Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations

TLDR

Synapse formation depends on coordinated pre‑ and postsynaptic differentiation, yet it is unclear which steps are cell‑autonomous versus requiring axon‑derived signals, a model that proposes axon‑released factors directly organize postsynaptic sites beneath the nerve terminal. The study tests whether axon‑derived signals are required for postsynaptic differentiation by examining zebrafish primary motor system neuromuscular junctions. The authors examined zebrafish primary motor NMJs, using time‑lapse imaging to track acetylcholine receptor cluster dynamics as axons extend. They found that AChR clusters pre‑form in the myotome before axon arrival, are incorporated into NMJs as axons advance, but axons are required for remodeling and selective stabilization of synaptic clusters, indicating that motor axons are dispensable for initial postsynaptic differentiation yet essential for later stages, with heterogeneity in signaling mechanisms across muscle fiber types.

Abstract

Synapse formation requires the coordination of pre- and postsynaptic differentiation. An unresolved question is which steps in the process require interactions between pre- and postsynaptic cells, and which proceed cell-autonomously. One current model is that factors released from presynaptic axons organize postsynaptic differentiation directly beneath the nerve terminal. Here, we used neuromuscular junctions (NMJs) of the zebrafish primary motor system to test this model. Clusters of neurotransmitter (acetylcholine) receptors (AChRs) formed in the central region of the myotome, destined to be synapse-rich, before axons extended and even when axon extension was prevented. Time-lapse imaging revealed that pre-existing clusters on early-born slow (adaxial) muscle fibers were incorporated into NMJs as axons advanced. Axons were, however, required for the subsequent remodeling and selective stabilization of synaptic clusters that precisely appose post- to presynaptic elements. Thus, motor axons are dispensable for the initial stages of postsynaptic differentiation but are required for later stages. Moreover, many AChR clusters on later-born fast muscle fibers formed at sites that had already been contacted by axons, suggesting heterogeneity in the signaling mechanisms leading to synapse formation by a single axon.