In VivoImaging of Preferential Motor Axon Outgrowth to and Synaptogenesis at Prepatterned Acetylcholine Receptor Clusters in Embryonic Zebrafish Skeletal Muscle

Abstract

Little is known about the spatial and temporal dynamics of presynaptic and postsynaptic specializations that culminate in synaptogenesis. Here, we imaged presynaptic vesicle clusters in motor axons and postsynaptic acetylcholine receptor (AChR) clusters in embryonic zebrafish to study the earliest events in synaptogenesis in vivo. Prepatterned AChR clusters are present on muscle fibers in advance of motor axon outgrowth from the spinal cord. Motor axon growth cones and filopodia are selectively extended toward and contact prepatterned AChR clusters, followed by the rapid clustering of presynaptic vesicles and insertion of additional AChRs, hallmarks of synaptogenesis. All initially formed neuromuscular synapses contain AChRs that were inserted into the membrane at the time the prepattern is present. Examination of embryos in which AChRs were blocked or clustering is absent showed that neither receptor activity or receptor protein is required for these events to occur. Thus, during initial synaptogenesis, postsynaptic differentiation precedes presynaptic differentiation, and prepatterned neurotransmitter clusters mark sites destined for synapse formation.

MuSK controls where motor axons grow and form synapses

“…These findings demonstrate that MuSK misexpression disrupts the AChR prepattern and indicate that approaching motor axons normally recognize and respond to the MuSK-dependent muscle prepattern. Consistent with these findings, live imaging of neuromuscular synapse formation in zebrafish suggested that motor axons sense and turn toward prepatterned AChR clusters (Flanagan-Steet et al, 2005;Panzer et al, 2006); our studies demonstrate that motor axons recognize a MuSK-dependent prepattern and are re-routed when the pattern of MuSK expression is altered. Further studies will be necessary to identify the signaling events downstream from MuSK that lead to the production of cell surface and/or secreted proteins that shape motor axon growth and promote synapse formation.…”

“…Recent studies have demonstrated that muscle is pre-specialized in the central, prospective synaptic region prior to and independent of innervation, and these findings have led to a revised view of the steps and mechanisms that regulate neuromuscular synapse formation (Flanagan-Steet et al, 2005;Lin et al, 2001;Panzer et al, 2006;Yang et al, 2001;Yang et al, 2000). Key features of postsynaptic differentiation are established in a muscleautonomous, MuSK-dependent manner and motor neurons refine and sharpen, rather than induce this prepattern of postsynaptic differentiation.…”

“…As a consequence, AChR clusters are selectively maintained at nascent synapses and dispersed at non-synaptic sites. These genetic studies in mice have been complemented by live-imaging of neuromuscular synapse formation in zebrafish (Flanagan-Steet et al, 2005;Panzer et al, 2006), which showed that this pattern of AChR expression is established prior to innervation and suggested that motor axon growth cones grow toward this prepatterned zone (Panzer et al, 2006). Important aspects of muscle pre-patterning, however, remain poorly understood.…”

Stabilization of Axon Branch Dynamics by Synaptic Maturation

“…These in vivo studies found that vesicle-associated membrane protein (VAMP)-GFP puncta constituted the principal sites of a new branch addition and that branches that failed to form VAMP-GFP puncta were more likely to be eliminated. Notably, a similar phenomenon occurs in motor neuron axon development in Xenopus (Javaherian and Cline, 2005) and zebrafish (Panzer et al, 2006). These observations suggest a central role for the synapse in axon arbor remodeling and raise the intriguing possibility that as synapses mature and strengthen, they may serve double duty as structural sites for axon branch stabilization.…”

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms

“…Interestingly, results obtained from studies of primary MNs in zebrafish AChRα1 mutants failed to show an effect on MN branching and terminal arborization, which may reflect species differences or unique characteristics of zebrafish primary MNs (29). Indeed, recent evidence suggests that the cues regulating primary and secondary motor axon branching in zebrafish are different (48,49).…”