Overexpression of Agrin Isoforms in Xenopus Embryos Alters the Distribution of Synaptic Acetylcholine Receptors during Development of the Neuromuscular Junction

摘要

Synapse formation involves a large number of macromolecules found in both presynaptic nerve terminals and postsynaptic cells. Many of the molecules involved in synaptogenesis of the neuromuscular junction have been discovered through morphological localization to the synapse and functional cell culture assays, but their role in embryonic development has been more difficult to study. One of the best understood of these molecules in agrin, a synaptic extracellular matrix protein secreted by both motor neurons and muscle cells, that organizes the postsynaptic apparatus, including high-density aggregates of acetylcholine receptors (AChRs), at the neuromuscular junction. We tested the specific hypothesis that different agrin isoforms made by neurons and muscle cells contribute to agrin's synapse organizing activity in the embryo. Agrin isoforms were overexpressed by injecting synthetic RNA into Xenopus laevis embryos at the one- or two-cells stage. To mark cells containing agrin RNA, green fluorescent protein (GFP) RNA was coinjected. The relative area of muscle AChR aggregates was measured by confocal microscopy and image analysis in GFP-positive segments of injected embryos. Innervated regions of myotomal muscles were compared in animals injected with a mixture of agrin and GFP RNAs or with GFP RNA alone. Overexpression of COOH-terminal 95-kDa fragments of a rat agrin isoform made only by neurons (4,8) and the major isoform (0,0) made by muscle cells both increased AChR cluster area by 100-200%. Rat agrin protein was colocalized with AChR aggregates in innervated regins of muscles in injected embryos. These results show that agrin derived from both the nerve terminal and the muscle cell could contribute to synaptic differentiation at the embryonic neuromuscular junction. They further demonstrate the usefulness of overexpression by RNA injection as an assay for molecular function in embryonic synapse formation.