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Fig. 3 Presynaptic Components Arrive Sequentially during Development (A) Left panel: lateral view of three segments of a 25 hpf embryo expressing ngn1:GFP (green) labeled with antibodies to synaptotagmin2b (magenta) and CoPA neurons (con-1, cyan). Notice synaptotagmin2b puncta were colocalized with CoPA cell bodies at the dlf. Right panels: zoom of box in left panel. Inset shows single plane of boxed region at the level of the dlf and the contacting region of the CoPA cell. Scale bar represents 10 μm. (B) Dorsal view of IF labeling for synaptotagmin2b (magenta) colocalized with CoPA cell bodies (cyan) in ngn1:GFP embryos from 1–28 hpf. Asterisk labels RB cell bodies. Scale bar represents 10 μm. (C) Dorsal view of IF labeling for MAGUKs (magenta) and synapsin 1/2 (cyan) from 19–28 hpf. Asterisk labels RB cell bodies. White arrowheads show sites of colocalization of synapsins and MAGUKs, white arrows indicate localization of synapsins only, and yellow arrowheads indicate localization of MAGUKs only. Outlines of CoPA cell bodies were generated by increasing the gain of the MAGUK IF. Scale bar represents 10 μm. (D) Histogram shows the number of synapsin 1/2, synaptotagmin2b, and MAGUK puncta per CoPA cell from 19–28 hpf. (E and F) Histograms show the percentage of MAGUK puncta colocalized with synapsin puncta, and the percentage of synapsin puncta colocalized with MAGUK puncta, respectively, from 19–28 hpf. Error bars show SEM. See also Figure S1 and Table S1.