Fig. 5.

Circadian variation in muscle growth and metabolism is required for volumetric growth of myotome. (A) Sibling larvae collected within 1 h to 2 h of fertilization were reared under LL or DD light regimes to prevent circadian entrainment and compared to LD, as schematized. Myotome volume was measured at 3 dpf (n = 29 to 63 fish, from three to five biological replicates). Symbols represent distinct lays (biological replicates). Orange bars represent mean ± SEM; black boxes represent global mean. Note consistent effect despite lay-to-lay variation in absolute size. (B) Circadian variation in growth of myotome (percent) in larvae raised under three cycles of LD and then switched to LL between 3 and 4 dpf (LD→LL) that were either unanesthetized (Control, dark blue) or anesthetized from 3 dpf to 4 dpf (Tricaine, dark red). Symbols represent distinct lays (n = 15 fish from three biological replicates). (C) Circadian variation in nascent protein synthesis visualized at CT0 to CT2 (subjective day) and CT12 to CT14 (subjective night) between 3 and 4 dpf, in larvae that were raised under LD→LL. Graph shows quantification of OPP−PA fluorescence of somite 17 (n = 9 fish from three biological replicates). Signals were normalized to Control CT0 to CT2 samples after subtraction of CHX background. Symbols represent distinct lays. (D) Persistent circadian variation in expression of clock genes, bmal1a and per1b, and atrophy-related genes murf1 and murf2, between 3 and 4 dpf in larvae raised under LD→LL (n = 3 biological replicates; black solid lines); ef1a was used for normalization. Representative traces from LD fish in separate experiments are shown as references (gray dashed lines).