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Circadian rhythm of retinotectal synaptogenesis during development in larval zebrafish in vivo. a Top, experimental procedure diagram. PGUSG larvae were entrained to 14–10 light-dark (LD) cycles during 0–4 days post fertilization (dpf) and then imaged every 6 hrs for 2 days from light onset (ZT0) at 4 dpf. Bottom, images obtained with a 6 h interval of a typical RGC axon arbor expressing Sypb-EGFP in the right hemisphere of the optic tectum (OT). Blue arrow, the primary axon; yellow arrow, a punctum at the first branch point of the axon arbor. L, lateral; R, rostral. Scale bars: 10 µm. b Summary of the total number (black) and growth rate (red) of Sypb-EGFP puncta on single-RGC axon arbor during 4–6 dpf. The larvae were under LD during 0–6 dpf. Gray line, linear-fitting line on the mean value of growth rate data. c Detrended growth rate of puncta shown in (b, red). It was obtained by subtracting the linear-fitting line from each raw data. d–g Detrended growth rate of puncta imaged during 4–6 dpf for wild type (WT) (d–f) or clocka−/− (g) larvae raised under different light conditions. d Blue and (g), LD during 0–4 dpf and constant darkness (DD) during 4–6 dpf; d Green, LD during 0–4 dpf and constant light (LL) during 4–6 dpf; e dark-light (DL) during 0–6 dpf; f DL during 0–4 dpf and DD during 4–6 dpf. Zebra stripes, day-night cycles; white stripe, daytime; gray stripe, subjective daytime; black stripe, nighttime or subjective nighttime. Colored curve, cosine-fitting waves. CT, circadian time; ZT, zeitgeber time. Numbers in brackets indicate the number of RGCs (n) and larvae (N) examined. n.s., not significant; ***P < 0.0001 in (b), **P = 0.001 in (c), *P = 0.01 and ***P = 0.0006 in (d), **P = 0.006 in (e), *P = 0.01 in (f) (one-way ANOVA for (b), one-tailed Fisher’s g-test for (c–g)). Error bars denote s.e.m.. Source data are provided as a Source Data file.
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