Factors influencing perpendicular elongation of embryonic frog muscle cells in a small applied electric field

In most vertebrate embryos, somites are formed by myoblasts radiating out from a central fluid-filled somatocoel, which eventually disappears. This form accounts for the familiar rosette appearance of transversely sectioned somites, for example in chick. In Xenopus laevis, however, a somite is formed when a segment of myoblasts is pinched off from the paraxial mesoderm and rotates en bloc through 90°. In doing this, the long axis of differentiated myoblasts shifts from being perpendicular to the notochord and neural tube to lying parallel to these structures (Hamilton, 1969). The mechanism(s) controlling these rotations are unclear, although contraction of myofibrils located asymmetrically within muscle cells in the ventro-cranial aspects of presumptive somites may be implicated (Burgess, 1988).

It is not known whether the initial orientation of myoblasts in vivo is subject to any environmental control. Orientation of myoblasts in culture, however, can be determined by both a diffusible chemical influence from the somites and the notochord (perhaps a catecholamine) (McCaig, 1986; McCaig, 1989a) and by a small applied electric field of a magnitude likely to exist in vivo (Hinkle et al. 1981; Hotary and Robinson, 1990). In an applied electric field of as little as 36mVmm⁻¹ (1 mV/celldiameter), myoblasts developed from spherical undifferentiated cells into elongated single muscle cells, with their long axes roughly perpendicular to the electric field vector (Hinkle et al. 1981). Myoblasts were firmly attached to the plastic substratum and neither migrated nor rotated once their axis of elongation had been established by the field. Since this original observation, a number of cell types has been shown both to assume a perpendicular form and to migrate, in most cases towards the cathode, when exposed to an applied electric field (Luther et al. 1983; Stump and Robinson, 1983; Erickson and Nuccitelli, 1984; Cooper and Keller, 1984). The mechanisms involved are unclear. One explanation proposed for the perpendicular RE-orientation seen in differentiated fish epidermal cells suggests that cells assume this orientation in order to expose their shortest dimension to the electric field. This would minimize the changes to the membrane potential across anodal- and cathodal-facing membranes (hyperpolarized and depolarized, respectively; Cooper and Keller, 1984). This hypothesis has been tested, but cannot explain oriented myoblast elongation, since this can continue once the electric field has been switched off.

A variety of pharmacological agents has been used to study the mechanisms by which a small externally applied electric field interacts with a single myoblast to induce differentiation perpendicular to the field. Perpendicular differentiation was blocked by two inhibitors of the inositol triphosphate second messenger system and by inorganic calcium channel blockers. In addition, an asymmetric distribution of organelles was induced, which may play a role in determining the oriented elongation of myoblasts in an applied electric field. Part of this work has been published in preliminary form (McCaig and Dover, 1989).

Xenopus laevis embryos at stages 19/20 were used for all experiments (Nieuwkoop and Faber, 1956). At this stage, the first six or seven somite pairs containing elongated myoblasts have segmented but usually lack innervation (Kullberg et al. 1977). Each somite comprises three different tissue types: myotome, dermatome and sclerotome. Differentiation and elongation of myoblasts to form somites begins around stage 18 (Hamilton, 1969). Those cells that differentiate in cultures of somites from stage 19/20 embryos are almost exclusively myoblasts. In vivo dermatome and sclerotome remain undifferentiated for at least a further 10–12 h. Very few fibroblasts develop in these cultures.

The dorsal third of the embryo was excised and soaked in collagenase for 10–15 min (1 mg ml⁻¹ Sigma type 1, in Steinberg’s solution, composition: 58 mM NaCl, 0.67 mM KC1, 0.44 mM Ca(NO₃)₂, 1.3 mM MgSO₄, 4.6 mM Tris-HCl, pH 7.9). Tissue comprising two to five somites was dissected free from each embryo and pipetted into a dissociating medium for 20–30 min (divalent ion-free Steinberg’s solution plus 0.4 mM EDTA). Flamedrawn Pasteur pipettes were used to pick up and disperse dissociated myoblasts into normal culture medium, present in the trough of a chamber constructed on the base of a tissue culture plastic dish (Falcon type 3003). Culture medium was Steinberg’s solution supplemented with 20 % Liebowitz L 15 solution, 1 % foetal bovine serum and 2% penicillin/streptomycin (5000i.u.ml⁻¹/5000; μgml⁻¹; Flow Laboratories, Irvine, Scotland). The total ionic strength of the culture fluid was 81 mM and the pH 7.9. Culture chambers were constructed by glueing two parallel strips of no. 1 coverglass (64 minx 11 mm) 1cm apart on the untreated surface of a tissue culture dish using silicone rubber. Cells were allowed 15–20 min to adhere to the dish before a roof of no. 1 coverglass was added and sealed to the side strips with silicon grease. This completed the culture chamber through which drug-containing medium could be perfused and/or constant current passed. Chamber dimensions were 64mm× 10mm×0.5mm.

Electric fields were applied using a pair of Agar-Steinberg’s salt bridges (15 cm long), which connected Ag/AgCl electrodes in beakers of Steinberg’s solution to the ends of the culture chambers. Since electric fields were applied for a limited time period (maximum of 18 h), these bridges were more than sufficient to prevent diffusion of electrode products into the cultures. Field strength was measured directly at the end of each experiment by bridging the culture chamber with Ag/AgCl electrodes at either end, connected to a voltmeter. A constant current source was used and the current supplied to the chambers was monitored periodically throughout the experiment.

The earliest myoblasts to differentiate in culture begin to elongate within 1-2 h of plating out, with additional numbers differentiating over a period of several hours. Testing the effects of pharmacological agents on the direction of cell elongation in an electric field required exchanging the culture medium as soon as the cells had attached and the chamber lid had been applied. Normal culture medium was replaced by perfusion of an excess of drug-containing medium through the chamber using a push-pull technique and hand-held Pasteur pipettes. All drugs were from Sigma with the exception of: the lipophilic fluorescent dye Di I, from Molecular Probes Inc.; phosphonovaleric acid (APV), a gift from Dr S. N. Davies; and Bay K8644, a gift from Bayer. Stock solutions of nifedipine, Bay K8644 and Di I were made in absolute ethanol; nocodazole was dissolved in dimethyl sulphoxide (DMSO). All other stocks were prepared in culture medium. Experiments with nifedipine and Bay K8644 were conducted in the dark.

The axis of myoblast elongation relative to the electric field vector was assessed as follows. Culture chambers were aligned such that their long edge, and thus the electric field vector, lay parallel to the photographic viewfinder of the microscope. The visual field was divided into quadrants by two lines lying at 45°, respectively, to the horizontal and the vertical. The long axis of elongation of each myoblast was assessed as forming an angle >45° or <45° to the horizontal electric field vector. An orientation index was calculated by dividing the difference between these values by their sum. If all cells were ‘perpendicular’ to the field, this ratio would be unity. If as many cells were perpendicular as were parallel, the ratio would be zero. Orientation indices of 0.2–0.25 (or higher) generally are derived from data that give clear statistical differences if paired t-tests are used on the raw data. Values of this order are taken to represent significant levels of orientation.

In some experiments, the effect of a unilateral source of the calcium channel agonists Bay K8644 (Bayer) on the direction of myoblast orientation was tested. One half of the culture chamber was filled with a thin layer of 1.3% agar made up with drugcontaining culture medium. Culture medium was plated into the other half of the chamber and contacted the agar slab. One hour later dispersed myoblasts were plated out in a column about 1 mm away from the agar-culture medium interface. A coverglass roof was applied, making the final volume of the chambers around 300 /d. Slow release from the agar slabs was checked by adding a few drops of ink to a test culture medium and preparing agar slabs with this. After 1 h a front of dye had extended about 1 mm into the culture medium. After 17 h this had advanced 9–10 mm from the agar-culture medium interface and the intensity of dye diminished with distance from the agar slab. Gradients of diffusible substances therefore are set up within the time course of these experiments and myoblasts plated 1 mm from the agar-culture fluid interface will differentiate and elongate while exposed to such gradients. A similar experimental design has shown that an agar source of β-nerve growth factor establishes a gradient within 24 h such that only 10% of the source concentration is present at a distance of 10 mm (Letourneau, 1978).

Rhodamine-phalloidin was used to stain selectively for F-actin. Myoblasts were fixed and stained simultaneously with a culture medium containing: rhodamine-phalloidin, 5 units ml⁻¹ (Molecular Probes Inc.), palmitoyl 50μgml⁻¹ (Sigma) and 3.7% formaldehyde. Staining was for 20 min at 4°C. Chambers were rinsed thoroughly with culture medium followed by glycerol and phosphate-buffered saline in a 1:1 ratio. The intensity of fluorescent staining on either side of myoblasts was assessed subjectively on coded culture dishes and the electric field orientation was determined after this assessment.

Myoblast cultures were fixed with 2% glutaraldehyde (2 h), then in 0.2% OsO₄ in phosphate buffer for 20 min at 4 °C. Standard dehydration in ethanol and embedding in Epon 812 resin followed. Plastic-embedded cultures were re-mounted and myoblasts sectioned longitudinally, parallel to the culture surface. In re-mounting the tissue, cell orientation with respect to the electric field vector was maintained. The distribution of organelles was assessed subjectively on either side of a midline bisecting the myoblasts.

When somites are removed from stage 19/20 embryos, many of the constituent myoblasts are single, elongated, unfused cells, which span the rostrocaudal length of the somite. Myoblasts therefore round up during dissociation and dispersal, since they are all spherical myoballs when first plated out. About 50% remain spherical even after many hours in culture. This does not indicate a failure to develop, since myofilament bundling and the appearance of Z-lines continue apparently on schedule in these cells (Dover and McCaig, unpublished). Muscle cells elongating in culture are therefore likely to be a mixture of cells expressing this behaviour de novo and those recapitulating their in vivo development.

In untreated cultures of myoblasts the orientation of cell elongation was random, with equivalent numbers of cells at angles >45° and <45° to the long axis of the growth chambers (Fig. 1; Table 1). There is nothing therefore about the design of the chambers per se that can induce an orienting influence on elongating myoblasts.

When exchanging media for drug solutions, five times more fluid was perfused through a chamber than its volume would hold. This ensured complete fluid exchange and homogeneous chamber contents. As for controls (no drug), there was no external orienting influence, therefore randomly directed elongation of muscle cells would be expected in all drug-containing media. Table 1 shows that this was the case for each of the drug-containing media tested alone (no electric field). Variations in the absolute numbers of elongating myoblasts per culture dish probably arose from differences in culture age at the time of assessment (5–18 h), slight differences in the amount of somitic tissue transferred to each chamber and the possibility that cell adhesion was influenced by drug treatment.

As reported previously (Hinkle et al. 1981), myoblasts showed a clear tendency to orient perpendicular to a continuous, small applied electric field (Fig. 2; Table 2). Roughly three times as many muscle cells elongated ‘perpendicular’ to the field as elongated ‘parallel’ to the field.

One of the actions of the aminoglycoside antibiotics neomycin and gentamycin is to bind avidly to polyphosphoinositides, making them unavailable to phospholipase C, and thus blocking this second messenger pathway prior to the formation of diacyl glycerol and inositol triphosphate (Lipsky and Leitman, 1982; Schwartz et al. 1984). Myoblasts continuously exposed to an applied electric field and concentrations of neomycin known to be inhibitory (for example, in PC 12 cells; 0.5 mM and 1 HIM; Paves et al. 1990), elongated randomly, showing no tendency towards perpendicular orientation. Lowering the concentration of neomycin to 0.1 mM was sufficient to permit expression of perpendicular elongation (Table 2; Fig. 3). Gentamycin also completely inhibited perpendicular orientation (Table 2). In addition to blocking this second messenger system, neomycin can compete with calcium for entry into presynaptic terminals and thus inhibit transmitter release. Raised external calcium is used to prevent this action (Fiekers, 1983) and with a 16fold increase of [Ca²⁺]₀ to 8mM, galvanic orientation of myoblasts still occurred (Table 2).

Lithium inhibits the synthesis of inositol from phosphoinositol and has been used to block this second messenger system (Berridge and Irvine, 1984). Exposure of myoblasts to lithium chloride (10 mM), however, did not prevent perpendicular orientation (Table 2). Galvanic orientation persisted also in the presence of the G protein inhibitor pertussis toxin and during muscarinic or nicotinic receptor blockade (Table 2).

Two types of experiment addressed the question of whether cells needed the continuous presence of a d.c. field during elongation in order to express oriented growth. Both involved field application before elongation had occurred, but no field while elongation was taking place. First, since the earliest muscle growth begins about 1 h after plating, this time window was used to give cells a brief exposure (between 5 and 15 min) to the field, which was then switched off before any cells had elongated.

Second, muscle cells do not elongate in 1μgml⁻¹ of the microfilament inhibitor cytochalasin D, but readily begin elongation when this is washed out. Myoblasts therefore were exposed simultaneously to 1μgml⁻¹ cytochalasin D and an applied electric field for 4 h. The drug was washed out with normal culture medium and the field switched off. Muscle cell elongation began about 1 h later. In both experiments, muscle elongation was biased towards the perpendicular, although the degree of orientation was smaller than when a field was present continuously (Figs 4 and 5; Table 2).

A possible role for an asymmetric distribution of integral membrane proteins in determining cell orientation arises from the observation that the plant lectin concanavalin A blocks both field-induced receptor asymmetry and nerve orientation in an applied electric field (Patel and Poo, 1982; McCaig, 1989b). Accordingly, several substances that perturb the distribution of integral membrane proteins in field-exposed myoblasts were tested for any influence on the direction of cell elongation.

Neither the lectin concanavalin A (Con A) nor the enzyme neuraminidase interfered with the normal perpendicular elongation in an applied electric field. The degree of orientation was quantitatively similar to that seen in an electric field alone (Table 2). The fluorescent, lipophilic carbocyanine dye Di I, however, totally inhibited oriented muscle cell elongation. The orientation index was no different from zero, indicating a random direction of growth (Table 2; Fig. 6). Stock solutions of Di I were made with ethanol, giving a final concentration of ethanol in culture medium of 1%. Vehicle alone, however, did not inhibit myoblast orientation, indicating a direct effect of the Di I per se. Indeed, myoblast orientation in 1 % ethanol culture medium was even more marked than in the electric field alone (Table 2).

A role for calcium in myoblast orientation. The orientation of nerves both to nerve growth factor and to an applied electric field may involve asymmetric calcium entry at the growth cone (Gundersen and Barrett, 1980; McCaig, 19896). Several methods of perturbing calcium homeostasis were tested for an effect on myoblast orientation. Diltiazem and nifedipine, organic blockers of calcium entry through dihydropyridine-sensitive L-type calcium channels, did not disrupt perpendicular orientation at concentrations known to block calcium entry in other cell types. Nor, in a single experiment, did the NMDA receptor antagonist APV, which blocks a specific class of receptor-activated, voltage-sensitive calcium channels, for example in hippocampal neurones (Collingridge and Bliss, 1987). Perpendicular development of myoblasts was inhibited totally by cobalt and less completely by lanthanum, inorganic blockers of calcium entry through N and T tvpe calcium channels (Table 2; Fig. 7).

A further indication of the involvement of calcium in myoblast orientation comes from experiments aimed at elevating calcium levels asymmetrically in developing myoblasts. Cells elongated predominantly perpendicular to an imposed gradient of the calcium channel agonist Bay K8644 (Table 2; Fig. 8).

In the presence of the microtubule inhibitor nocadazole, muscle cells retained an ability to express a The asymmetry index was derived by subtracting those with cathodal asymmetry from those with anodal asymmetry and dividing by the total number assessed.

bipolar axis that was oriented by an applied electric field. As outlined above, l μgml^–1 of cytochalasin completely inhibited muscle cell elongation; lower concentrations, however, permitted growth. Perpendicular elongation of myoblasts was evident in 0.1 μgml^–1 and 0.2 μgml^–1 cytochalasin but, interestingly, random direction of growth occurred in 0.5 μgml^–1 (Table 2; Fig. 9).

Control myoblasts were stained uniformly with rhodamine-phalloidin. In an applied electric field, however, staining of most cells was asymmetric, with the anodalfacing half of cells staining more intensely (Table 3; Fig. 10). Such a response was evident in spherical cells around the time that the earliest myoblasts were elongating.

Myoblasts treated with cytochalasin D (0.2 μg ml^–1) and an electric field showed punctate fluorescence that also was more prominent at the anodal-facing membrane (Fig. 11).

Ultrastructural analysis also revealed organelle asymmetries within elongated myoblasts. The most consistently striking was an accumulation of polyribosomes running as a band subjacent to the plasma membrane in the anodal-facing half of myoblasts after 18 h in culture (Fig. 12). No such obvious asymmetry was seen in spherical myoblasts cultured for only Ih in an electric field. No attempt was made to quantify this, but a striking asymmetry was evident in 10/18 myoblasts (round and elongated) examined after 18 h in an applied field (100 – 150 mV mm^–1).

In addition there was a noticeable shift of yolk granules towards the anodal side of myoblasts. Control cells had equal numbers of yolk granules in either half of the cell (8.5±1.7, left half; 8.1 ± 1.1, right half). By contrast, there were 40 % more yolk granules in anodal-facing cell halves, compared with the number seen on the cathodal sides (anodal, 8.3±1.7; cathodal, 5.9±1.4; P<0.02).

Neomycin and gentamycin bind to polyphosphoinositides and prevent their breakdown by phospholipase C, thus blocking the inositol phospholipid second messenger system (Lipsky and Leitman, 1982; Schwartz et al. 1984). Both substances abolished perpendicular elongation; neomycin did so in a dose-dependent manner. Another action of these positively charged aminoglycoside antibiotics is to competitively prevent calcium entry into cells. In presynaptic nerve terminals, for example, adding excess external calcium completely blocks their effect (Fiekers, 1983). However, since neomycin still blocked perpendicular orientation of myoblasts even with a 16-fold increase of external calcium, it seems more likely that the second messenger blockade may be the critical action in this case.

Lithium also may block this second messenger system, by inhibiting the phosphatase that regenerates inositol from InsP (Fisher and Agranoff, 1987). However, lithium did not affect polarised myoblast elongation, perhaps because muscle cells contain ample stores of PtdIns(4,5)P₂ from which to generate InsP₃ and DAG (diacylglycerol) and do not require the cyclic regeneration of the phospholipids.

Calcium entry is essential for field-induced perpendicular reorientation of mouse embryo fibroblasts (Onuma and Hui, 1988) and may regulate the orientation of nerves both to a source of nerve growth factor and to an electric field (Gundersen and Barrett, 1980; McCaig, 19896). In myoblasts, the inorganic calcium channel blockers cobalt and, to a less complete extent, lanthanum inhibited perpendicular elongation, indicating the involvement of T, N and L-type voltage-sensitive calcium channels (Miller, 1987). Blocking only the L-type channels with diltiazem or nifedipine did not inhibit oriented growth, presumably because calcium could still enter through T and N-type channels. The ability to mimic the electric field induced perpendicular elongation using a unilateral source of the dihydropyridine agonist Bay K8644, which opens L-type channels, implies that asymmetrically elevated cytoplasmic calcium may underlie oriented elongation. However, blocking NMDA (N-methyl-D-aspartate type glutamate receptor)-activated calcium channels with APV did not inhibit orientation.

Perpendicular elongation of myoblasts occurred with the microtubule inhibitor nocadazole and an applied field. Alignment of microtubule polymerization therefore cannot direct this response. By contrast, directed microfilament polymerization may be critical. Cells elongated with random orientation in 0.5 μgml⁻¹ of the microfilament inhibitor cytochalasin D (CD). Higher or lower concentrations permitted no elongation, or oriented elongation, respectively. Perhaps a critical, limited amount of F-actin polymerization is necessary for orientation.

Two effects of an externally applied electric field are the redistribution of integral membrane proteins, and depolarization and hyperpolarization of cathodal- and modal-facing membranes, respectively. The voltage drop across the outside of a cell in an electric field will be 10³ to 10⁶ times that across the cell cytoplasm, because of the high resistance of the cell membrane (Jaffe and Nuccitelli, 1977). Charged integral membrane proteins move in response to external fields as small as 1 mV/cell diameter (Jaffe, 1977; Poo and Robinson, 1977). The side of the cell that accumulates integral proteins depends on the proteins involved, their electrostatic properties and the net charge on the cell surface (McLaughlin and Poo, 1981). Receptors for the plant lectin Con A, for the neurotransmitter acetylcholine (ACh), and for epidermal growth factor, despite bearing a net negative charge, accumulate asymmetrically at the cathodal edge of Xenopus myoblasts (Poo and Robinson, 1977; Orida and Poo, 1978; Stollberg and Fraser, 1988, 1990; Giugni et al. 1987). Indeed, cathodal accumulation of ACh receptors continues when the field is removed, due to the activation of a diffusiontrap mechanism (Stollberg and Fraser, 1988, 1990). This may apply also to receptors for molecules other than ACh and for specific anchoring proteins on the inner aspect of the muscle cell membrane (Rochlin and Peng, 1989). This type of irreversible receptor and submembranous protein clustering phenomenon may underlie the ability of myoblasts to ‘remember’ exposure to an electric field and respond galvanotropically when the field is no longer present. Brief exposure to a field before elongation (5 – 15 min) and field exposure during pharmacological arrest of growth both resulted in perpendicular elongation, once the field was turned off.

Several drugs perturb surface receptor movements in an applied electric field. Pretreatment with Con A before field application immobilizes receptors by crosslinking and prevents receptor redistribution; it also stops cathodally oriented nerve growth (Patel and Poo, 1982; McCaig, 19896). Muscle cells treated with Con A, however, elongated perpendicular to an applied field. Redistribution of Con A receptors is therefore unlikely to be essential for myoblast orientation.

Con A receptors accumulate anodally when cell surface negativity is reduced pharmacologically. Both neuraminidase, which removes sialic acid residues from the cell surface, and the positively charged lipophilic fluorescent dye Di I reversed the direction of electromigration of Con A receptors on myoblasts (McLaughlin and Poo, 1981). Nevertheless, myoblasts in neuraminidase elongated perpendicularly in an applied field. By contrast, Di I totally blocked oriented elongation. One mechanism for perpendicular elongation involving membrane receptor asymmetry is outlined below. In this scheme, receptors may accumulate cathodally or anodally; therefore, neither drug would be expected to abolish perpendicular orientation. Di I, however, has a conjugated bridge structure that lies parallel to the phospholipid bilayer surface, while its hydrocarbon tails are incorporated into the membrane to lie parallel to those of the bilayer (Axelrod, 1979). This conformation may interfere with the membrane-associated enzymic events of the inositol triphosphate pathway and thus inhibit perpendicular orientation.

Thus, oriented myoblast elongation requires a field-induced mechanism, which may activate the inositol phosphate second messenger system, depends both on calciurfi entry and on microfilament polymerization and may involve redistribution of a number of cell surface receptor types. One scenario could involve the cathodal accumulation of voltage-gated calcium channels (VGCC). Many receptor types redistribute to the cathodal-facing cell surface (Poo and Robinson, 1977; Orida and Poo, 1978; Stollberg and Fraser, 1988, 1990), whilst L-type calcium channel hotspots locally raise cytoplasmic calcium concentration in neuronal growth cones and promote local membrane expansion (Silver et al. 1990). Increased Ca²⁺ influx would occur both through the increased number of channels and because the cathodal-facing membrane will be depolarized (Cooper and Keller, 1984), thus opening VGCCs. Locally elevated cell calcium may promote cell adhesion (Schubert et al. 1978) predominantly along the cathodal side of round myoblasts. Increased cell adhesion running perpendicularly to the electric field (along the cathodal-facing side) would create tensions within the cytoplasm and promote microfilament polymerization along the lines of tension (Kolega, 1986). The end result would be elongation of myoblasts perpendicular to the electric field. (Weakened cell adhesions parallel to an applied electric field have been proposed recently to explain perpendicular reorientation of spread chick fibroblasts (Harris et al. 1990).)

A predominant role for the cathodal side of the cell is proposed, both because observations of integral membrane protein displacements mostly report cathodal accumulation and because VGCCs would open on the depolarized cathodal side. However, in mouse macrophages, receptors for the lectin from Phaseolus vulgaris accumulate anodally (Orida and Feldman, 1982). A subpopulation of anodally accumulating receptors tethered to the cortical cytoskeleton might explain the anodal accumulation of Factin staining seen in Xenopus myoblasts. Consequently, an asymmetric cytoskeletal meshwork may physically impose asymmetry on other cell organelles: ribosomes and yolk granules; both predominated anodally. The implications for cellular metabolism of organelle asymmetries are unclear.

Inositol phospholipid second messenger involvement in galvanic orientation may arise because (asymmetric) calcium entry through VGCCs can activate phospholipase C (Eberhard and Holz, 1988). This produces inositol triphosphate, which further raises calcium by release from intracellular stores. Calcium entry through ligand-gated channels also may activate this system (Eberhard and Holz, 1988). The inositol phosphate system with calcium may regulate the activities of the actin-binding proteins profilin and gelsolin, with elevated PtdIns(4,5)P₂ promoting actin polymerization locally. PtdIns(4,5)P₂ promotes the dissociation of profilactin complexes at the cytoplasmic membrane surface, thus releasing monomeric actin for local polymerization (Lassing and Lindberg, 1985), whilst gelsolin severs actin filaments in high calcium media before PtdIns(4,5)P₂-promoted assembly occurs at the newly exposed barbed ends of filaments (Janmey and Stossel, 1987; see also Forscher, 1989).

Rat PC12 cells and rabbit neutrophils, respectively, respond to nerve growth factor (NGF) and to a chemotactic factor (fMet-Leu-Phe) with rapid polymerization of microfilaments. Both responses depend on the inositol triphosphate system (Paves et al. 1990; Sha’afi et al. 1986). Moreover, both substances can induce directed growth (Gundersen and Barrett, 1980; Zigmond, 1977). Since galvanically oriented myoblast growth also may require the inositol phosphate system, perhaps a unitary pathway transduces chemical and electrical gradients into directed growth. Moreover, electric field activation of a single receptor type, coupled to phospholipase C, may be sufficient to initiate oriented growth. However, since neither nicotinic or muscarinic antagonists, nor the G-protein inhibitor pertussis toxin prevented galvanic orientation of myoblasts, cholinergic receptors and G-protein-linked receptors are unlikely to be involved.

Could electrically directed perpendicular cell growth occur in vivo? Currents around 1 – 10 μ A cm^{– 2} enter across the skin of the early Xenopus embryo and leave through the blastopore (Robinson and Stump, 1984). Thus an electrical field perpendicular to elongating myoblasts may exist in differentiating mesoderm, and could initiate oriented cell elongation. A lower limit estimation of field strength within the Xenopus embryo has been calculated (0.18mVmm^–1) using a current density of 1 μAcm^–2 and a resistivity of 1800 Qcm, that of muscle (Robinson and Stump, 1984). This is two orders of magnitude lower than the threshold for myoblast orientation in vitro (36 mV mm^–1; Hinkle et al. 1981) and may raise doubts about a physiological role for endogenous electric fields in orienting myoblast elongation. However, other cell types (quail fibroblasts and chick neural crest cells), which both assume a perpendicular orientation and migrate cathodally in applied fields, experience endogenous fields that are more than sufficient to control the directions of cell orientation and migration (Erickson and Nuccitelli, 1984; Hotary and Robinson, 1990). Also, mouse cerebellar neurones reorient and migrate perpendicularly on cultured parallel bundles of granule cell neurites (Hekmat et al. 1989; Nakatsuji and Nagata, 1989). This has been described as a novel form of contact guidance. It would be interesting to know whether this behaviour persists in the presence of action potential blockers, in other words in the absence of local external electric fields!

We thank the Wellcome Trust and Action Research for the Crippled Child for their financial support.