The tunicate embryo, which proceeds from fertilization to swimming larva in about 18 hours (in the case of Ciona intestinalis), provides a remarkable preparation for the study of the role of intercellular communication in development. Preliminary data indicate compartmentation of dye coupling; the mechanisms whereby these compartments are formed will be investigated. Gap junctions in the ascidians show voltage dependence and the blastomeres exhibit two stable resting potentials sufficiently far apart to shut down junctional conductance and uncouple cells; these properties could lead to transient or reversible compartment formation. Determination of regional differences in resting potential, already indicated by differences in intensity of fluorescence observed with slow voltage sensitive dyes, will be validated with microelectrode measurement. Such regional differences must occur if voltage dependence of junctional conductance accounts for compartment formation. Alternatively the distribution of gap junctions might be the controlling factor. Measurement of coupling coefficients along with resting potentials would establish this mechanism of compartment formation. Some inductive interactions are known; electrical coupoing between participating cells will be assayed over time and compared to that between other cells where developmental information is not being transmitted. Effects of antibodies on junctional properties will be assayed and their use in exploration of the role of gap junctions in development explored. Studies of gap junctions from other embryos will be continued. Sensitivity to H and Ca will be assayed as part of an ongoing comparative study. Permeability will be correlated with conductance during treatments modulating junctional conductance. The results will provide information about the permeation process and indicate whether channel closure is all-or-none or graded. Reconstitution will be attempted in a three, rather than a two, compartment system. Improvements in perfusion media will be sought with a view to understanding cellular control of junction formation and removal, and parallel studies will be carried out on intact cells. Phosphorylation of junction protein has been demonstrated and pharmacological treatments of intact cells suggest an involvement in control of junctional conductance in a number of systems. The availability of antibodies and the imminent availability of the cDNA will make the cell biology of gap junctions accessible.
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