Curran KL et al. (2014), Circadian genes, xBmal1 and xNocturnin, modulat...

XB-ART-49436

PLoS One 2014 Jan 01;99:e108266. doi: 10.1371/journal.pone.0108266.

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Circadian genes, xBmal1 and xNocturnin, modulate the timing and differentiation of somites in Xenopus laevis.

Curran KL, Allen L, Porter BB, Dodge J, Lope C, Willadsen G, Fisher R, Johnson N, Campbell E, VonBergen B, Winfrey D, Hadley M, Kerndt T.

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We have been investigating whether xBmal1 and xNocturnin play a role in somitogenesis, a cyclic developmental process with an ultradian period. Previous work from our lab shows that circadian genes (xPeriod1, xPeriod2, xBmal1, and xNocturnin) are expressed in developing somites. Somites eventually form the vertebrae, muscles of the back, and dermis. In Xenopus, 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 Notch signaling has been implicated in the somite clock. Disruption of Notch 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 (xESR9; 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.

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Species referenced: Xenopus laevis
Genes referenced: axin2l bmal1 clock creb1 cry1 cry2 csnk1e dkk1 dll1 egfr hes1 hes5.6 hes5.7 hes6.2 myod1 noct notch1 per1 rora sp5 tnfrsf9
???displayArticle.antibodies??? Arntl Ab1 Noct Ab1 Somite Ab3 Tuba4b Ab15
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	Figure 1. xClock, xCry1, and xCry2 are expressed in developing somites of tailbud stage embryos. Expression of each gene in the whole embryo and somites is provided. Panels A and B show xClock expression in the developing somites of a stage 35/36 embryo. Panels C and D show xCry1 expression in the developing somites of a stage 37/38 embryo. Panels E and F show xCry2 expression in a stage 37/38 embryo. White arrow heads indicate the anterior-posterior borders at the ventral extent of one somite. doi:10.1371/journal.pone.0108266.g001
	Figure 2. Reduction in xBMAL1 and xNOCTURNIN protein by morpholino injection. 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 xNocturnin MO (Noc; 2 ng total). Significant reduction of xBMAL1 protein (69Kd) was observed with injection of 2 ng or 1 ng xBmal1 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 xNocturnin 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 [28]. Alpha tubulin (100Kd) was used as a loading control for each lane. doi:10.1371/journal.pone.0108266.g002
	Figure 3. Depletion of xBMAL1 or xNOCTURNIN results in fewer somites on the injected side (asterisk). Results of injection of 1 ng of either control, xBmal1, or xNocturnin 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 xESR9 (B). The percent of embryos with equal, less, or more xESR9 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, xBmal1MO, and xNocturninMO, 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 xESR9 expression. Black arrowheads in F show normal xESR9 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 xESR9 expression were evaluated. White arrowheads show an example of decreased expression of xESR9 in the eye on the side injected with xNocturnin MO. doi:10.1371/journal.pone.0108266.g003
	Figure 4. Depletion of xBMAL1 or xNOCTURNIN results in smaller, disorganized somites with disrupted somite boundaries. Panel A represents the results of analyzing the somite area (uninjected side vs injected) of pairs of somites 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. Somites injected with either xBmal1 MO (1 ng) or xNocturnin MO (1 ng) showed a significant decrease in somite area (*) when compared to the control MO (1 ng). Images in panels B-F are oriented with the anterior of the embryo to the left and dorsal up. Panel B shows an extreme phenotype where somite boundaries have been eliminated on the injected side (1 ng xBmal1 MO). Panels C (uninjected) and E (injected) show effects on somite size, organization and boundaries in an embryo injected with 1 ng (xBmal1 MO). Expression from the uninjected side of the embryo can be seen through the cleared embryo in E, grey dots separate foreground image from the background. Panels D and F show similar results were observed with injection of xNocturnin MO (1 ng) although there is usually less disruption of somite borders (D, uninjected; F, xNocturnin MO injected). All images were taken at the same magnification (scale bar = 10 Âµm). doi:10.1371/journal.pone.0108266.g004
	Figure 5. Overexpression of xBMAL1 or xNOCTURNIN results in fewer somites on the injected side (asterisk). 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 xESR9 (B). The percent of embryos with equal, less, or more xESR9 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 xESR9 expression. A GFP RNA injected embryo (500 pg) is shown in panels C, D, E, and F. xBmal1 RNA injected embryos (500 pg) are shown in panels G, H, I, and J. xNocturnin RNA (500 pg) injected embryos is shown in panels K, L, M, and O. Black arrowheads show an example where the posterior xESR9 stripes were aligned (F) or not aligned (J, O) between the injected and uninjected sides. doi:10.1371/journal.pone.0108266.g005
	Figure 6. Overexpression of xBMAL1 or xNOCTURNIN protein results in larger, disorganized somites with disrupted somite boundaries. 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 xBmal1 RNA injection (; p<0.02) and 500 pg xNocturnin 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 Âµm). doi:10.1371/journal.pone.0108266.g006
	Figure 7. Here we speculate on the possible interactions between cell autonomous circadian and somite oscillator components during somite formation and differentiation. 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 (â€œdimer cloudâ€) [13]. 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 (MyoD, MRF5). 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. doi:10.1371/journal.pone.0108266.g007
	Figure S1. Circadian genes are expressed in the somites during tailbud stages. Co-localization of the mRNA expression and 12/101 protein (somite marker) are shown in each pair of panels, such as A and Aâ€². The white dotted lines were drawn on the borders of the in situ expression pattern for each gene and positioned in the exact same position over the 12/101 expression. In all cases the circadian genes were present throughout the somite and excluded from the myocoel.
	Figure S2. A summary of the developmental expression of xClock, xCry1, and xCry2. In situ hybridization was performed on stage 35â€“38 embryos. Dorsal view of the head and lateral views of the entire embryo are shown for xClock (A,B), xCry1 (C,D), and xCry2 (E,F). Black arrowheads highlight the otic vesicle while white arrows show expression in the olfactory bulb. Red arrowheads highlight the pronephric tubules while red arrows show the pronephric duct if visible. The cement gland is indicated by a green arrowhead. A developmental time series is provided (G) below the images to show the earliest we were able to detect each gene's expression in various embryonic organs and tissues.
	Figure S3. Depletion of xBMAL1 or xNOCTURNIN results in fewer somites on the injected side. The percent of embryos with equal, less, or more somites on the injected side when compared to the uninjected side is indicated on the vertical axis. The concentration and type of MO injected is shown on the horizontal axis. Injection of 500 pg of xBmal1 MO (Nâ€Š=â€Š54) consistently resulted in fewer somites when compared to control MO Injection (500 pg; Nâ€Š=â€Š26). Injection of 1.5 ng of xNocturnin MO (Nâ€Š=â€Š33) consistently resulted in fewer somites when compared to the more variable phenotype displayed by control MO injection (Nâ€Š=â€Š30).
	Figure S4. In some cases, depletion of xBMAL1 and xNOCTURNIN protein affected xESR9 expression in the developing eye and central nervous system. In panel A, depletion of xBMAL1 protein (500 pg xBmal1MO injection; ) decreased expression of xESR9 in the eye (white arrowhead). Comparison of the width of xESR9 expression in the hindbrain and spinal cord shows a wider expression of xESR9 on the injected side, indicated by the width of the white line, when compared to the uninjected side (width of black line). Panels B and C show the effects of depletion of xNOCTURNIN (1 ng, ). Depletion of xNOCTURNIN decreased expression of xESR9 in the eye (white arrow head) and decreased xESR9 expression in the brain and spinal cord. The embryo in panel C was also anencephalic.
	cry1 (cryptochrome circadian clock 1) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.
	cry2 (cryptochrome circadian clock 2) gene_symbol (gene_name) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 36, lateral view, anterior left, dorsal up.

References [+] :

Andrews, CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function. 2010, Pubmed