Presumptive Muscle Research Articles

Anabolic steroid treatment increases myofiber damage in mdx mouse muscular dystrophy

In order to study whether myofiber size is an important determinant of the severity of dystrophic injury, mdx and control mice were treated with an anabolic steroid, nandrolone decanoate, for 3 weeks. Treatment resulted in a population of significantly smaller fibers in both strains, and was accompanied by an increase in the proportionate area or the number of foci of dystrophic injury in mdx soleus (slow-twitch) or tibialis anterior plus extensor digitorum longus (fast-twitch) muscles, respectively. As well, serum creatine kinase activity was increased in steroid-treated mdx mice. Fiber centronucleation, an index of accumulated injury and repair, in steroid-treated mdx soleus was doubled compared to that observed in soleus muscles from untreated mdx mice. There was no change in the distribution of immunoreactive basic fibroblast growth factor, important in muscle cell proliferation, with the increased damage from treatment. However, presumptive muscle precursor cells (identified by immunoperoxidase histochemistry for neural cell adhesion molecule), appeared to be more abundant in foci of very recent fiber damage in muscles from steroid-treated than untreated mdx mice. Results show that mdx dystrophy is worsened by anabolic steroid treatment, possibly by altered influences on muscle use patterns and muscle precursor fusion, and is not accompanied by an increase in fiber size.

Immunocytochemical investigations of muscle differentiation in the Atlantic herring (Clupea harengus: Teleostei)

The myotomes in yolk-sac larvae of the Atlantic herring (Clupea harengus: Teleostei) contain a single layer of small-diameter superficial muscle fibres surrounding an inner mass of around 280 larger-diameter muscle fibres. The fraction of muscle fibre volume occupied by mitochondria is dependent on temperature, and in larvae reared at 8°C was 41% for the superficial fibres, and 25% for the inner muscle fibres. The inner muscle fibres of larvae share some myofibrillar proteins with adult white muscle, but contain unique isoforms of myosin heavy chains, troponin T, troponin I and myosin light chain 2. A monoclonal antibody has been produced which is specific to myosin light chain 3 (MLC3). Immunocytochemical studies have shown that the expression of MLC3 is switched off in the superficial muscle fibres at the start of metamorphosis when larvae reach 28–30 mm total length (TL). Metamorphosis to the juvenile stage is complete in fish 35–40 mm TL and is also associated with the development of gill filaments and the production of presumptive slow muscle fibres which form externally to the larval superficial muscle fibres in the region of the lateral line nerve.

Role of cell interactions in ascidian muscle and pigment cell specification.

Muscle and brain pigment cell specification was studied by disrupting cell adhesion, cell dissociation, and reaggregation in embryos of the ascidianStyela clava. Treatment of embryos with Ca2+-free sea water between the 2-cell and gastrula stages disrupted blastomere adhesion but did not prevent acetylcholinesterase or muscle actin expression in presumptive muscle cells. Similar treatments initiated between the 2- and 32-cell stages caused more ectoderm cells to express tyrosinase and develop pigment granules than expected from the cell lineage. Whereas 2 pigment cells become the otolith and ocellus sensory organs in normal embryos, up to 33 pigment cells could differentiate in embryos after disruption of cell adhesion. Replacement of Ca2+-free sea water with normal sea water restored cell adhesion and usually resulted in development of embryos containing the conventional number of pigment cells. Dissociation of embryos into single cells between the 2- and 64-cell stages and culture of these cells beyond the fate restricted stage had no effect on the accumulation of muscle actin mRNA and muscle actin synthesis, but blocked pigment cell differentiation. Reaggregation of the dissociated cells did not enhance the number of cells that developed muscle features, but rescued pigment cell development. The results indicate that ascidian muscle cell specification occurs by an autonomous mechanism, whereas pigment cell specification occurs by a conditional mechanism involving cell interactions. In addition, the results suggest that negative cell interactions may restrict the potential for pigment cell development in the ectoderm of cleaving ascidian embryos.

Basement membrane lectin binding sites are decreased in the esophageal endoderm during the arrival of presumptive muscle mesenchyme in the developing asteroid Pisaster ochraceus.

Basement membranes (BMs) of vertebrates and invertebrates have been shown to contain glycoproteins and proteoglycans, which include oligosaccharides and glycosaminoglycans. Lectin binding sites were characterized in the BM of gastrulating embryos of the starfish, Pisaster ochraceus. In early and mid-gastrulae, the fluorescein isothiocyanate (FITC)-lectin conjugates of concanavalin A (Con A) and wheat germ agglutinin (WGA) reveal the presence of mannose/glucose and glucosamine/sialic acid residues in the BM of all regions of the embryos. However, in the late gastrula embryo, an apparent reduction of these components is observed over the esophageal BM. Ultrastructural studies using the lectin-gold conjugates Con A, Limax flavus agglutinin (LFA), specific for sialic acid, and Dolichos biflorus agglutinin (DBA), specific for galactosamine, demonstrate that most mannose/glucose and galactosamine-containing residues lie in the lamina densa, whereas most sialic acid residues are located over the lamina lucida. In addition, a statistical analysis of lectin binding in the late gastrula embryo reveals that the amount of labelling with both Con A and LFA is significantly reduced in the esophageal region, suggesting that mannose/glucose and sialic acid residues are reduced in this region. These results confirm the observations of the FITC-lectin studies described above. They also confirm earlier studies that demonstrated a difference in BM morphology of the esophageal region (Crawford, '88). Mesenchyme cells, some of which arise from the forming coeloms (Crawford, '90), and which may represent a distinct population, colonize exclusively on this esophageal BM, where they later differentiate into muscle. Quantitative differences in BM glycoconjugates may act to direct the presumptive muscle cells to the region of the esophagus.

Widespread expression of MyoD genes in Xenopus embryos is amplified in presumptive muscle as a delayed response to mesoderm induction.

The MyoD gene codes for an important regulatory factor in skeletal myogenesis. To explore the relationship between mesoderm induction in Xenopus embryos and expression of MyoD, I have monitored MyoD mRNA levels in normal embryos and cultured explants by RNase protection. Transcription from the two Xenopus MyoD gene copies is activated weakly across the whole embryo at the midblastula transition, and this activation occurs in the absence of mesoderm induction. In response to induction this basal expression is amplified 50- to 100-fold, but in animal-pole explants 6-10 hr elapse before induced mRNAs appear, and this induction requires prior protein synthesis. The promiscuous transcripts disappear from animal explants at a time when induction "competence" is lost, suggesting a link between these events. The data highlight a broad, but transient, permissiveness for MyoD expression in embryos, which is propagated and amplified only in presumptive muscle in response to induction. Moreover, muscle-specific MyoD expression is a relatively late (postgastrulation) event in the mesoderm-induction cascade.

Development of hindlimb muscle spindles in the marsupial Macropus eugenii (tammar wallaby)

The development of muscle spindles was studied in the hindlimb of pouch young of the marsupial Macropus eugenii (tammar wallaby) from birth to 100 days. Primary myogenesis was evident in hindlimb muscles at birth. Presumptive muscle spindles were identified as early as day 30 postnatally. The intrafusal muscle fibres developed sequentially, to form the full juvenile complement by day 50, followed by development of the periaxial space. The formation of muscle spindles in the marsupial's hindlimb after birth was in contrast to the mainly prenatal development of spindles noted in placental mammals.

Correlated muscle and nerve development in the bullfrog cutaneous pectoris

The development of the cutaneous pectoris muscle was studied and compared with the differentiation of its peripheral nerve in bullfrog ( Rana catesbeiana) tadpoles and frogs by light and electron microscopic techniques. This muscle preparation was chosen for this study because it possesses a number of advantages for (and has become a model system for) the study of correlated nerve-muscle development. At the earliest stage examined (stage XI) the presumptive muscle did not contain any contractile or morphologically distinguishable myotubes, but was contacted by the well-defined cutaneous pectoris nerve trunk. Myotubes were present at stage XII, the same time that nerve-associated acetylcholine receptor aggregations and nerve-evoked muscle contractions were first observed. The adult number of axons was present in the cutaneous pectoris nerve at stage XII, but no axons were myelinated. Gradually thereafter, the number of muscle fibers increased and the cutaneous pectoris axons became myelinated. By stages XX and XXI, but prior to metamorphic climax (stage XXV), the adult numbers of cutaneous pectoris muscle fibers and myelinated and unmyelinated nerve fibers were present. These numbers did not change significantly between stages XX and XXI, through metamorphosis, and in the adult, even during the period of the most rapid loss of multiple innervation in the first 2 weeks after metamorphosis. These results show that the nerve was present and in contact with the cutaneous pectoris muscle from the earliest stages of development prior to muscle differentiation, at a time when the muscle was a disorganized mass of undifferentiated cells. Such early contact suggests that the nerve may have a significant influence on muscle maturation.

The specificity of motor innervation of the chick wing does not depend upon the segmental origin of muscles.

In vertebrate embryos, motor axons originating from a particular craniocaudal position in the neural tube innervate limb muscles derived from myoblasts of the same segmental level. We have investigated whether this relationship is important for the formation of specific nerve-muscle connections, by altering the segmental origin of muscles and examining their resulting innervation. First, by grafting quail wing somites to a new craniocaudal position opposite the chick wing, we established that the segmental origin of a muscle can be altered: presumptive muscle cells migrated according to their new, rather than their original, somitic level, colonizing a different subset of muscles. However, after reversal of a length of brachial somitic mesoderm along the craniocaudal axis, or exchange or shift of brachial somites, the craniocaudal position of wing muscle motoneurone pools within the spinal cord was undisturbed, despite the new segmental origin of the muscles themselves. While not excluding the possibility that muscles and their motor nerves are labelled segmentally, we conclude that specific motor axon guidance in the wing does not depend upon the existence of such labels.

Autonomous fluorescence of eggs of the ascidian Ciona intestinalis

AbstractUnder ultraviolet illumination, eggs of the ascidians Ciona intestinalis and C. savignyi were found to emit specific autonomous fluorescence in the myoplasmic region of egg cytoplasm, the vacuoles of test cells, and the follicular vesicles of follicle cells. The fluroescent egg cytoplasm formed a crescent after ooplasmic segregation and was distributed mainly to presumptive muscle cells. The fluorescence of the test cells appeared at early stages of oogenesis and remained constant during oogenesis and embryogenesis. The number of test cells within the perivitelline space of Ciona unfertilized eggs was estimated to be 1,154 ± 182 (S.D.); at the tailbud stage the number was 1,089 ± 149 (S.D.). These counts suggest that there is no proliferation of test cells from fertilization to at least the tailbud stage. The fluorescence of follicle cells also appeared at early stages of oogenesis and did not change during embryogenesis. The number of follicle cells attached to the chorions of eggs was estimated to be 63 ± 8 (S.D.).

The differentiation of normal and muscle-free distal chick limb bud mesenchyme in micromass culture

Distal chick wing bud mesenchyme from stages 19 to 27 embryos has been grown in micromass culture. The behavior of cultures comprising mesenchyme located within 350 μm of the apical ectodermal ridge (distal zone mesenchyme) was compared to that of cultures of the immediately proximal mesenchyme (subdistal zone cultures). In cultures of the distal mesenchyme from stages 21–24 limbs, all of the cells stained immunocytochemically for type II collagen within 3 days, indicating ubiquitous chondrogenic differentiation. At stage 19 and 20, this behavior was only observed in cultures of the distal most 50–100 μm of the limb bud mesenchyme. Between stages 25 and 27, distal zone cultures failed to become entirely chondrogenic. At all stages, subdistal zone cultures always contained substantial areas of nonchondrogenic cells. The different behavior observed between distal zone and corresponding subdistal zone cultures appears to be a consequence of the presence of somite-derived presumptive muscle cells in the latter, since no such difference was observed in analagous cultures prepared from muscle-free wing buds. The high capacity of the distal zone for cartilage differentiation supports a view of pattern formation in which inhibition of cartilage is an important component. However, its consistent behavior in vitro indicates that micromass cultures do not reflect the in vivo differences between the distal zones at different stages. The subdistal region retains a high capacity of cartilage differentiation and the observed behavior in micromass rellects interactions with a different cell population.

An ultrastructural analysis of coelomogenesis in the hoplonemertine Prosorhochmus americanus and the polychaete Magelona sp.

Formation of lateral vessels in the esophageal region of Prosorhochmus americanus embryos and coelomogenesis in the pygidial region of larval Magelona sp. are examined and compared. Earliest vessel rudiments of P. americanus are composed of a compact band of mesodermal cells (mesodermal band), lying on a layer of extracellular matrix (ECM) and lacking intercellular junctions. Rudiments are surrounded by presumptive muscle cells. Rudiments at later stages of differentiation possess lumina of differing sizes formed by a separation of apposing cell apices (schizocoely). Aohagrens junctions are apparent between lining cels of vessels following cavitation, and overlying muscle cells exhibit many myofilaments. Mesodermal bands of the recognized coelomate, Magelona sp. consist of glycogen-rich, mesodermal cells resting on ECM and joined by adhaerens junctions. Some of the cells possess a rudimentary cilium. Coelom formation occurs as a splitting of the cell band as is the case for P. americanus. Recognition of an accepted mode of coelomogenesis in P. americanus, correlated with morphological details of adult nemertine vessels, affirms the view that nemertine vessels are coelomic homologues.

Histospecific acetylcholinesterase development in the presumptive muscle cells isolated from 16-cell-stage ascidian embryos with respect to the number of DNA replications

The presumptive muscle cells (B5.1 blastomeres) were isolated from 16-cell-stage embryos of the ascidian, Ciona intestinalis. The isolated cells were allowed to divide either twice or three times thereafter. Then further divisions of the cells were continuously inhibited by a simultaneous treatment with aphidicolin (a specific inhibitor of DNA synthesis) and cytochalasin B (an inhibitor of cytokinesis). When development of muscle-specific acetylcholinesterase in these division-arrested progeny cells of B5.1 blastomeres was examined histochemically, the B5.1 blastomeres which had been allowed two further divisions did not produce any detectable acetylcholinesterase activity. Whereas those which had been allowed three further divisions showed the tissue-specific enzyme activity. These results provide further evidence for the presence of a quantal DNA replication cycle for the tissue-specific enzyme development, which is qualitatively different from the other DNA replication cycles.

Quantitative regulation of acetylcholinesterase development in the muscle lineage cells of cleavage-arrested ascidian embryos

Some embryos of Ciona intestinalis which were permanently cleavage-arrested with cytochalasin B at the 1-cell, 4-cell, or 8-cell stages produced, after 12 or 16 h of development time (18 degrees C), a level of muscle acetylcholinesterase activity equal to that found in normal early and later larval stage embryos of the same age. Enzyme activity was measured quantitatively in single whole embryos by a colorimetric procedure using microdensitometry. Quantitative regulation of a differentiation end product indicated that the usual transcriptional and translational control mechanisms for that histospecific protein continued to operate normally in the cleavage-arrested embryos. Acetylcholinesterase expression was apparently regulated independently of the usual cell cytoplasmic volume in the muscle lineage cells and possibly also independently of the normal nuclear number in the lineage. There is an egg cytoplasmic determinant that is segregated into the muscle lineage cells during cleavage and which appears to specify the pathway of larval muscle development. Quantitative control of muscle acetylcholinesterase is possibly one of the consequences of how the agent releases genetic expression in the presumptive muscle cells. Quantitative regulation was not, however, a general functional activity of cleavage-arrested embryos. Mitochondrial cytochrome oxidase, an enzyme whose development is believed to be unaffected by cytoplasmic determinants, was not regulated quantitatively in cleavage-arrested embryos. Cytochrome oxidase activity of cleavage-arrested embryos, measured in single whole embryos by a colorimetric microdensitometry assay, increased only slightly during 16 h of development time whereas the activity in normal control embryos doubled during that time.

A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development

In this investigation, Triton X-100 extraction was utilized to examine the cytoskeleton of ascidian eggs and embryos. The cytoskeleton contained little carbohydrate or lipid and only about 20–25% of the total cellular protein and RNA. It was enriched in polypeptides of molecular weight ( M r ) 54, 48, and 43 × 10 3. The 43 × 10 3 M r polypeptide was identified as actin based on its M r , isoeletric point, and affinity for DNase I. Electron microscopy of the detergent-extracted eggs showed that they contained cytoskeletal domains corresponding to colored cytoplasmic regions of specific morphogenetic fate in the living egg. A yellow crescent cytoskeletal domain in the myoplasm was examined and shown to consist of a plasma membrane lamina (PML) and a deeper lattice of filaments which appeared to connect the yellow crescent pigment granules to the PML. The PML probably consists of integral membrane proteins stabilized by an underlying network of actin filaments since NBD-phallacidin stained this area of the egg cortex and the PML was extracted from the cytoskeleton by DNase I treatment. The yellow crescent cytoskeletal domain was found throughout the cortex of the unfertilized egg. During ooplasmic segregation it progressively receded into the vegetal hemisphere and was subsequently partitioned to the presumptive muscle and mesenchyme cells of the 32-cell embryo. It is suggested that contraction of the actin network in the yellow crescent cytoskeletal domain is the motive force for ooplasmic segregation. This structure may also serve as a framework for the positioning of morphogenetic determinants involved in muscle cell development.

Myogenic Events in Compound Ascidian Larvae

SYNOPSIS. Comparative studies on the compound ascidians Distaplia occidentalis and Diplosonta macdonaldi indicate that larval myogenesis is divisible into three periods. In the prolifnative phase , the presumptive muscle cells divide by mitosis, assume specific relationships to the notochordal and epidermal rudiments of the embryo, and assemble the synthetic machinery to support the ensuing period of myogenesis. In the synthetic phase , the differentiating muscle cells increase in volume, establish the contractile apparatus, and augment the cisternae of agranular sarcoplasmic reticulum. The sarcoplasmic reticulum and sarcolemma form local adhesions that foreshadow structural couplings. In the elaborative phase , the caudal muscle cells refine the contractile apparatus, envelop it with perifibrillar cisternae of sarcoplasmic reticulum, and anchor it to the sarcolemma at the transverse cellular boundaries. Dyadic couplings link the perifibrillar sarcoplasmic reticulum to the sarcolemma. Subsarcolemmal specializations appear at future synaptic sites shortly before hatching.

Development of sodium, calcium and potassium channels in the cleavage-arrested embryo of an ascidian.

1. The cleavage of the embryos of an ascidian, Halocynthia roretzi, was arrested with cytochalasin B (1 microgram/ml.) at the 16- or 64-cell stage. These cleavage-arrested embryos were still able to develop membrane excitability. 2. In the cleavage-arrested 64-cell embryos at the time when control embryos became hatched larvae, Ca spikes were evoked in the presumptive muscle blastomeres, and Na- and Ca-dependent action potentials were induced in some ectodermal blastomeres. 3. Membrane currents of the cleavage-arrested 16-cell embryos were recorded with the voltage-clamp technique and analysed as a function of developmental time at 15 degrees C. For this purpose, intact eggs, 4- and 8-cell embryos were also used. The cleavage-arrested embryos behaved electrically like single cells, due to tight electrical coupling between blastomeres. After the 25-hr stage decoupling occurred. 4. Both Na and Ca currents decreased during the initial 10 hr. Na current became less than one third and Ca current almost disappeared. At 17 hr both Na and Ca currents increased again. 5. The potential-dependence of the Na and Ca currents after 17 hr was similar to that in the egg, although substantial parallel shifts in the current-voltage relations were observed: a 5 mV positive shift for the Na current and a 15-20 mV negative shift for the Ca current. 6. Delayed (outward) K current developed gradually until 20 hr and then increased abruptly. The activation level for the delayed rectification was markedly negative (around -10 mV) in comparison with that of the egg (around + 100 mV). Anomalous (inward) K current, on the other hand, increased gradually without changes in the potential-dependence throughout development. 7. The results suggest that the differentiation of excitable membranes in the ascidian embryo does not involve changes in the properties of the individual channels, but rather changes in the numbers of various kinds of ion channels.

A definite number of aphidicolin-sensitive cell-cyclic events are required for acetylcholinesterase development in the presumptive muscle cells of the ascidian embryos

ABSTRACT In order to determine whether or not a crucial number of DNA replications are prerequisite for cellular differentiation, we have studied development of a tissue-specific enzyme, muscle acetylcholinesterase (AChE) in the presumptive muscle cells of cleavage-arrested ascidian embryos. Embryos were cleavage-arrested with cytochalasin B (an inhibitor of cytokinesis) and aphidicolin (an inhibitor of DNA synthesis). The 64-ceIl-stage embryos which had been permanently cleavage-arrested with cytochalasin B developed AChE in all the eight presumptive muscle cells, but the same stage embryos which had been prevented from undergoing further divisions by simultaneous treatment with aphidicolin and cytochalasin did not produce AChE at all. Cytochalasin-arrested 76-cell-stage embryos were able to differentiate AChE in the ten presumptive muscle cells, while aphidicolin-cytochalasin-arrested 76-cell stages in as many as four cells. The early gastrulae which had been arrested with cytochalasin B produced AChE in all the sixteen presumptive muscle cells, while the same stage embryos arrested with aphidicolin and cytochalasin in as many as twelve cells. Cytochalasin-arrested late gastrulae developed AChE in twenty blastomeres, while aphidicolin-cytochalasin-arrested late gastrulae in eighteen cells. The presumptive muscle cells at these four stages consist of those of three different (seventh, eighth, and ninth) generations, and the relative positions of the blastomeres in the cleavage-arrested embryos remained fixed. Judging from the relative positions of the blastomeres, the AChE-producing cells in aphidicolin-cytochalasin-arrested embryos were always at eighth or ninth generation, while those with no AChE activity were certainly at seventh generation. Based on these findings it was supposed that aphidicolin-sensitive cell-cyclic events, presumably DNA replication, are closely associated with AChE development and that the eighth cleavage cycle may be ‘quantal’ for the histospecific enzyme development.

On the ‘clock’ mechanism determining the time of tissue-specific enzyme development during ascidian embryogenesis

ABSTRACT During ascidian embryogenesis a tissue-specific enzyme, muscle acetylcholinesterase (AChE) may first be detected histochemically in the presumptive muscle cells of the neurula. In order to investigate the ‘clock’ or counting mechanism that is determining the time when AChE first appears, Whittaker’s experiment (1973) has been repeated using eggs of the ascidian, Halocynthia roretzi. Embryos that had been permanently cleavage-arrested with cytochalasin B were able to differentiate AChE in their muscle lineage blastomeres. The time of first AChE occurrence in embryos that had been cleavage-arrested in the 32-cell stage with cytochalasin B was about the same as in normal embryos. This result indicates that the clock is not apparently regulated by the events of cytokinesis. The early gastrulae which had been arrested with colchicine or with colcemid could develop AChE activity, although no histochemically detectable AChE activity was observed in the cleavage-stage embryos that had been arrested with either drug. Therefore the clock does not seem to be controlled by the mitotic cycle of the nucleus. It is suggested that the cycle of DNA replication may be related to the regulation of the clock that is determining the time of development of histospecific protein.

Viral transformation of chick myogenic cells: The relationship between differentiation and the expression of the SRC gene

When chick embryo presumptive muscle cells are transformed at 35 °C with a temperature-sensitive mutant of Rous sarcoma virus, tsLA29, they do not undergo myogenic differentiation. When these cultures are shifted to 41 °C the cells revert to a phenotypically normal state and fuse into myotubes. The synthesis of myosin, the appearance of myosin mRNA active in vitro, and the synthesis of acetylcholinesterase (AChE) were all activated following a shift from 35 °C to 41 °C. The activation of myosin synthesis also occurred in cultures prevented from fusing by calcium deprivation. After myosin synthesis had been initiated at 41 °C, however, it could not be suppressed by shifting the cultures back to 35 °C. [ 3H]Thymidine labeling and autoradiography demonstrated that DNA synthesis in tsLA29-infected myoblasts ceased within 24 h after the shift to 41°C. A kinetic analysis of the withdrawal of these cells from the cell cycle indicates that at least a fraction of the cells do not need to traverse a complete cell cycle prior to terminal differentiation.

Development of neuromuscular transmission in a larval tunicate

1. The time sequence of the development of acetylcholinesterase (AChE), acetylcholine (ACh) receptors and functional synapses on the embryonic muscle membrane in a tunicate larva (Halocynthia roretzi) was investigated in vivo.2. The fertilized tunicate egg was incubated in natural sea water at 9 degrees C. Sixty-eight hr after fertilization the free-swimming larva was hatched, which had six striated muscle fibres in the tail. The developmental stage of the embryo was indicated by the developmental hours after fertilization.3. The transmitter at the neuromuscular junction in the hatched larva is ACh. (i) Neuromuscular transmission was completely blocked by D-tubocurarine (1-5 x 10(-5)M). (ii) Eserine (5-10 x 10(-7)M) approximately doubled the time constant of the falling phase of miniature excitatory junctional currents (e.j.c.s). (iii) The reversal potential of the membrane response to iontophoretically applied ACh was -10 mV and similar to that of e.j.c.s. (iv) AChE was present on the muscle membrane surface.4. AChE activity became visible histochemically on the embryonic cell membrane in the presumptive muscle region as early as the late gastrula stage (27 hr after fertilization, 12 hr before the ACh response appeared).5. The response to iontophoretically applied ACh was present at 39 hr after fertilization but could not be evoked at 38 hr.6. Between 39 and 41 hr after fertilization, the ACh responses increased rapidly, then remained relatively unchanged until larval hatching.7. The stage of the initial appearance of the ACh response corresponded to the stage when the Ca current abruptly increased in the muscle membrane.8. The first sign of neuromuscular transmission was appearance of a giant excitatory junctional potential (e.j.p.) with uniform amplitude (about 15-20 mV) and slow time course (time constant of the falling phase of a giant e.j.c. was 23.4 +/- 6.9 msec, mean and S.D., at -60 mV and 11 degrees C).9. Within a few hours, these giant e.j.p.s disappeared and were successively replaced by medium-sized e.j.p.s and then e.j.p.s similar to those seen in hatched larvae (time constant of the falling phase of a miniature e.j.c. was 8.5 +/- 1.8 msec at -60 mV and 11 degrees C).