Journal of Neuroscience, Vol 12, 2982-2992, Copyright © 1992 by Society for Neuroscience
Early appearance of and neuronal contribution to agrin-like molecules at embryonic frog nerve-muscle synapses formed in culture
MW Cohen and EW Godfrey
Department of Physiology, McGill University, Montreal, Quebec, Canada.
Antibodies against chicken and Torpedo agrin were used for
immunofluorescent staining in order to assess the spatial distribution and
temporal appearance of agrin-like molecules at newly formed synaptic
contacts in cultures of embryonic Xenopus nerve and muscle cells. The
antibodies stained Xenopus neuromuscular junctions and removed ACh receptor
(AChR)-aggregating activity from extracts of Xenopus brain.
Immunofluorescence was observed at almost all nerve- induced AChR
aggregates, even at microaggregates in cocultures as young as 7.5 hr and at
nerve-muscle contacts less than 2 hr old. Microdeposits of
immunofluorescence extended as far distally as, or farther than, the
microaggregates of AChRs along young nerve-muscle contacts. They also
occurred along portions of growing neurites that were not in contact with
muscle. By contrast, immunofluorescence was rarely observed at the
nonsynaptic aggregates of AChRs that form on noninnervated muscle cells.
These results raise the possibility that neuronally derived microaggregates
of agrin-like molecules may be primary sites of nerve-induced clustering of
AChRs, and they indicate that these molecules are present at embryonic
nerve-muscle synapses from the very onset of AChR aggregation. The cellular
origin of the agrin-like molecules at synapses was examined in
cross-species cocultures in which the neurons and muscle cells were
obtained from embryos of Xenopus laevis and Rana pipiens. Immunofluorescent
staining with anti-agrin antibodies reactive at both Rana and Xenopus
neuromuscular junctions revealed immunofluorescence at AChR aggregates
along nerve-muscle contacts involving both cross-species combinations.
Immunofluorescent staining with an anti-agrin antibody reactive at Rana but
not at Xenopus neuromuscular junctions was positive only at cross- species
nerve-muscle contacts involving Rana neurons. These results provide the
first demonstration that embryonic neurons supply agrin- like molecules to
the synapses they form with embryonic muscle cells.
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