Developmental changes in branchiomotor axon branching and synaptic structures at the jaw neuromuscular junctions. Ventral views with anterior to the left of the jaw musculature. (A,D,G,J) Axon (magenta) outgrowth on jaw muscles (green) in 3, 5, 7, and 9 dpf Tg (α-actin:GFP); Tg (zCREST1:mRFP) larvae. 3D rendering of muscles and axons were overlaid to determine axon position relative to the muscles. Fine axon branches (white arrowheads) across the jaw muscles increased as larvae aged. (B,E,H,K) SV2a antibody (magenta) labeled presynaptic structures on the jaw muscles (green) in 3, 5, 7, and 9 dpf Tg(isl1:GFP); Tg(α-actin:GFP) larvae. 3D rendering was used to visualize the presynaptic regions in contact with the muscles (arrowheads), and calculate their volumes. (M) Presynaptic volumes increased greatly from 3 to 7 dpf larvae, especially for ima/imp and ih muscles. There was a significant increase from 5 to 7 dpf in the ima/imp, ih, and hh muscles. Both ih and hh muscles had a significant decrease in presynaptic volume from 7 to 9 dpf larvae. (C,F,I,L) Ventral view with anterior to the left. Acetylcholine receptor (AChR) clusters (arrowheads) were labeled with alpha-bungarotoxin (αBTX) (magenta) on the jaw muscles (green) in live 3, 5, 7, and 9 dpf Tg(α-actin:GFP) larvae. (N) Although AChR cluster volumes on all muscles tend to increase from 3 to 7 dpf, these changes were not significant. Data pooled from 3 to 5 experiments (number of larvae in parenthesis). Statistical analysis was carried out with Chi-square test and One-way analysis of variance (ANOVA) with post hoc Tukey’s HSD (honest significance difference). NS, not significant; *p < 0.05, **p < 0.01. ima/imp, intermandibularis anterior/intermandibularis posterior; ih, interhyal; hh, hyohal; sh, sternohyoideus.
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