Fig. 4

Fig. 4 Circadian rhythm of synaptogenesis affects the arrangement of retinotectal synapses and the refinement of visual function.

a Experimental procedure diagram. Larvae reared under LD during 0–4 dpf and DD afterwards were assayed at 6 dpf. b Left, projection view of a typical RGC axon arbor expressing Sypb-EGFP in WT, clocka^−/− or hcrtr2^−/−. Right, boxplots of the arbor territory/area (yellow polygon) (**P = 0.006; *P = 0.02). c Boxplots of the punctum density of single-RGC axon arbor calculated by total punctum number dividing arbor territory (***P = 0.0006; **P = 0.001). d Left, heat maps of the images in (b) color-coded by the distance of each pixel to its nearest punctum. Right, boxplots of the standard deviation of pixel intensities within the arbor territory (white polygon) (P = 0.02). e–g The same measurements as in (b–d) but larvae were raised in DD from 0 dpf. h Cumulative distribution of the receptive field (RF) size of tectal neurons (TNs) in WT (5976 TNs, 12 larvae), clocka^−/− (5416 TNs, 12 larvae) and hcrtr2^−/− (4859 TNs, 10 larvae) (P_clocka^-/-_{vs WT} = 5.2 × 10⁻⁸; P_hcrtr2^-/-_{vs WT} = 1.2 × 10⁻⁴). Inset, zoom-in view of the dashed black square. i Working model. Left, during brain development, endogenous circadian clock drives retinotectal synapses (made by RGC axons on TN dendrites) to grow faster at daytime and slower at nighttime, a process due to rhythmic synapse formation (red puncta) but not synapse elimination (yellow puncta). The HCRT neural system is involved in this circadian retinotectal synaptogenesis rhythm. Right, disruption of the circadian synaptogenesis rhythm causes defects in the development of retinotectal synapses, RGC axon arbors and TN function. Scale bars, 10 μm. Numbers in brackets indicate the numbers of RGCs (top) and fish (bottom) examined. The same datasets were used in (b–d) or in (e–g). n.s., not significant; *P < 0.05, **P < 0.01, ***P < 0.001 (nonparametric two-tailed Mann–Whitney test in (b–g); one-tailed two-sample Kolmogorov-Smirnov test in (h)). Boxplots in (b–g): centre, median; bounds of box, first and third quartiles; whiskers, minimum and maximum values. Source data are provided as a Source Data file.