0255/88. We thank Gianni Gragnaniello for help with the electron microscopy

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