Depletion of xBMAL1 or xNOCTURNIN results in fewer somites on the injected side (asterisk).

<p>Results of injection of 1 ng of either control, <i>xBmal1</i>, or <i>xNocturnin</i> MO are shown. Panel A shows the percent of embryos with equal, less, or more somites on the injected side when compared to the uninjected side. Embryos were also analyzed for effects on the posterior striping pattern of <i>xESR9</i> (B). The percent of embryos with equal, less, or more <i>xESR9</i> stripes on the injected side when compared to the uninjected side is indicated on the vertical axis while the type of MO is shown on the horizontal axis. All pictures shown in panels C-N are displayed with anterior to the left. Panels C, G, and K display the uninjected side for each treatment. Panels D, H, and L display the injected side for control MO, <i>xBmal1</i>MO, and <i>xNocturnin</i>MO, respectively. Panels E, I, and M show a dorsal view of each embryo for somite staining while panels F,J, and N show a dorsal view of <i>xESR9</i> expression. Black arrowheads in F show normal <i>xESR9</i> expression in the posterior. Arrowheads in J show an example where no stripes are visible but the posterior border was different between injected and uninjected sides of the embryo. The embryo in panel N experienced slight exogastrulation, but somite expression and <i>xESR9</i> expression were evaluated. White arrowheads show an example of decreased expression of <i>xESR9</i> in the eye on the side injected with <i>xNocturnin</i> MO.</p

Circadian Genes, <i>xBmal1</i> and <i>xNocturnin</i>, Modulate the Timing and Differentiation of Somites in <i>Xenopus laevis</i>

<div><p>We have been investigating whether <i>xBmal1</i> and <i>xNocturnin</i> play a role in somitogenesis, a cyclic developmental process with an ultradian period. Previous work from our lab shows that circadian genes (<i>xPeriod</i>1, <i>xPeriod2</i>, <i>xBmal1</i>, and <i>xNocturnin</i>) are expressed in developing somites. Somites eventually form the vertebrae, muscles of the back, and dermis. In <i>Xenopus</i>, a pair of somites is formed about every 50 minutes from anterior to posterior. We were intrigued by the co-localization of circadian genes in an embryonic tissue known to be regulated by an ultradian clock. Cyclic expression of genes involved in <i>Notch</i> signaling has been implicated in the somite clock. Disruption of <i>Notch</i> signaling in humans has been linked to skeletal defects in the vertebral column. We found that both depletion (morpholino) and overexpression (mRNA) of xBMAL1 protein (bHLH transcription factor) or xNOCTURNIN protein (deadenylase) on one side of the developing embryo led to a significant decrease in somite number with respect to the untreated side (p<0.001). These manipulations also significantly affect expression of a somite clock component (<i>xESR9</i>; p<0.05). We observed opposing effects on somite size. Depletion of xBMAL1 or xNOCTURNIN caused a statistically significant decrease in somite area (quantified using NIH ImageJ; p<0.002), while overexpression of these proteins caused a significant dose dependent increase in somite area (p<0.02; p<0.001, respectively). We speculate that circadian genes may play two separate roles during somitogenesis. Depletion and overexpression of xBMAL1 and NOCTURNIN both decrease somite number and influence expression of a somite clock component, suggesting that these proteins may modulate the timing of the somite clock in the undifferentiated presomitic mesoderm. The dosage dependent effects on somite area suggest that xBMAL1 and xNOCTURNIN may also act during somite differentiation to promote myogenesis.</p></div

Overexpression of xBMAL1 or xNOCTURNIN protein results in larger, disorganized somites with disrupted somite boundaries.

<p>Panel A represents the results of analyzing paired somite area (uninjected side vs injected) using NIH Image J. The average ratio of the area of injected to uninjected sides of the embryos analyzed is shown with error bars representing standard deviation. A significant difference in area between 150 pg <i>xBmal1</i> RNA injection (*; p<0.02) and 500 pg <i>xNocturnin</i> RNA injection was observed (*; ANOVA posthoc LSD compared to control MO, p<0.009). Panels B-G compare muscle actin (12/101) of the uninjected (B, C, F) and injected sides (D, E, G) of embryos overexpressing xBMAL1 (D, 500 pg; G, 150 pg) or xNOCTURNIN (E, 500 pg). All images were taken at the same magnification (scale bar = 10 µm).</p

<i>xClock</i>, <i>xCry1</i>, and <i>xCry2</i> are expressed in developing somites of tailbud stage embryos.

<p>Expression of each gene in the whole embryo and somites is provided. Panels A and B show <i>xClock</i> expression in the developing somites of a stage 35/36 embryo. Panels C and D show <i>xCry1</i> expression in the developing somites of a stage 37/38 embryo. Panels E and F show <i>xCry2</i> expression in a stage 37/38 embryo. White arrow heads indicate the anterior-posterior borders at the ventral extent of one somite.</p

Here we speculate on the possible interactions between cell autonomous circadian and somite oscillator components during somite formation and differentiation.

<p>The somite and circadian clocks both consist of negative feedback loops requiring the transcriptional activation (BMAL1, CLOCK) and repression (HES6/7, HER1; PERIOD1/2, CRY1/2) of clock components. The period of the somite clock is thought to be regulated by the balance of bHLH transcription factors present in the cell (“dimer cloud”) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Schroter1" target="_blank">[13]</a>. Bmal1 may upset the balance by hetero-dimerizing with proteins in the dimer cloud (HES6/7, HER1) or by competing with HES6/7/HER1 for binding to the H-box (green arrow). NOCTURNIN may also impact the balance of proteins present in the dimer cloud by inhibiting translation of dimer cloud components (red line). BMAL1-CLOCK heterodimers are known to positively activate genes involved in myogenesis (<i>MyoD</i>, <i>MRF5</i>). Perhaps, NOCTURNIN inhibits translation of repressors of myogenesis (blue arrow). We hypothesize that phosphorylation of CREB protein (CREB-P) may also act to coordinate components of the circadian and somite clock.</p

Reduction in xBMAL1 and xNOCTURNIN protein by morpholino injection.

<p>Both cells of a two celled embryos were injected with 1 ng Control Morpholino (Ctrl; 2 ng total), 1 ng or 500 pg of xBmal1 MO (Bmal1; 2 ng and 1 ng total) and 1 ng of <i>xNocturnin</i> MO (Noc; 2 ng total). Significant reduction of xBMAL1 protein (69Kd) was observed with injection of 2 ng or 1 ng <i>xBmal1</i> MO compared to control MO injection (0.11 and 0.26 relative to control MO injected protein levels). An approximate 50% reduction of NOCTURNIN (43Kd, indicated) protein was observed when embryos were injected with a total of 1 ng <i>xNocturnin</i> MO (0.49 relative to control MO injected protein levels). The Nocturnin antibody also recognizes a larger (62Kd) band which likely represents a postranslationally modified form of xNOCTURNIN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Green2" target="_blank">[28]</a>. Alpha tubulin (100Kd) was used as a loading control for each lane.</p

Effects of morpholino injection on somite number in stage 24–28 embryos.

<p>Effects of morpholino injection on somite number in stage 24–28 embryos.</p

Effect of RNA injection on somite number in stage 24–28 embryos.

<p>Effect of RNA injection on somite number in stage 24–28 embryos.</p

A compilation of results from microarray analyses of temporal expression of Notch, WNT, and circadian genes in the somites [34] and suprachiasmatic nucleus, liver, and heart [35], [36], [37], [38].

<p>*CREB1 phosphorylation rhythm in <i>Xenopus</i> retina <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Li1" target="_blank">[42]</a>.</p><p>A compilation of results from microarray analyses of temporal expression of Notch, WNT, and circadian genes in the somites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Curran1" target="_blank">[34]</a> and suprachiasmatic nucleus, liver, and heart <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Dequeant1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Panda1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Storch1" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108266#pone.0108266-Oishi1" target="_blank">[38]</a>.</p

Overexpression of xBMAL1 or xNOCTURNIN results in fewer somites on the injected side (asterisk).

<p>Panel A shows the percent of embryos with equal, less, or more somites on the injected side when compared to the uninjected side. The concentration and type of RNA injected is shown on the horizontal axis. Embryos were also analyzed for effects on the posterior striping pattern of <i>xESR9</i> (B). The percent of embryos with equal, less, or more <i>xESR9</i> stripes on the injected side when compared to the uninjected side is indicated on the vertical axis while the concentration and type of RNA is shown on the horizontal axis. All pictures shown in panels C-O are displayed with anterior to the left and dorsal up. Panels C, G, K display the uninjected side for each treatment. Panels D, H, and L display the injected side. Panels E, I, and M show a dorsal view of each embryo for somite staining while panels F, J, and O show a dorsal view of <i>xESR9</i> expression. A GFP RNA injected embryo (500 pg) is shown in panels C, D, E, and F. <i>xBmal1</i> RNA injected embryos (500 pg) are shown in panels G, H, I, and J. <i>xNocturnin</i> RNA (500 pg) injected embryos is shown in panels K, L, M, and O. Black arrowheads show an example where the posterior <i>xESR9</i> stripes were aligned (F) or not aligned (J, O) between the injected and uninjected sides.</p