Daily Rhythmicity of Clock Gene Transcripts in Atlantic Cod Fast Skeletal Muscle

<div><p>The classical notion of a centralized clock that governs circadian rhythmicity has been challenged with the discovery of peripheral oscillators that enable organisms to cope with daily changes in their environment. The present study aimed to identify the molecular clock components in Atlantic cod (<i>Gadus morhua</i>) and to investigate their daily gene expression in fast skeletal muscle. Atlantic cod clock genes were closely related to their orthologs in teleosts and tetrapods. Synteny was conserved to varying degrees in the majority of the 18 clock genes examined. In particular, <i>aryl hydrocarbon receptor nuclear translocator-like 2</i> (<i>arntl2), RAR-related orphan receptor A</i> (<i>rora</i>) and <i>timeless</i> (<i>tim</i>) displayed high degrees of conservation. Expression profiling during the early ontogenesis revealed that some transcripts were maternally transferred, namely <i>arntl2</i>, <i>cryptochrome 1b</i> and <i>2</i> (<i>cry1b</i> and <i>cry2</i>), and <i>period 2a</i> and <i>2b</i> (<i>per2a</i> and <i>per2b</i>). Most clock genes were ubiquitously expressed in various tissues, suggesting the possible existence of multiple peripheral clock systems in Atlantic cod. In particular, they were all detected in fast skeletal muscle, with the exception of neuronal PAS (Per-Arnt-Single-minded) domain-containing protein (<i>npas1</i>) and <i>rora</i>. Rhythmicity analysis revealed 8 clock genes with daily rhythmic expression, namely <i>arntl2</i>, circadian locomotor output cycles kaput (<i>clock</i>), <i>npas2</i>, <i>cry2</i>, <i>cry3 per2a</i>, nuclear receptor subfamily 1, group D, member 1 (<i>nr1d1</i>), and <i>nr1d2a</i>. Transcript levels of the myogenic genes <i>myogenic factor 5</i> (<i>myf5</i>) and <i>muscleblind-like 1</i> (<i>mbnl1</i>) strongly correlated with clock gene expression. This is the first study to unravel the molecular components of peripheral clocks in Atlantic cod. Taken together, our data suggest that the putative clock system in fast skeletal muscle of Atlantic cod has regulatory implications on muscle physiology, particularly in the expression of genes related to myogenesis.</p></div

Rhythmicity parameters of clock gene transcription in Atlantic cod fast skeletal muscle.

<p>Rhythmicity parameters of clock gene transcription in Atlantic cod fast skeletal muscle.</p

Expression of clock genes in fast skeletal muscle during a daily cycle.

<p>The values are mean<u>+</u>SEM (n = 6) of the normalized transcript levels of each clock gene. Statistical difference between time points is indicated by different letter notations. The broken line is the periodic sinusoidal function of gene expression in a circadian cycle constructed from the periodicity parameters calculated by COSINOR. An asterisk (*) besides the gene name indicates significance of daily rhythmicity. The photoperiod regime is represented by the composite block above the graph. White, black and gray represent the light phase, the dark phase and the light-dark transition phase, respectively.</p

Early ontogenetic expression of clock genes in Atlantic cod.

<p>The cDNAs used in semi-quantitative RT-PCR were from the pooled RNA of approximately 50 specimens from each stage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099172#pone.0099172-Ruangsri1" target="_blank">[26]</a>. <i>Luciferase</i> (<i>luc</i>) was used as an external reference.</p

Presence of clock gene transcripts in different tissues of Atlantic cod.

<p>Expression of clock genes in various tissues (blood, liver, spleen, mid-intestine, kidney, brain, pituitary, heart, gills, eyes, dorsal skin, ventral skin, testis, ovary, and fast muscle) was analyzed by semi-quantitative RT-PCR. <i>Eef1a</i> gene was used as an endogenous reference.</p

Correlation of clock and muscle-related transcript levels.

<p>Correlation of clock and muscle-related transcript levels.</p

Changes in the plasma melatonin level during a daily cycle.

<p>Plasma melatonin levels were quantified by competitive ELISA. Values shown are mean<u>+</u>SEM of plasma melatonin (n = 6). The background represents the photoperiod regime: the light background is the light phase, the dark background is the dark phase and light gray indicates the light-dark transition phase.</p

Molecular components of the clock system identified in fast skeletal muscle of Atlantic cod and myogenic genes with daily rhythmic expression.

<p>The peripheral clock components in Atlantic cod fast skeletal muscle that have been identified in this study are shown inside the green circle, which represents a muscle fiber. They comprise members of the transcriptional activator arm (in red: <i>arntl1, arntl2, clock</i> and <i>npas2</i>), transcriptional repressor arm (in blue: c<i>ry1a, cry1b, cry-dash, cry2, cry3, per1, per2a, per2b</i> and <i>tim</i>) and the stabilizing loop (in yellow: <i>nr1d1, nr1d2a</i> and <i>nr1d2b</i>). Clock genes with a colored background displayed a daily rhythmic expression in fast skeletal muscle. The daily rhythmicity of <i>myf5</i>, a gene for myogenic lineage specification, and <i>mbnl1</i>, a gene for terminal muscle differentiation, suggests a possible circadian clock control of myogenesis in Atlantic cod. The grey box indicates that the mechanism underlying this regulatory process remains to be identified.</p

Daily expression of muscle-related genes.

<p>The values presented here are mean<u>+</u>SEM (n = 6) of normalized expression. Statistical differences between time points are indicated by different letter notations. The broken line is the periodic sinusoidal function of the gene expression in a daily cycle constructed from the rhythmicity parameters provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099172#pone.0099172.s005" target="_blank">Table S3</a>. An asterisk (*) beside the gene name indicates that the expression is daily rhythmic. The photoperiod regime is represented by the composite block above the graph. White, black and gray represent the light phase, the dark phase and the light-dark transition phase, respectively.</p