When a spherical muscle cell (myoball) was manipulated into contact with either the soma or the neurite of an isolated neuron in 2-day-old Xenopus nerve-muscle cultures, depolarizations similar to miniature endplate potentials (MEPPs) were frequently detected in the muscle cell. These depolarizations occurred within minutes after myoball-soma contact and within seconds after myoball-neurite contact. They had time course and amplitude distribution similar to those of the MEPPs recorded from naturally occurring neuromuscular synapses between neurites and muscle cells found in the same cultures, but they occurred at a lower frequency and had smaller average amplitudes. These depolarizations were induced by acetylcholine (ACh) since they were reversibly blocked by addition of d-tubocurarine into the culture, and they were abolished in muscle cells pretreated with alpha-bungarotoxin before contact with the neuron. Greater than 60% of the neuronal population in these cultures released ACh upon this direct muscle contact. The appearance of MEPP-like potentials in the myoball upon contact with an isolated neuron suggests that the cellular machinery responsible for ACh release is present throughout the neuron and that packages of ACh molecules are available for release prior to nerve-muscle synapse formation. We also found that neurons which had previously made synapse with other muscle cells in the culture all failed to release ACh from the soma and showed reduced release capability at the neurite for the first 30 min to 1 hr of contact with a myoball. This finding suggests that, during synapatogenesis, there is a depletion of ACh molecules and/or substances responsible for the triggering of their release in the extrasynaptic regions of the neuron.

SUMMARY1. The acquisition and distribution of nerve fibres and of acetylcholine (ACh) receptors were examined in the myotomes of Xenopus laevis during normal development. This muscle is well-suited for investigating temporal relationships during neuromuscular synaptogenesis because the age of the Xenopus embryo at the onset of innervation can be assessed with an accuracy of about one hour. Myotomal nerve fibres were visualized after staining them with nitroblue tetrazolium and ACh receptors were examined after exposure to x-bungarotoxin labelled with 1251 or fluorescent dye.2. Nerve fibres were seen in the myotomes of some embryos as early as stage 19(20'75 hr) and in virtually all embryos by stage 24 (26-25 hr). From the outset they were located mainly at the ends of the myotomes, but some myotomes also exhibited nerve fibres in more central regions.3. ACh receptors were already present in myotomes by stage 19 (20-75 hr) and initially had a widespread, uniform distribution. The density of extrajunctional ACh receptors increased until stage 36 (50 hr) and then declined less than 3-fold over the next 10 days of development.4. Discrete patches of high ACh receptor density began to appear at the ends of the myotomes at stage 22 (24 hr) and were seen in almost all embryos by stage 26 (29-5 hr). ACh receptor patches were also seen in central regions of some myotomes and these were usually aligned in patterns which resembled the course of nerve fibres.5. The present findings suggest that myotomal muscle cells in Xenopus embryos begin to acquire ACh receptors shortly before the arrival of nerve fibres and that discrete patches of ACh receptors begin to form at presumptive synaptic sites on the average about 3 hr after the arrival of the nerve fibres. The latter delay is considerably shorter than that in developing rat muscle.6. The temporal and spatial relationships between nerve fibres and the development of ACh receptor patches in Xenopus myotomes in vivo are consistent with findings in Xenopus cell cultures which indicate that nerve fibres can rapidly induce ACh receptor localization at sites of nerve-muscle contact.

Cultured mammalian ciliated cells from the respiratory tract respond to mechanical stimulation of their cell surface by displaying a rapid transient increase in beat frequency. Surrounding adjacent and more distal neighboring ciliated cells display a similar frequency response after a short delay that is proportional to their distance from the stimulated cell. To characterize the progression of this communicated response we developed an automated computer-assisted image-analysis system to examine high-speed films of responding cells. Transmission of the frequency response between cells occurs at 0.63 cells/s at 25 degrees C and 1.54 cells/s at 37 degrees C. We have also confirmed that gap junctions exist between cells in both epithelial explants and outgrowths and that adjacent or nonadjacent ciliated, as well as nonciliated, cells are electrically coupled. We postulate that mechanical stimulation and intercellular communication provide a mechanism to regulate beat frequency between ciliated cells in order to facilitate efficient ciliary function and mucus transport.

Cell surface lectin receptors underwent rapid redistribution after embryonic Xenopus myotomal muscle cells were manipulated into contact in culture. Soybean agglutinin (SBA) receptors became highly concentrated at the contact area and concanavalin A (Con A) and ricin receptors were depleted at the same region. The accumulation of SBA receptors was greatly reduced by the presence of SBA specific sugars in the incubating medium, by precontact binding of SBA to the surface and by lowering the temperature, but it was unaffected by prolonged treatments with metabolic inhibitors. It is culture-age dependent: older cultures showed a markedly reduced extent of accumulation, and the high accumulation resulting from contact made in younger cultures disappeared with time in culture. These findings are consistent with the notion that specific molecular interaction between the contacting surfaces results in a redistribution of preexisting rapidly diffusing surface receptors. In support of this notion, ligand-free SBA and Con A receptors were shown to be laterally mobile in the membrane, and at least a subpopulation of the SBA receptors contains physically distinct molecules from the Con A receptors. We suggest that such contact-induced redistribution of various surface components may play a role in the interaction between embryonic cells.Cell surface components play an important role in cell-cell recognition, intercellular communication, and cellular differentiation during embryonic development (for reviews, see 2,7,[9][10][11]19). Specific interaction between molecules on the surfaces of contacting cells within the tissue seems to be an essential step for many of these developmental processes. Previous studies of selective cell-cell adhesion have clearly implicated the involvement of specific cell surface glycoproteins (8,11,20,21). During the past decade, it has also become quite clear that the physical state of cell surface glycoproteins is highly dynamic. Many glycoproteins, especially those in the embryonic plasma membrane, are capable of rapid lateral migration in the plane of the membrane (5,12,16,17). Two questions immediately arise: does lateral migration of membrane components play any role in establishing specific cellcell interaction; or conversely, can specific intercellular interaction be responsible for inducing lateral rearrangement of membrane components into specific patterns of membrane topography that is characteristic of many differentiated cells?As a first step in studying these problems, we examined the distribution of plasma membrane glycoproteins on cultured embryonic muscle before and after the cells were manipulated into contact. Preliminary findings indicated that many surface soybean agglutinin receptors, presumably glycoproteins containing n-galactose and/or N-acetyl-t~-D-galactosamine residues, are induced to accumulate at the site of cell-cell contact. The rate of this accumulation of surface receptors is consistent with the idea that the contact site serves as a trap for rapidly diff...

Blastomere lineages are differentially biased to produce different neurotransmitter subtypes of amacrine cells (Huang and Moody, 1995, 1997,). To elucidate when this bias is acquired, we examined amacrine lineages at different early developmental times. Our experiments demonstrate that the bias to express dopamine and neuropeptide Y amacrine fates involves several steps before the formation of the definitive optic cup. At cleavage stages, a retinal progenitor that contributes large numbers of cells is already biased to produce its normal repertoire of dopamine amacrine cells, as revealed by transplantation to a new location, whereas the amacrine fate of a progenitor that contributes fewer cells is modified by its new position. At neural plate stages, not all retinal progenitors are multipotent. Nearly one-half populate only the inner nuclear layer and are enriched in amacrine cells. During early optic vesicle stages, an appropriate mitotic tree is required for dopamine and neuropeptide Y, but not serotonin, amacrine cell clusters to form. Thus, the acquisition of amacrine fate bias involves intrinsic maternal factors at cleavage, fate restriction in the neural plate, and specified mitotic patterns in the optic vesicle. At each of these steps only a subset of the embryonic retinal progenitors contributing to amacrine subtypes is biased; the remaining progenitors maintain multipotency. Thus, from the earliest embryonic stages, progenitors of the retina are a dynamic mosaic. This is the first experimental demonstration of amacrine fate decisions that occur during early embryonic periods in advance of the events described in the later, committed retina.