The Journal of Neuroscience, January 18, 2006, ():
In Vivo Imaging of Preferential Motor Axon Outgrowth to and Synaptogenesis at Prepatterned Acetylcholine Receptor Clusters in Embryonic Zebrafish Skeletal Muscle
J. Neurosci. Panzer et al.
26: 934
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Fig. 1 VAMP-GFP marks sites of presynaptic vesicle clusters and does not alter motor axon outgrowth.
Transient transgenic embryos at 24 hpf in which some motor neurons express VAMP-GFP (green) and postsynaptic AChRs are labeled with rhodamine ?BTX (red).
A: VAMP-GFP+ CaP primary motor neurons (green), one in each of two adjacent caudal segments. The boxed regions 1 and 2 are shown at higher magnification to the right. Post-imaging immunostaining shows that VAMP-GFP is colocalized with SV2 (blue), demonstrating that VAMP-GFP marks clusters of synaptic vesicles. Scale bar = 10 ?m.
B: The left of 2 adjacent rostral segments is innervated by a VAMP-GFP+ CaP motor neuron (green). Comparison with segments innervated by GFP- motor neurons (right) immunostained for SV2 to mark axons and presynaptic terminals (blue) shows that the extent of motor axon outgrowth and branching is unaffected by VAMP-GFP expression. Scale bar = 10 ?m.
C: VAMP-GFP+ primary motor neuron CaP imaged over time at 24-72 hpf. At ca. 24 hfp, relatively few motor axon branches or neuromuscular synapses are present, whereas by ca. 72 hpf axons have branched into their appropriate, cell specific territory and many neuromuscular synapses are present. These data show that the imaging procedures used do not interfere with axon outgrowth or neuromuscular synaptogenesis. Scale bar = 10 microns.
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Supplemental Fig. 2 Blockade of sodium channels with tricaine does not affect dispersal of prepatterned AChR clusters or initial motor axon outgrowth.
A-B: SV2+ presynaptic axons and terminals (green) and rhodamine ?BTX labeled AChRs (red) were analyzed.
A: Motor axon outgrowth, prepatterned AChRs and initial neuromuscular synaptogenesis in wild type, unmanipulated control embryos at 21 hpf.
B: Motor axon outgrowth, prepatterned AChRs and initial neuromuscular synaptogenesis in embryos in which AChRs were blocked with tricaine from 10-21 hpf are indistinguishable from that observed in control embryos. Scale bar = 10 microns.
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Supplemental movie 1
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Supplemental movie 2
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Supplemental movie 3
