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Ionic mechanism of the action potential and of its disappearance after fertilization in the Dentalium egg
DEVELOPMENTAL
BIOLOGY
122,516-521 (1987)
Ionic Mechanism of the Action Potential and of Its Disappearance after Fertilization in the Dentalium Egg C. BAUD,* M. MOREAU,~ AND P. GUERRIER-~ tStation Biologique,
29211
Roscofl, France, and *Laboratoire de Neurobiologie Cellulaire et Moleculaire, C.N.R.S. 91190, Gif-SW-Yvette, France Received December 2, 1986; accepted in revised form March 25, 1987
The membrane electrical properties of the Dentalium egg have been studied under voltage-clamp, before and after fertilization, up to the trefoil stage. The egg has an action potential with two rapid rising phases and one steady component. Most of the current is carried by calcium ions. All inward currents are blocked by cobalt. After fertilization, excitability disappears within about 50 min. This is mainly due to the appearance of a new steady K conductance, which provides a shunt to the calcium current. This new conductance appears shortly after emission of the second polar body and slightly before the formation of the polar lobe of the embryo. It is also blocked by cobalt. o 1987 Academic press. IX INTRODUCTION
Membrane excitability, i.e., the ability to produce an action potential, is a well-documented property of many eggs of invertebrates and vertebrates (see Hagiwara and Jaffe, 1979, and Hagiwara, 1983, for reviews). The production of action potentials depends on the presence of voltage-gated ion channels. Studies of the fate of these channels after fertilization show that they generally disappear during the first few cleavages; they reappear later in differentiated cell lines (Hagiwara and Miyazaki, 1977; Spitzer, 1979; Takahashi and Yoshii, 1981; Hirano and Takahashi, 1984; Hirano et al., 1984; Mitani, 1985). Excitability has been demonstrated in the early embryo of the scaphopod mollusc Dental&m, (Jaffe and Guerrier, 1981). The early embryo of Dentalium goes through a “trefoil” stage before completion of the first cellular division (see Materials and Methods for a description of the embryo). Jaffe and Guerrier (1981) showed that the trefoil embryo is not excitable, but the isolated polar lobe, the vegetal part of the embryo, is excitable. This has been, so far, the only direct demonstration of spatial heterogeneity in membrane excitability of an embryo at the time of first cleavage. In Dentalium the significance of membrane excitability in the egg is not understood: there appears to be no electrical block to polyspermy, although spontaneous action potentials are generated in the period between 5 and 40 min following fertilization (Moreau and Guerrier, submitted for publication). The localized excitability of the early embryo does not seem to be the early expression of future differentiation since both animal and vegetal halves give rise to excitable tissues of the larva (Wilson, 1904). However, ionic channel segregation might play a role by generating currents through the egg and/or early developing embryo (Jaffe, 1982). 0012-1606/W Copyright All rights
$3.00
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
516
As a first step toward the understanding of the role of the ionic currents in the egg, we have followed the properties of the unfertilized egg through fertilization up to the trefoil stage. The unfertilized egg has a typical action potential due mainly to calcium ions. After fertilization, the excitability disappears within about 50 min. This is mainly due to an increased steady permeability to K ions. This new conductance will have to be taken into consideration when dealing further with the establishment of the polarity of excitability in the embryo. MATERIALS
Preparation
AND
METHODS
of the Eggs and Sperm
Experiments were performed at the Station Biologique in Roscoff during the summers of 1984, 1985, and 1986, the breeding season extending from July to August. The animals, which normally live buried in the sand, were collected weekly from the sea floor at 20-25 m water depth and kept in sand-filled containers under running seawater. Shedding of eggs and sperm was induced by exposing the animal to natural light (and sometimes to a temperature shock at 25°C) in bowls containing natural seawater. The eggs were dejellied by gentle stirring in several rinses of filtered seawater. The eggs had a diameter of about 230 pm; they were pipetted up and down in a narrow glass tube (about 100 pm) to remove their eggshells. Eggs were in better condition if freshly dejellied before impalement. For the fertilization experiments, a drop of sperm suspension was diluted about a hundred times in ASW. One drop of this solution was then added to the beaker containing the eggs (final dilution about 10,000). Occasional eggs which underwent
BAUD, MOREAU, AND GUERRIER
spontaneous polar body emission, before addition of sperm, were discarded. Electrophysiological
Recording Setup
For voltage-clamp experiments, 3 to 10 MO electrodes were pulled from thin-walled glass (150TF from Clark Electromedical Instruments) on a horizontal puller (BBCH, Geneva). For prolonged recordings during and after fertilization a single electrode was used, made from thick-walled glass tube and with resistance ranging between 20 and 40 MO. The membrane potential was measured by a microprobe (M707, WPI) and fed to a clamp amplifier (Biologic, Grenoble, France) for voltage-clamp of the cell. The current was injected by a second electrode and measured by an amplifier before the current injecting electrode, as the voltage dropped through a lOO-KR resistor. The bath was directly connected to the ground by a chlorided Ag wire. Voltage and current control steps were applied by a pulse generator (WPI). The current and voltage were recorded on a pen recorder (Brush, Gould, Inc.; flat response up to 80Hz) and in parallel on a tape recorder (Euromag3, Enertec) for storage and analysis of faster transients. Resistance and capacitance measurements were obtained from current-clamp experiments. The membrane potential changes exponentially when small square pulses of current are applied. The time course was fitted to a single exponential by a least-squares regression analysis. The time constant of this exponential divided by the input resistance of the cell gives the capacitance of the cell. For each capacitance value, five successive measurements were averaged. The bath was continuously perfused by a peristaltic pump (Gilson Minipuls) and maintained at 17-19°C by cooling the solutions in the inlet tube to the chamber. The temperature was monitored with a small thermistor in the egg chamber. Solutions
The artificial seawater (4.4 Ca ASW) had the following composition in millimolar: NaCl 452, KC1 10, MgClz 25, MgS04 1’7, CaClz 4.4, buffered to pH 8.2 by 5 mM Tris. High calcium solution (22 Ca SW) was obtained by adding CaClz up to 22 mM to standard ASW. Low-sodium solutions were prepared by replacing NaCl with equimolar N-methyl-D-glucamine chloride or Tris-Cl. Cobalt was added to 22 Ca ASW at a concentration of 20 mlM. Descm’ption of Dental&m Stage
Development
up to the Trefoil
The two polar bodies are emitted at about 20-min intervals, indicating completion of meiosis. About 30 min
Action
Potential
in Dental&m
517
Egg
after emission of the second polar body, a protrusion appears at the vegetal pole, this is the first indication of polar lobe formation. This protrusion expands further giving the embryo its characteristic “pear” shape. A constriction follows which isolates the polar lobe from the rest of the embryo. At the same time, cell division occurs in the animal half. As a result of these two processes, the embryo obtains the characteristic “trefoil” shape. This stage lasts for about 10 min. The polar lobe then fuses with one of the blastomeres forming an asymetrical two-cell embryo, in which most of the cytoplasm and membrane of the vegetal pole of the egg is segregated into one of the two first blastomeres. Deletion experiments as well as experimental equalization of the first division have shown that the polar lobe contains “determinants” necessary to the formation of the apical tuft and the posterior part of the trochophore larva (Wilson, 1904; Guerrier et al., 1978; Verdonk and Cather, 1983; Render and Guerrier, 1984). RESULTS
Electrophysiological
Properties
of the Unfertilized
Egg
When penetrated with a single high-resistance microelectrode, the eggs had a resting potential around -80 mV. However, when two lower resistance microelectrodes were used for voltage-clamp experiments, the resting potential in many batches was around -10 mV. Although we disregarded these batches at an early stage of this work, we used them later, especially in the summer of 1986, when almost all the eggs had a resting potential of -10 mV. The -10 mV resting potential is due to an inflection in the I-V curve which makes the resting potential very sensitive to ionic leak around the electrodes (see below). In some cases, the membrane hyperpolarized spontaneously with time. Eggs with a resting potential around -80 mV showed a typical action potential when stimulated by a small depolarizing pulse. The rising phase of the action potential showed two distinct components, one regenerative depolarization starting around -60 mV and a second one starting around -30 mV, as illustrated in Fig. 1. The peak potential slightly exceeded 0 mV. After the peak, the membrane remained depolarized for a few hundred milliseconds before returning to its resting potential. In Fig. 2A, current traces during imposed voltage steps are shown. The egg was maintained at a holding potential of -90 mV. During depolarizing pulses starting from -60 mV, there was a transient inward current; its activation became faster as the depolarizing step became larger. In the lower panel of Fig. 2A, membrane current is plotted against voltage. The filled circles show the steady-state current (the current at the end of a 300msec pulse). This curve was not linear around the resting
518
DEVELOPMENTAL BIOLOGY 0.5s
!iy r---l
-50.
-100’
-.;
\
rl
(2.5nA
FIG. 1. Typical action potential in an unfertilized egg in normal ASW. Arrows point to the 2 inflection points in the rising phase of the a.p.
potential (-10 mV in this egg); therefore, no attempt was made to correct for the “leakage” current in any of the data shown. There was an inflection in the steadystate current around -30 mV, suggesting that a noninactivating or a slowly inactivating inward current was present at this potential. The open circles show the peak of the transient inward current. Two components were clearly apparent. One had a threshold around -60 mV and the second had a threshold around -30 mV. The current traces in Fig. 2B show the effect of increasing the external calcium concentration five times in the same egg as in Fig. 2A. In this case, the resting potential was +15 mV. A large steady-state inward current was seen at potentials more negative than +15 mV, and the peak amplitude of the inactivating inward current was also increased. The corresponding I-V curves are plotted in the lower panel. The amplitude of the first current peak was little affected, whereas that of the second one increased. From six similar experiments, the maximum inward current in the second peak was increased by a factor 2.4 + 0.13 (SD), while the first peak was not significantly altered. In Fig. 2C, 20 mMcobalt was added to a high Ca solution in the same egg as in Figs. 2A and 2B. The corresponding I/V curve shows that all inward current components were blocked. The block was reversible; it was half-maximal for a cobalt concentration of 4 m&f (not shown). It appears that the high threshold rapidly inactivating inward current was mainly a calcium current. The question arises whether the steady inward component, which also increased in high calcium, was due to the same calcium channels. First, the steady component was truely inactivating since all eggs in 22 Ca SW had a resting potential of +15, +20 mV after firing one spike. The channels responsible for this steady current were also truly voltage-dependent since at the end of each pulse the current went back to its original level, after a slow inward tail current (see traces in Figs. 2A and 2B). The coexistence of an inactivating component and a steady component may also be due to a single channel with a
VOLUME 122, 1987
Ca-dependent inactivation mechanism (Chad et ak, 1984). However, the rate of inactivation is then expected to depend on the amplitude of the inward current, the more calcium that enters the cell the faster the inactivation. This was not observed in Dental&m eggs, rather the inactivation was voltage-dependent; this can be seen, for instance, in the two upper traces in Fig. 2B where the inward currents are about equal (owing to the leakage current), whereas the inactivation is much faster at +20 mV than at +5 mV. It seems therefore likely that the inactivating and the noninactivating components are due to two different calcium conductances. The possibility remained that part of the conductances were calcium-activated rather than a calcium conductance as is often the case in invertebrates eggs (Lansman, 1983; Jaffe et al., 1986). One possible carrier was sodium. External sodium was therefore replaced totally or partially by N-methyl-D-glucamine or by Tris. This did not affect any of the current components described previously (not shown). It should be noted that the removal of external sodium often induced a nonreversible modification of the egg membrane. The same modification was also observed in 6
C
22CaSW
22Ca+20Co
-7 ,r---r,
-
SW +30
1 OOms
d -80
&f
-40
-10
mV P,, l 40
I,
I,
T
FIG. 2. Current traces during voltage steps to different potentials as indicated on each trace. Holding potential, -90 mV. The lower panel represents the I/V relations. (A) Normal ASW; (B) in high (22 mM) external calcium; (C) after the addition of 20 mM cobalt to the high Ca solution. Scales and symbols in the I/V curves are the same in all cases: (0) current at the end of the 300-msec pulse; (0) peak inward current.
BAUD, MOREAU, AND GUERRIER
Action
Potential
in Dentalium
519
Egg
ferent from the loss of excitability tilization (see below).
almost all eggs after several series of voltage-clamp depolarizing steps, even if no ionic modification of the solution was performed. It was characterized by a loss of excitability. This artifactual state of the membrane had a large slow inward rectifying current at potentials negative to -60 mV, and the second peak of inward current was decreased (Baud and Moreau, unpublished observations). These features made this artifact clearly dif-
D’zsappearance of Excitability
occurring after fer-
after Fertilization
Jaffe and Guerrier (1981) have reported that the trefoil embryo is not excitable. Since the unfertilized egg is excitable, we have followed the changes occurring after
minispikes
-root
L 0
I 20
I 40
I 60
I
a’ t (mn)
loo
FIG. 3. Variation of input resistance (Rm), membrane capacitance (Cm), and resting potential (Em) measured at intervals during and after fertilization. The arrival of the spermatozoa (spz) is indicated by an arrow on each trace. On top of the first trace, the morphological events are illustrated with approximate timing. About 10 min after fertilization, a series of small depolarizations (minispikes) of lo-20 mV amplitude was observed (they do not appear on the potential trace, because only the potential at the time of calculation of Rm and Cm is plotted). After emission of the second polar body (pb2), Rm dropped by two-thirds, while Cm stayed around 15 nF. In the lower panel, the inset shows examples of potential transients from which Rm and Cm were calculated, before and 40,64, and 82 min after fertilization; pulse amplitude, 3nA; duration 700 ms; vertical calibration 20 mV. The vertical line in the potential trace around 32 min indicates a spontaneous action potential. The vertical dotted line indicates the moment at which depolarizing current pulses no longer elicited an action potential in normal ASW.
520
DEVELOPMENTAL BIOLOGY
fertilization. All experiments were performed in normal ASW. Only those experiments where a normal trefoil stage was attained were considered here, because this ensured that the embryo was monospermic and able to pursue normal development. Figure 3 shows V,, Ri,, and C in an egg representative of seven similar recordings. Typically, the membrane resistance was unchanged while the two polar bodies were emitted. It started to decrease sharply after emission of the second polar body and before the appearance of the polar lobe. It decreased steadily during the pear-shaped stage and, after about 35 min, stabilized at a new level. The embryo then reached the trefoil stage with no further changes. Figure 3 also shows that during the same period, the cell capacitance was constant. Data from seven embryos showed that the input resistance dropped from 15.8 MO + 1.9 (SD) in the fertilized egg to 2.0 MQ + 1.0 (SD) in the trefoil embryo; during that time, the capacitance was constant. Therefore the decrease in resistance was not correlated with an increase in membrane area. The eggs were also tested for generation of an action potential and were found to become “nonexcitable” when the input resistance reached about 9 MQ. Ionic Conductance of the Trefoil Stage Membrane Figure 4 shows current traces from an embryo at the trefoil stage, in high calcium (22 mM) solution. Both electrodes were introduced in the same blastomere; at this stage, the blastomeres are still connected by cytoplasmic bridges and are electrically coupled (Jaffe and A
6
22 casw
22 Ca+20CoSW
I,/
C
r
+25
Ifn/l
-50 f-
5---y-‘LA---4 s N I
-,
-10 -20 -7 -40 -50
-
t I&-,..“-I _a-6 l -t -----%-I1OOmS
+10b 07 -10
p
-20
c
-40
/i
c-10
1OOms
FIG. 4. Current traces from an embryo at the trefoil stage in (A) 22 Ca SW and (B) after addition of 20 mM cobalt. Potential steps are to the values indicated on each trace. (C) Current-voltage relation from the traces in (A) and (B); in 22 Ca SW: current at the end of the 300ms pulse (a) and peak inward current (0); in 22 Ca + 20 Co SW (m).
VOLUME 122, 1987
Guerrier, 1981). Figure 3A shows the current traces in 22 Ca SW. The peak inward current and the steady state current were plotted against voltage in Fig. 3C. They appeared qualitatively similar to those described in the unfertilized egg. Both inactivating components as well as the noninactivating component are present. However, the resting conductance is much larger, preventing the establishment of a net inward current and therefore the generation of an action potential. This new conductance had a reversal potential around -75 mV; we assumed that it was mainly a potassium conductance. No new transients due to this additional conductance were observed in the current traces; therefore it is either a steady conductance, present at all potentials, or it activates and deactivates in less than 5 msec, which was the average settling time of the clamp amplifier. When cobalt (20 mM) was added to 22 Ca SW, the membrane depolarized rapidly, and in voltage-clamp, all currents disappeared, including the new steady K current (Figs. 4B and 4C). These changes were rapidly reversible upon removal of cobalt. DISCUSSION
The unfertilized egg of Dental&m can generate an action potential when stimulated by a depolarizing current. This action potential shows two rising phases and a plateau. There is so far one other report of action potential in Molluscans eggs, the mud snail Ilyanassa (Moreau and Guerrier, 1981). There exist a number of reports on action potentials in other phyla. In many cases, the action potential is sensitive to both sodium and calcium. This is the case in all the starfish species studied (Miyazaki et al., 1975; Shen and Steinhardt, 1976; Moody and Lansman, 1983), in the marine worm Urechis (Jaffe et ah, 1979), in tunicates (Miyazaki et al., 1974; Okamoto et al, 1976; Thompson and Knier, 1983), and in a ctenophore (Barish, 1984). By contrast, purely calcium-dependent spikes are observed in the annelid Chaetopterus (Hagiwara and Miyazaki, 1977) and in mouse and hamster (Okamoto et aZ.,1977; Georgiou et ah, 1984). Finally, purely sodium-dependent action potentials are observed in amphibians (Baud et ah, 1982; Schlichter, 1983; Baud and Barish, 1984). In Dentalium the ionic nature of the small component of inward current with low threshold has not been elucidated. This current is little affected by increasing external calcium and is not affected by removing external sodium. It is blocked by 20 mMcobalt. It is still possible that it is a calcium conductance, already saturating at normal calcium level. The second peak and the steady current are attributed to calcium ions because their amplitude is markedly increased when external calcium is increased and they are not affected by removal of ex-
BAUD, MOREAU, AND GUERRIER
Action
ternal sodium. Therefore the action potential in DentaZium is mainly due to calcium ions. It is not possible to decide yet whether the two components come from the same conductance, but a calcium-induced inactivation mechanism seems to be ruled out by the observation that the rate of inactivation is not dependent on the amplitude of the inward current. After fertilization the input resistance decreases sharply by about sevenfold in 40 min, before reaching the trefoil stage. At that time, action potentials can no longer be generated in the embryo. Considering the IV curves, it can be predicted that a cell will produce a regenerative response to depolarization if there exists a potential range in which the sum of the inward currents is larger than the sum of the outward currents. In the unfertilized egg, the net inward current is maximum around -10 mV and never exceeds 10 nA. In the unfertilized egg, assuming an input resistance of 15 MQ, a resting potential of -80 mV, and linear resting and leak conductances, the outward current will be 4.6 nA at -10 mV. The balance is then in favor of the inward current and the egg is excitable. After fertilization the outward current will reach 10 nA when the resistance reaches about 7 MQ. Cells with lower input resistance are not expected to be excitable anymore. We have shown that at the trefoil stage all inward current components are present, supporting the above interpretation. REFERENCES BARISH, M. E. (1984). Calcium-sensitive action potential of long duration in the fertilized egg of the Ctenophore Mnemiopsis leidyi. Dev. BioL 105,29-40. BAUD, C., and BARISH, M. E. (1984). Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes. Dev. Biol. 105,423-434. BAUD, C., KADO, R. T., and MARCHER, K. S. (1982). Sodium channels induced by depolarization of the Xenopus laevis oocyte. Proc. NatL Acad. Sci. USA. 79,3188-3192. CHAD, J., ECKERT, R., and EWALD, D. (1984). Kinetics of calcium-dependent inactivation of calcium currents in voltage-clamped neurones of Aplysia califokca. J. PhysioL 347, 279-300. GEORGIOIJ, P., BOUNTRA, C., BLAUD, K. P., and HOUSE, C. R. (1984). Calcium action potentials in unfertilized eggs of mice and hamsters. Q. J. Exp. Physiol. 69, 365-380. GUERRIER, P., VAN DEN BIGGELAAR, J. A. M., VAN DONGEN, C. A. M., and VERDONK, N. M. (1978). Significance of the polar lobe for the determination of dorso-ventral polarity in Dental&m vulgare (Da Costa). Dev. BioL 63,233-242. HAGIWARA, S. (1983). Membrane potential-dependent ion channels in cell membrane. Raven Press, New York. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8,385-416. HAGIWARA, S., and MIYAZAKI, S. (1977). Changes in excitability of the cell membrane during “differentiation without cleavage” in the egg of the annelid, Chaetopterus pergamentaceus. J. Physiol. 272, 197216.
Potential
in Dentalium
Egg
521
HIRANO, T., and TAKAHASHI, K. (1984). Comparison of properties of calcium channels between the differentiated l-cell embryo and the egg cell of ascidians. J. Physiol. 347,327-344. HIRANO, T., TAKAHASHI, K., and YAMASHITA, N. (1984). Determination of excitability types in blastomeres of the cleavage-arrested but differentiated embryos of an ascidian. J. PhysioL 347,301-325. JAFFE, L. F. (1982). Developmental currents, voltage and gradients. In “Developmental Order: Its Origin and Regulation.” (S. Subtelny, Ed.), pp. 183-215. A. R. Liss, New York. JAFFE, L. A., and GUERRIER, P. (1981). Localization of electrical excitability in the early embryo of Dentalium. Dew. BioL 83,370-373. JAFFE, L. A., GOULD-SOMERO, M., and HOLLAND, L. (1979). Ionic mechanism of the fertilization potential of the marine worm Urechti caupo (Echiura). J. Gen. Physiol 73,469-492. JAFFE, L. A., KADO, R. T., and KLINE, D. (1986). A calcium-activated sodium conductance produces a long-duration action potential in the egg of a nemertean worm. J. Physiol. 381,263-278. LANSMAN, J. B. (1983). Components of the starfish fertilization potential: Role of calcium and calcium-dependent inward current. Zn “The Physiology of Excitable Cells,” (A. D. Grinnell and W. J. Moody Jr., Eds), pp. 233-246, A. R. Liss, New York. MITANI, S. (1985). The reduction of calcium current associated with early differentiation of the murine embryo. J. PhysioL 363, 71-86. MIYAZAKI, S., OHMORI, H., and SASAKI, S. (1975). Action potential and non-linear current-voltage relation in starfish oocytes. J PhysioL 246,37-54. MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1974). Electrical excitability in the egg cell membrane of the tunicate. J Physiol. 238,3754. MOODY, W. J., and LANSMAN, J. B. (1983). Developmental regulation of Ca and K currents during hormone-induced maturation of starfish oocytes. Proc. Natl. Acad. Sci. USA 80,3096-3100. MOREAU, M., and GUERRIER, P. (1981). Absence of regional differences in the membrane properties from the embryo of the mud snail Ilyanassa obsoleta. BioL Bull. 161, 320. OKAMOTO, H., TAKAHASHI, K., and YOSHII, M. (1976). Membrane currents of the tunicate egg under the voltage-clamp conditions. J. PhysioL 254, 607-638. OKAMOTO, H., TAKAHASHI, K., and YAMASHITA, N. (1977). Ionic currents through the membrane of the mammalian oocyte and their comparison with those in the tunicate and sea urchin. J. Physiol. 267, 465-495. RENDER, J. A., and GUERRIER, P. (1984). Size regulation and morphogenetic localization in the Dentalium polar lobe. J. Exp. ZooL 232, 79-86. SCHLICHTER, L. C. (1983). Spontaneous action potentials produced by Na and Cl channels in maturing Rana p$iens oocytes. Dew. BioL 98, 47-59. SHEN, S., and STEINHARDT, R. A. (1976). An electrophysiological study of the membrane properties of the immature and mature oocyte of the batstar Patiria miniata. Dev. Biol. 48, 148-162. SPITZER, N. C. (1979). Ion channels in development. Annu. Rev. Newosci. 2,363-397. TAKAHASHI, K., and YOSHII, M. (1981). Development of sodium, calcium and potassium channels in the cleavage arrested embryo of an ascidian. .I PhysioL 315, 515-529. THOMPSON, S., and KNIER, J. (1983). Spontaneous action potentials and resting potential shifts in fertilized eggs of the tunicate Clavelina. Dev. BioL 99, 121-131. VERDONK, N. H., and CATHER, J. N. (1983). Morphogenetic determination and differentiation. In “The Mollusca Development” (N. H. Verdonk, J. A. M. van den Biggelaar, and A. S. Tompa, Eds.), Vo13, pp. 215-252. Academic Press, New York. WILSON, E. B. (1904). Experimental studies on germinal localization. I. The germ regions in the egg of Dental&m. J. Exp. ZooL 1, l-72.
BIOLOGY
122,516-521 (1987)
Ionic Mechanism of the Action Potential and of Its Disappearance after Fertilization in the Dentalium Egg C. BAUD,* M. MOREAU,~ AND P. GUERRIER-~ tStation Biologique,
29211
Roscofl, France, and *Laboratoire de Neurobiologie Cellulaire et Moleculaire, C.N.R.S. 91190, Gif-SW-Yvette, France Received December 2, 1986; accepted in revised form March 25, 1987
The membrane electrical properties of the Dentalium egg have been studied under voltage-clamp, before and after fertilization, up to the trefoil stage. The egg has an action potential with two rapid rising phases and one steady component. Most of the current is carried by calcium ions. All inward currents are blocked by cobalt. After fertilization, excitability disappears within about 50 min. This is mainly due to the appearance of a new steady K conductance, which provides a shunt to the calcium current. This new conductance appears shortly after emission of the second polar body and slightly before the formation of the polar lobe of the embryo. It is also blocked by cobalt. o 1987 Academic press. IX INTRODUCTION
Membrane excitability, i.e., the ability to produce an action potential, is a well-documented property of many eggs of invertebrates and vertebrates (see Hagiwara and Jaffe, 1979, and Hagiwara, 1983, for reviews). The production of action potentials depends on the presence of voltage-gated ion channels. Studies of the fate of these channels after fertilization show that they generally disappear during the first few cleavages; they reappear later in differentiated cell lines (Hagiwara and Miyazaki, 1977; Spitzer, 1979; Takahashi and Yoshii, 1981; Hirano and Takahashi, 1984; Hirano et al., 1984; Mitani, 1985). Excitability has been demonstrated in the early embryo of the scaphopod mollusc Dental&m, (Jaffe and Guerrier, 1981). The early embryo of Dentalium goes through a “trefoil” stage before completion of the first cellular division (see Materials and Methods for a description of the embryo). Jaffe and Guerrier (1981) showed that the trefoil embryo is not excitable, but the isolated polar lobe, the vegetal part of the embryo, is excitable. This has been, so far, the only direct demonstration of spatial heterogeneity in membrane excitability of an embryo at the time of first cleavage. In Dentalium the significance of membrane excitability in the egg is not understood: there appears to be no electrical block to polyspermy, although spontaneous action potentials are generated in the period between 5 and 40 min following fertilization (Moreau and Guerrier, submitted for publication). The localized excitability of the early embryo does not seem to be the early expression of future differentiation since both animal and vegetal halves give rise to excitable tissues of the larva (Wilson, 1904). However, ionic channel segregation might play a role by generating currents through the egg and/or early developing embryo (Jaffe, 1982). 0012-1606/W Copyright All rights
$3.00
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
516
As a first step toward the understanding of the role of the ionic currents in the egg, we have followed the properties of the unfertilized egg through fertilization up to the trefoil stage. The unfertilized egg has a typical action potential due mainly to calcium ions. After fertilization, the excitability disappears within about 50 min. This is mainly due to an increased steady permeability to K ions. This new conductance will have to be taken into consideration when dealing further with the establishment of the polarity of excitability in the embryo. MATERIALS
Preparation
AND
METHODS
of the Eggs and Sperm
Experiments were performed at the Station Biologique in Roscoff during the summers of 1984, 1985, and 1986, the breeding season extending from July to August. The animals, which normally live buried in the sand, were collected weekly from the sea floor at 20-25 m water depth and kept in sand-filled containers under running seawater. Shedding of eggs and sperm was induced by exposing the animal to natural light (and sometimes to a temperature shock at 25°C) in bowls containing natural seawater. The eggs were dejellied by gentle stirring in several rinses of filtered seawater. The eggs had a diameter of about 230 pm; they were pipetted up and down in a narrow glass tube (about 100 pm) to remove their eggshells. Eggs were in better condition if freshly dejellied before impalement. For the fertilization experiments, a drop of sperm suspension was diluted about a hundred times in ASW. One drop of this solution was then added to the beaker containing the eggs (final dilution about 10,000). Occasional eggs which underwent
BAUD, MOREAU, AND GUERRIER
spontaneous polar body emission, before addition of sperm, were discarded. Electrophysiological
Recording Setup
For voltage-clamp experiments, 3 to 10 MO electrodes were pulled from thin-walled glass (150TF from Clark Electromedical Instruments) on a horizontal puller (BBCH, Geneva). For prolonged recordings during and after fertilization a single electrode was used, made from thick-walled glass tube and with resistance ranging between 20 and 40 MO. The membrane potential was measured by a microprobe (M707, WPI) and fed to a clamp amplifier (Biologic, Grenoble, France) for voltage-clamp of the cell. The current was injected by a second electrode and measured by an amplifier before the current injecting electrode, as the voltage dropped through a lOO-KR resistor. The bath was directly connected to the ground by a chlorided Ag wire. Voltage and current control steps were applied by a pulse generator (WPI). The current and voltage were recorded on a pen recorder (Brush, Gould, Inc.; flat response up to 80Hz) and in parallel on a tape recorder (Euromag3, Enertec) for storage and analysis of faster transients. Resistance and capacitance measurements were obtained from current-clamp experiments. The membrane potential changes exponentially when small square pulses of current are applied. The time course was fitted to a single exponential by a least-squares regression analysis. The time constant of this exponential divided by the input resistance of the cell gives the capacitance of the cell. For each capacitance value, five successive measurements were averaged. The bath was continuously perfused by a peristaltic pump (Gilson Minipuls) and maintained at 17-19°C by cooling the solutions in the inlet tube to the chamber. The temperature was monitored with a small thermistor in the egg chamber. Solutions
The artificial seawater (4.4 Ca ASW) had the following composition in millimolar: NaCl 452, KC1 10, MgClz 25, MgS04 1’7, CaClz 4.4, buffered to pH 8.2 by 5 mM Tris. High calcium solution (22 Ca SW) was obtained by adding CaClz up to 22 mM to standard ASW. Low-sodium solutions were prepared by replacing NaCl with equimolar N-methyl-D-glucamine chloride or Tris-Cl. Cobalt was added to 22 Ca ASW at a concentration of 20 mlM. Descm’ption of Dental&m Stage
Development
up to the Trefoil
The two polar bodies are emitted at about 20-min intervals, indicating completion of meiosis. About 30 min
Action
Potential
in Dental&m
517
Egg
after emission of the second polar body, a protrusion appears at the vegetal pole, this is the first indication of polar lobe formation. This protrusion expands further giving the embryo its characteristic “pear” shape. A constriction follows which isolates the polar lobe from the rest of the embryo. At the same time, cell division occurs in the animal half. As a result of these two processes, the embryo obtains the characteristic “trefoil” shape. This stage lasts for about 10 min. The polar lobe then fuses with one of the blastomeres forming an asymetrical two-cell embryo, in which most of the cytoplasm and membrane of the vegetal pole of the egg is segregated into one of the two first blastomeres. Deletion experiments as well as experimental equalization of the first division have shown that the polar lobe contains “determinants” necessary to the formation of the apical tuft and the posterior part of the trochophore larva (Wilson, 1904; Guerrier et al., 1978; Verdonk and Cather, 1983; Render and Guerrier, 1984). RESULTS
Electrophysiological
Properties
of the Unfertilized
Egg
When penetrated with a single high-resistance microelectrode, the eggs had a resting potential around -80 mV. However, when two lower resistance microelectrodes were used for voltage-clamp experiments, the resting potential in many batches was around -10 mV. Although we disregarded these batches at an early stage of this work, we used them later, especially in the summer of 1986, when almost all the eggs had a resting potential of -10 mV. The -10 mV resting potential is due to an inflection in the I-V curve which makes the resting potential very sensitive to ionic leak around the electrodes (see below). In some cases, the membrane hyperpolarized spontaneously with time. Eggs with a resting potential around -80 mV showed a typical action potential when stimulated by a small depolarizing pulse. The rising phase of the action potential showed two distinct components, one regenerative depolarization starting around -60 mV and a second one starting around -30 mV, as illustrated in Fig. 1. The peak potential slightly exceeded 0 mV. After the peak, the membrane remained depolarized for a few hundred milliseconds before returning to its resting potential. In Fig. 2A, current traces during imposed voltage steps are shown. The egg was maintained at a holding potential of -90 mV. During depolarizing pulses starting from -60 mV, there was a transient inward current; its activation became faster as the depolarizing step became larger. In the lower panel of Fig. 2A, membrane current is plotted against voltage. The filled circles show the steady-state current (the current at the end of a 300msec pulse). This curve was not linear around the resting
518
DEVELOPMENTAL BIOLOGY 0.5s
!iy r---l
-50.
-100’
-.;
\
rl
(2.5nA
FIG. 1. Typical action potential in an unfertilized egg in normal ASW. Arrows point to the 2 inflection points in the rising phase of the a.p.
potential (-10 mV in this egg); therefore, no attempt was made to correct for the “leakage” current in any of the data shown. There was an inflection in the steadystate current around -30 mV, suggesting that a noninactivating or a slowly inactivating inward current was present at this potential. The open circles show the peak of the transient inward current. Two components were clearly apparent. One had a threshold around -60 mV and the second had a threshold around -30 mV. The current traces in Fig. 2B show the effect of increasing the external calcium concentration five times in the same egg as in Fig. 2A. In this case, the resting potential was +15 mV. A large steady-state inward current was seen at potentials more negative than +15 mV, and the peak amplitude of the inactivating inward current was also increased. The corresponding I-V curves are plotted in the lower panel. The amplitude of the first current peak was little affected, whereas that of the second one increased. From six similar experiments, the maximum inward current in the second peak was increased by a factor 2.4 + 0.13 (SD), while the first peak was not significantly altered. In Fig. 2C, 20 mMcobalt was added to a high Ca solution in the same egg as in Figs. 2A and 2B. The corresponding I/V curve shows that all inward current components were blocked. The block was reversible; it was half-maximal for a cobalt concentration of 4 m&f (not shown). It appears that the high threshold rapidly inactivating inward current was mainly a calcium current. The question arises whether the steady inward component, which also increased in high calcium, was due to the same calcium channels. First, the steady component was truely inactivating since all eggs in 22 Ca SW had a resting potential of +15, +20 mV after firing one spike. The channels responsible for this steady current were also truly voltage-dependent since at the end of each pulse the current went back to its original level, after a slow inward tail current (see traces in Figs. 2A and 2B). The coexistence of an inactivating component and a steady component may also be due to a single channel with a
VOLUME 122, 1987
Ca-dependent inactivation mechanism (Chad et ak, 1984). However, the rate of inactivation is then expected to depend on the amplitude of the inward current, the more calcium that enters the cell the faster the inactivation. This was not observed in Dental&m eggs, rather the inactivation was voltage-dependent; this can be seen, for instance, in the two upper traces in Fig. 2B where the inward currents are about equal (owing to the leakage current), whereas the inactivation is much faster at +20 mV than at +5 mV. It seems therefore likely that the inactivating and the noninactivating components are due to two different calcium conductances. The possibility remained that part of the conductances were calcium-activated rather than a calcium conductance as is often the case in invertebrates eggs (Lansman, 1983; Jaffe et al., 1986). One possible carrier was sodium. External sodium was therefore replaced totally or partially by N-methyl-D-glucamine or by Tris. This did not affect any of the current components described previously (not shown). It should be noted that the removal of external sodium often induced a nonreversible modification of the egg membrane. The same modification was also observed in 6
C
22CaSW
22Ca+20Co
-7 ,r---r,
-
SW +30
1 OOms
d -80
&f
-40
-10
mV P,, l 40
I,
I,
T
FIG. 2. Current traces during voltage steps to different potentials as indicated on each trace. Holding potential, -90 mV. The lower panel represents the I/V relations. (A) Normal ASW; (B) in high (22 mM) external calcium; (C) after the addition of 20 mM cobalt to the high Ca solution. Scales and symbols in the I/V curves are the same in all cases: (0) current at the end of the 300-msec pulse; (0) peak inward current.
BAUD, MOREAU, AND GUERRIER
Action
Potential
in Dentalium
519
Egg
ferent from the loss of excitability tilization (see below).
almost all eggs after several series of voltage-clamp depolarizing steps, even if no ionic modification of the solution was performed. It was characterized by a loss of excitability. This artifactual state of the membrane had a large slow inward rectifying current at potentials negative to -60 mV, and the second peak of inward current was decreased (Baud and Moreau, unpublished observations). These features made this artifact clearly dif-
D’zsappearance of Excitability
occurring after fer-
after Fertilization
Jaffe and Guerrier (1981) have reported that the trefoil embryo is not excitable. Since the unfertilized egg is excitable, we have followed the changes occurring after
minispikes
-root
L 0
I 20
I 40
I 60
I
a’ t (mn)
loo
FIG. 3. Variation of input resistance (Rm), membrane capacitance (Cm), and resting potential (Em) measured at intervals during and after fertilization. The arrival of the spermatozoa (spz) is indicated by an arrow on each trace. On top of the first trace, the morphological events are illustrated with approximate timing. About 10 min after fertilization, a series of small depolarizations (minispikes) of lo-20 mV amplitude was observed (they do not appear on the potential trace, because only the potential at the time of calculation of Rm and Cm is plotted). After emission of the second polar body (pb2), Rm dropped by two-thirds, while Cm stayed around 15 nF. In the lower panel, the inset shows examples of potential transients from which Rm and Cm were calculated, before and 40,64, and 82 min after fertilization; pulse amplitude, 3nA; duration 700 ms; vertical calibration 20 mV. The vertical line in the potential trace around 32 min indicates a spontaneous action potential. The vertical dotted line indicates the moment at which depolarizing current pulses no longer elicited an action potential in normal ASW.
520
DEVELOPMENTAL BIOLOGY
fertilization. All experiments were performed in normal ASW. Only those experiments where a normal trefoil stage was attained were considered here, because this ensured that the embryo was monospermic and able to pursue normal development. Figure 3 shows V,, Ri,, and C in an egg representative of seven similar recordings. Typically, the membrane resistance was unchanged while the two polar bodies were emitted. It started to decrease sharply after emission of the second polar body and before the appearance of the polar lobe. It decreased steadily during the pear-shaped stage and, after about 35 min, stabilized at a new level. The embryo then reached the trefoil stage with no further changes. Figure 3 also shows that during the same period, the cell capacitance was constant. Data from seven embryos showed that the input resistance dropped from 15.8 MO + 1.9 (SD) in the fertilized egg to 2.0 MQ + 1.0 (SD) in the trefoil embryo; during that time, the capacitance was constant. Therefore the decrease in resistance was not correlated with an increase in membrane area. The eggs were also tested for generation of an action potential and were found to become “nonexcitable” when the input resistance reached about 9 MQ. Ionic Conductance of the Trefoil Stage Membrane Figure 4 shows current traces from an embryo at the trefoil stage, in high calcium (22 mM) solution. Both electrodes were introduced in the same blastomere; at this stage, the blastomeres are still connected by cytoplasmic bridges and are electrically coupled (Jaffe and A
6
22 casw
22 Ca+20CoSW
I,/
C
r
+25
Ifn/l
-50 f-
5---y-‘LA---4 s N I
-,
-10 -20 -7 -40 -50
-
t I&-,..“-I _a-6 l -t -----%-I1OOmS
+10b 07 -10
p
-20
c
-40
/i
c-10
1OOms
FIG. 4. Current traces from an embryo at the trefoil stage in (A) 22 Ca SW and (B) after addition of 20 mM cobalt. Potential steps are to the values indicated on each trace. (C) Current-voltage relation from the traces in (A) and (B); in 22 Ca SW: current at the end of the 300ms pulse (a) and peak inward current (0); in 22 Ca + 20 Co SW (m).
VOLUME 122, 1987
Guerrier, 1981). Figure 3A shows the current traces in 22 Ca SW. The peak inward current and the steady state current were plotted against voltage in Fig. 3C. They appeared qualitatively similar to those described in the unfertilized egg. Both inactivating components as well as the noninactivating component are present. However, the resting conductance is much larger, preventing the establishment of a net inward current and therefore the generation of an action potential. This new conductance had a reversal potential around -75 mV; we assumed that it was mainly a potassium conductance. No new transients due to this additional conductance were observed in the current traces; therefore it is either a steady conductance, present at all potentials, or it activates and deactivates in less than 5 msec, which was the average settling time of the clamp amplifier. When cobalt (20 mM) was added to 22 Ca SW, the membrane depolarized rapidly, and in voltage-clamp, all currents disappeared, including the new steady K current (Figs. 4B and 4C). These changes were rapidly reversible upon removal of cobalt. DISCUSSION
The unfertilized egg of Dental&m can generate an action potential when stimulated by a depolarizing current. This action potential shows two rising phases and a plateau. There is so far one other report of action potential in Molluscans eggs, the mud snail Ilyanassa (Moreau and Guerrier, 1981). There exist a number of reports on action potentials in other phyla. In many cases, the action potential is sensitive to both sodium and calcium. This is the case in all the starfish species studied (Miyazaki et al., 1975; Shen and Steinhardt, 1976; Moody and Lansman, 1983), in the marine worm Urechis (Jaffe et ah, 1979), in tunicates (Miyazaki et al., 1974; Okamoto et al, 1976; Thompson and Knier, 1983), and in a ctenophore (Barish, 1984). By contrast, purely calcium-dependent spikes are observed in the annelid Chaetopterus (Hagiwara and Miyazaki, 1977) and in mouse and hamster (Okamoto et aZ.,1977; Georgiou et ah, 1984). Finally, purely sodium-dependent action potentials are observed in amphibians (Baud et ah, 1982; Schlichter, 1983; Baud and Barish, 1984). In Dentalium the ionic nature of the small component of inward current with low threshold has not been elucidated. This current is little affected by increasing external calcium and is not affected by removing external sodium. It is blocked by 20 mMcobalt. It is still possible that it is a calcium conductance, already saturating at normal calcium level. The second peak and the steady current are attributed to calcium ions because their amplitude is markedly increased when external calcium is increased and they are not affected by removal of ex-
BAUD, MOREAU, AND GUERRIER
Action
ternal sodium. Therefore the action potential in DentaZium is mainly due to calcium ions. It is not possible to decide yet whether the two components come from the same conductance, but a calcium-induced inactivation mechanism seems to be ruled out by the observation that the rate of inactivation is not dependent on the amplitude of the inward current. After fertilization the input resistance decreases sharply by about sevenfold in 40 min, before reaching the trefoil stage. At that time, action potentials can no longer be generated in the embryo. Considering the IV curves, it can be predicted that a cell will produce a regenerative response to depolarization if there exists a potential range in which the sum of the inward currents is larger than the sum of the outward currents. In the unfertilized egg, the net inward current is maximum around -10 mV and never exceeds 10 nA. In the unfertilized egg, assuming an input resistance of 15 MQ, a resting potential of -80 mV, and linear resting and leak conductances, the outward current will be 4.6 nA at -10 mV. The balance is then in favor of the inward current and the egg is excitable. After fertilization the outward current will reach 10 nA when the resistance reaches about 7 MQ. Cells with lower input resistance are not expected to be excitable anymore. We have shown that at the trefoil stage all inward current components are present, supporting the above interpretation. REFERENCES BARISH, M. E. (1984). Calcium-sensitive action potential of long duration in the fertilized egg of the Ctenophore Mnemiopsis leidyi. Dev. BioL 105,29-40. BAUD, C., and BARISH, M. E. (1984). Changes in membrane hydrogen and sodium conductances during progesterone-induced maturation of Ambystoma oocytes. Dev. Biol. 105,423-434. BAUD, C., KADO, R. T., and MARCHER, K. S. (1982). Sodium channels induced by depolarization of the Xenopus laevis oocyte. Proc. NatL Acad. Sci. USA. 79,3188-3192. CHAD, J., ECKERT, R., and EWALD, D. (1984). Kinetics of calcium-dependent inactivation of calcium currents in voltage-clamped neurones of Aplysia califokca. J. PhysioL 347, 279-300. GEORGIOIJ, P., BOUNTRA, C., BLAUD, K. P., and HOUSE, C. R. (1984). Calcium action potentials in unfertilized eggs of mice and hamsters. Q. J. Exp. Physiol. 69, 365-380. GUERRIER, P., VAN DEN BIGGELAAR, J. A. M., VAN DONGEN, C. A. M., and VERDONK, N. M. (1978). Significance of the polar lobe for the determination of dorso-ventral polarity in Dental&m vulgare (Da Costa). Dev. BioL 63,233-242. HAGIWARA, S. (1983). Membrane potential-dependent ion channels in cell membrane. Raven Press, New York. HAGIWARA, S., and JAFFE, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8,385-416. HAGIWARA, S., and MIYAZAKI, S. (1977). Changes in excitability of the cell membrane during “differentiation without cleavage” in the egg of the annelid, Chaetopterus pergamentaceus. J. Physiol. 272, 197216.
Potential
in Dentalium
Egg
521
HIRANO, T., and TAKAHASHI, K. (1984). Comparison of properties of calcium channels between the differentiated l-cell embryo and the egg cell of ascidians. J. Physiol. 347,327-344. HIRANO, T., TAKAHASHI, K., and YAMASHITA, N. (1984). Determination of excitability types in blastomeres of the cleavage-arrested but differentiated embryos of an ascidian. J. PhysioL 347,301-325. JAFFE, L. F. (1982). Developmental currents, voltage and gradients. In “Developmental Order: Its Origin and Regulation.” (S. Subtelny, Ed.), pp. 183-215. A. R. Liss, New York. JAFFE, L. A., and GUERRIER, P. (1981). Localization of electrical excitability in the early embryo of Dentalium. Dew. BioL 83,370-373. JAFFE, L. A., GOULD-SOMERO, M., and HOLLAND, L. (1979). Ionic mechanism of the fertilization potential of the marine worm Urechti caupo (Echiura). J. Gen. Physiol 73,469-492. JAFFE, L. A., KADO, R. T., and KLINE, D. (1986). A calcium-activated sodium conductance produces a long-duration action potential in the egg of a nemertean worm. J. Physiol. 381,263-278. LANSMAN, J. B. (1983). Components of the starfish fertilization potential: Role of calcium and calcium-dependent inward current. Zn “The Physiology of Excitable Cells,” (A. D. Grinnell and W. J. Moody Jr., Eds), pp. 233-246, A. R. Liss, New York. MITANI, S. (1985). The reduction of calcium current associated with early differentiation of the murine embryo. J. PhysioL 363, 71-86. MIYAZAKI, S., OHMORI, H., and SASAKI, S. (1975). Action potential and non-linear current-voltage relation in starfish oocytes. J PhysioL 246,37-54. MIYAZAKI, S., TAKAHASHI, K., and TSUDA, K. (1974). Electrical excitability in the egg cell membrane of the tunicate. J Physiol. 238,3754. MOODY, W. J., and LANSMAN, J. B. (1983). Developmental regulation of Ca and K currents during hormone-induced maturation of starfish oocytes. Proc. Natl. Acad. Sci. USA 80,3096-3100. MOREAU, M., and GUERRIER, P. (1981). Absence of regional differences in the membrane properties from the embryo of the mud snail Ilyanassa obsoleta. BioL Bull. 161, 320. OKAMOTO, H., TAKAHASHI, K., and YOSHII, M. (1976). Membrane currents of the tunicate egg under the voltage-clamp conditions. J. PhysioL 254, 607-638. OKAMOTO, H., TAKAHASHI, K., and YAMASHITA, N. (1977). Ionic currents through the membrane of the mammalian oocyte and their comparison with those in the tunicate and sea urchin. J. Physiol. 267, 465-495. RENDER, J. A., and GUERRIER, P. (1984). Size regulation and morphogenetic localization in the Dentalium polar lobe. J. Exp. ZooL 232, 79-86. SCHLICHTER, L. C. (1983). Spontaneous action potentials produced by Na and Cl channels in maturing Rana p$iens oocytes. Dew. BioL 98, 47-59. SHEN, S., and STEINHARDT, R. A. (1976). An electrophysiological study of the membrane properties of the immature and mature oocyte of the batstar Patiria miniata. Dev. Biol. 48, 148-162. SPITZER, N. C. (1979). Ion channels in development. Annu. Rev. Newosci. 2,363-397. TAKAHASHI, K., and YOSHII, M. (1981). Development of sodium, calcium and potassium channels in the cleavage arrested embryo of an ascidian. .I PhysioL 315, 515-529. THOMPSON, S., and KNIER, J. (1983). Spontaneous action potentials and resting potential shifts in fertilized eggs of the tunicate Clavelina. Dev. BioL 99, 121-131. VERDONK, N. H., and CATHER, J. N. (1983). Morphogenetic determination and differentiation. In “The Mollusca Development” (N. H. Verdonk, J. A. M. van den Biggelaar, and A. S. Tompa, Eds.), Vo13, pp. 215-252. Academic Press, New York. WILSON, E. B. (1904). Experimental studies on germinal localization. I. The germ regions in the egg of Dental&m. J. Exp. ZooL 1, l-72.