Skip to main content
. 2015 Feb 11;35(6):2572–2587. doi: 10.1523/JNEUROSCI.2551-14.2015

Figure 2.

Figure 2.

Identification of a zebrafish per1b insertional mutant. A, B, Insertion of the retroviral sequence into the first intron of zebrafish per1b. Arrows show the locations of PCR primers used to identify homozygous per1b mutants, heterozygotes, or wild-type fish. C, D, qRT-PCR analysis showed that per1b is rhythmically expressed in wild-type fish but its expression is nearly abolished in homozygous per1b mutants under both LD and DD conditions. One-way ANOVA test. Error bars are ±SEM. E, Whole-mount in situ hybridization experiments showed rhythmic expression of per1b in wild-type fish, but abolition of per1b expression in homozygous mutants under LD conditions. F, The homozygous per1b mutant lacks the Per1b protein. Proteins were extracted from wild-type and per1b mutant brains and eyes. Western blotting was done with tubulin antibody as an internal control. G, Immunofluorescence staining of Per1b indicated that the Per1b protein is primarily expressed in the inner nuclear layer of the retina (white arrow) and there is no detectable signal in the per1b mutant. Scale bar, 40 μm. H, Colocalization of the PER1 (green) and TH (red) proteins in dopaminergic neurons in the SN of mice, shown by immunofluorescence. Scale bar, 100 μm. I–K, Under DD conditions, per1a (I), per2 (J), and per3 (K) are all upregulated in per1b mutant zebrafish during both subjective daytime and nighttime. Expression ratios were normalized according to β-actin (RTPrimerDB ID:705) as an internal control using the 2−ΔΔCT method, and analyzed by one-way ANOVA test. L, Homozygous per1b mutants (5–10 dpf) exhibited ∼1.2 h shorter period under DD conditions. M, The relative amplitude of per1b mutants was significantly higher than wild types under DD conditions. Wild-type larvae, n = 48; per1b mutant larvae, n = 48. Student's t test. N, The per1b mutant displays a phase-advanced phenotype compared with wild types under DD conditions. The average time advanced was ∼2 h. Wild-type larvae, n = 48; per1b mutant larvae, n = 48. The number on the right side of the star indicates the p values.