Fig. 4

Fig. 4 Optogenetic Stimulation of plod3?/? Zebrafish CaP Neurons Rescues Their Axon Guidance Defect, Allowing Juxtaposition of Pre- and Post-synaptic Machinery within the Ventral Myotome (A) PA-Rac1 expression (red) in spinal motor neurons of plod3+/? zebrafish at 28 hpf (scale bar, 50 ?m). (B) Left: an age-matched plod3?/? mutant sibling prior to stimulation fails to extend CaP axons into the ventral myotome past the horizontal myoseptum (dashed white line). Right: In plod3?/? fish, a single PA-Rac1+ CaP axon extended into the ventral myotome after illumination, while unilluminated axons remain arrested at the horizontal myoseptum (blue circle, region of illumination; scale bar, 50 ?m). (C) Enlarged image of plod3?/? CaP axon following stimulation (solid white box in B; scale bar, 50 ?m). (D) Illumination of PA-Rac1+ CaP axons in plod3?/? mutant fish induced growth significantly farther past the horizontal myoseptum than unilluminated axons. There was no significant difference in the distance grown past the horizontal myoseptum in illuminated plod3?/? fish compared with plod+/? fish (FDR adjusted, difference of least square means from mixed linear model, n = 8 axons, one axon per fish, ???p < 0.001, mean and 95% CIs shown). (E) Immunohistochemistry for mCherry (red, left), SV2 (green, middle left), and ?BT (cyan, middle right) showing colocalization of pre- and post-synaptic markers (merge, right) in the ventral myotome of the stimulated axon (bottom), but not in the unstimulated axon (top; scale bar, 5 ?m). Stimulated and unstimulated regions correspond to solid white boxes in (C). See also Video S4.