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ARIA is concentrated in the synaptic basal lamina of the developing chick neuromuscular junction
A D Goodearl1, A G Yee, A W Sandrock
1Neurobiology Department, Harvard Medical School, Boston, Massachusetts 02115, USA.
Insights
Amphiregulin (ARIA) is a growth factor found in motor neurons that helps form neuromuscular junctions. This study shows ARIA protein concentrates at these junctions, suggesting its role in synapse maturation.
Area of Science:
- Neuroscience
- Molecular Biology
- Developmental Biology
Background:
- Amphiregulin (ARIA) is part of a family of growth factors, including glial growth factor (GGF).
- ARIA mRNA is present in cholinergic neurons, including spinal cord motor neurons in rats and chicks.
- ARIA enhances acetylcholine receptor incorporation into muscle cell membranes in vitro.
Purpose of the Study:
- To investigate the role of ARIA in regulating the synthesis of junctional synaptic acetylcholine receptors in chick embryos.
- To examine the expression and distribution of ARIA in motor neurons and at the neuromuscular junction.
Main Methods:
- Development of riboprobes and polyclonal antibody reagents for ARIA isoforms.
- Analysis of ARIA mRNA expression in motor neurons during embryonic development.
- Immunoelectron microscopy to determine ARIA protein localization at the neuromuscular junction.
Main Results:
- ARIA mRNA was detected in motor neurons by embryonic day 5, coinciding with initial motor axon contact.
- ARIA protein was found concentrated in the synaptic cleft of neuromuscular junctions in older embryos and postnatal animals.
- Immunoelectron microscopy revealed ARIA immunoreactivity within the synaptic basal lamina, associated with presynaptic components.
Conclusions:
- ARIA is expressed in motor neurons early in development and transported to nerve terminals.
- ARIA protein localizes to the neuromuscular junction, suggesting a role in synapse formation.
- These findings support ARIA's function as a trophic factor regulating mature neuromuscular synapse development.
Abstract:
ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.