The immediate goal of these experiments is to understand the role of agrin in the formation of the embryonic neuromuscular junction. Agrin is a protein made and externalized by the motor nerve terminal that causes the postsynaptic apparatus to form on muscle cells. Currently we are using Xenopus (frog) embryos as an assay system for agrin and other molecules thought to be involved in formation of this synapse. We are assaying the function of agrin isoforms and domains by overexpressing the proteins in the embryo. RNA encoding agrin proteins is injected into early blastomeres of the embryo; it is mixed with RNA encoding the green fluorescent protein (GFP) as a marker. After about 38 hours of development, tadpoles are screened for expression of GFP in the ryot6mal muscles or spinal cord of the trunk. Embryos are fixed and the acetylcholine receptors (AChRs) are stained with rhodamine-bungarotoxin. The AChR aggregates in GFP-positive regions of the embryos are imaged by confocal microscopy. Several Z-series are taken from each group of embryos in an experiment. The volume of AChR aggregates in the myotomes; imaged is quantitated using Metamorph image analysis software. Results to date indicate that overexpression of both neuronal and muscle isoforms of agrin result in a significant increase in AChR aggregate volume. These results suggest that both isoforms of agrin may be important in the formation of the neuromuscular synapse in the developing embryo.
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