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Fig. 15 TEF is required for the light-induced per2 promoter activation in vivo.
Transgenic Tg(-0.43per2:EGFP)tlv1 embryos were injected with tef-MO or control MO. Injected and uninjected embryos were entrained for two LD cycles. At the beginning of the third day of development, embryos were either exposed to light or kept in darkness. After 2 h (ZT 2) embryos were fixed and subjected to whole mount ISH for egfp or otx5 mRNA. (A) Tef (E2I2) MO injection altered tef mRNA splicing. Sequence analysis of the PCR products indicate that tef (E2I2) MO caused the integration of the 226 nucleotides of intron 2, which creates a premature stop-codon (at position +1,303), generating a truncated, non-functional protein lacking the proline and acidic amino acid-rich region (PAR), DNA binding domain (DBD), and the basic leucine zipper domain (bZIP). Grey boxes represent exons, and a red box represents the integrated intron 2. Translation start site and integrated stop codon are marked by arrows. (B) Signal intensities, determined using ImageJ software, revealed a ∼10-fold increase in pineal egfp mRNA levels after a 2 h light pulse in uninjected (n = 20, p<0.001) and control MO injected (n = 16, p<0.001) embryos (see also Figure 3). Tef-MO-injected embryos (n = 22) showed no significant increase in pineal signal after a 2 h light pulse suggesting that tef is required for the light-induced expression driven by the -0.43per2 promoter. Error bars represent SE. Dorsal views of representative whole mounts are shown below. Red arrows indicate the location of the pineal gland. (C) Pineal zotx5 mRNA levels of tef-MO injected, control-MO-injected, and uninjected transgenic embryos were similar, indicating that the injected MOs did not affect the development of the pineal gland. Statistical analysis was performed by Kruskal-Wallis test followed by Mann-Whitney test.