Figure 6

Identification of molecular mechanisms underlying the reduced activity of DKO and TKO zebrafish. (A) Left and middle panels: WT (solid line) and DKO (dotted line) zebrafish were raised in DD conditions and exposed to 3-h (left panel) or 12-h (middle panel) light at the beginning of 6 dpf. The expression level of zAanat2 was examined by RT-PCR analysis. Right panel: WT (solid line) and TKO (dotted line) zebrafish were raised in DD conditions and exposed to 12-h light at the beginning of 6 dpf. The expression level of zAanat2 was examined by RT-PCR analysis. In all experiments, the value at time point 0 was set to 1. Values are mean ± S.E.M. of three independent experiments. (B) Zebrafish were raised in DD conditions and exposed to a 12-h light pulse at the beginning of 6 dpf. At indicated time points after the onset of light, melatonin levels in each WT (solid line) and TKO (dotted line) zebrafish were examined by LC-MS/MS. Values are mean ± S.E.M. of three independent experiments. (C) Heatmap analysis of microarray data showing hierarchical clustering of differentially expressed genes in WT zebrafish kept in darkness relative to WT, DKO, and TKO zebrafish exposed to 3-h or 12-h light. Red or green colors indicate differentially up- or downregulated genes, respectively. Hierarchical cluster analysis of the microarray data identified seven gene clusters. (D) Measurement of ATP levels in WT, DKO and TKO zebrafish. WT, DKO and TKO animals raised in constant darkness were exposed to light for 12-h at the beginning of 6 dpf. They were then kept in constant darkness for 12-h and prepared for the ATP assay. In both experiments for DKO and TKO zebrafish, the average ATP level in WT zebrafish was set to 100%. Left panel; WT (n = 7), DKO (n = 5). Right panel; WT (n = 8), TKO (n = 8). P < 0.01 and P < 0.05 (Student’s t-test).