Systematic Identification of Rhythmic Genes Reveals <em>camk1gb</em> as a New Element in the Circadian Clockwork

<div><p>A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, <em>camk1gb</em>, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of <em>camk1gb</em> disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that <em>camk1gb</em> plays a role in linking the pineal master clock with the periphery.</p> </div

Effect of <i>camk1gb</i> knockdown on pineal <i>aanat2</i> and <i>otx5</i> mRNA rhythms.

<p>Zebrafish embryos injected with either control morpholino (black line) or <i>camk1gb</i> morpholino (gray line) were subjected to DD during their third day of development and pineal <i>aanat2</i> and <i>otx5</i> mRNA levels were determined by whole mount ISH. A) Embryos were sampled at 4-hr intervals for <i>aanat2</i>. Statistical differences in <i>aanat2</i> mRNA levels between the control morpholino and <i>camk1gb</i> morpholino injected embryos were determined by two-tailed <i>t</i>-test with Bonferroni correction (* P-value<0.05, ** P-value<0.01, *** P-value<0.001). B) Whole-mount ISH for <i>aanat2</i> in the heads (dorsal views) of representative larvae from each group, at CT14 and CT18. Arrows indicate pineal <i>aanat2</i> mRNA expression. C) <i>otx5</i> expression at CT18. Error bars represent SE (n = 10–15). CT = circadian time.</p

Genes detected as circadian using both DNA microarray and RNA–seq.

<p>Genes detected as circadian using both DNA microarray and RNA–seq.</p

<i>camk1gb</i> spatio-temporal expression.

<p>A) Rhythmic expression of <i>camk1gb</i> exclusively in the pineal glands (indicated by black arrows) of 48–72 hpf embryos as detected by whole mount ISH under constant darkness. B) Rhythmic expression of <i>camk1gb</i> in the zebrafish embryo (right) is correlated with the RNA-seq (solid line, left vertical axis) and the microarray data (dashed line, right vertical axis) from the adult (left). Correlation coefficients between the whole mount ISH results and the data obtained by microarrays and RNA-seq were determined by Pearson correlation (r = 0.89 and 0.77, respectively). For whole mount ISH, statistical differences in mRNA levels were determined by one-way ANOVA followed by a Tukey test (P-value<0.05). Error bars represent SE (n = 10–15). CT = circadian time. Gray and black bars represent subjective day and subjective night, respectively.</p