Fig. 6.

Dominant negative clock (ΔCLK) reduces daytime and enhances nighttime growth of the myotome. Control EGFP (light blue) and ΔCLK (dark blue) mRNA-injected larvae were raised under LD and analyzed between 3 and 4 dpf. (A) Design of EGFP (Control) and dominant negative CLOCK-5xMyc (ΔCLK) constructs, and their corresponding EGFP accumulation at 4 dpf after mRNA microinjection at one-cell stage. (B) Western analysis between 3 and 4 dpf using c-Myc antibody showing persistent presence of ΔCLK protein in ΔCLK-expressing larvae, but not in EGFP Control larvae. Histone H3 was used as loading control. (C) Diurnal variation in level of per1b mRNA prevented by ΔCLK (n = 3 biological replicates); ef1a was used for normalization. Note that Control strengthens result shown in Fig. 5D and SI Appendix, Fig. S7B. (D) Absolute myotome volume measured at 3 dpf in α-actin:mCherryCAAX larvae (shown in SI Appendix, Fig. S9A) is reduced by ΔCLK (n = 29 fish from three biological replicates). (E) Growth of the myotome (percent) in either unanesthetized (blue) or anesthetized from 3 dpf to 4 dpf (red) in ΔCLK (dark colors) or Control (light colors) (n = 14 or 15 fish from three biological replicates). Symbols represent distinct lays. Note that Control data strengthen the result in Fig. 1B.