Correction: Live Imaging of Innate Immune Cell Sensing of Transformed Cells in Zebrafish Larvae: Parallels between Tumor Initiation and Wound Inflammation

<p>Correction: Live Imaging of Innate Immune Cell Sensing of Transformed Cells in Zebrafish Larvae: Parallels between Tumor Initiation and Wound Inflammation</p

Temperature Steps Regulate Clock Gene Expression Levels

<div><p>(A) Larvae were raised in DD at 21 °C for 7 d and then shifted to 29 °C and harvested at the indicated times relative to the temperature shift (h). Controls remained at 21 °C and were harvested in parallel with the temperature shift larvae. RPA analysis of the indicated genes was then performed. “t” represents a tRNA control sample.</p> <p>(B) As in (A), except that 5-d-old larvae were shifted from 29 °C to 21 °C, and controls remained at 29 °C.</p> <p>All data are representative of at least three independent experiments.</p></div

Rhythmic Clock Gene Expression under LD and Temperature Cycles

<div><p>Graphical summary of RPA assays are described:</p> <p>(A) <i>Per4</i> (solid line) and <i>cry3</i> mRNA expression (dashed line) in zebrafish larvae raised for 6 d either in a light (12 h) or dark (12 h) cycle at a constant temperature (25.3 °C).</p> <p>(B) <i>Per4</i> (solid line) and <i>cry3</i> mRNA expression (dashed line) in zebrafish larvae raised for 6 d in DD, under a temperature cycle of 4 °C (23.5 °C/11 h, 27.5 °C/11 h, plus 1 h for each heating and cooling phase). RNA samples were harvested during the seventh day (ZT0 is defined as the beginning of the heating and light periods).</p> <p>(C and D) Equivalent analysis of <i>clock1</i> (solid line) and <i>cry2a</i> (dashed line) expression in (C) LD, and (D) temperature cycle larvae.</p> <p>(E) <i>Per2</i> expression was assayed in LD (dashed line) or temperature cycle (ΔT) larvae (solid line). By linear regression analysis, the slope of the ΔT trace has no significant deviation from zero (R² = 0.033 and <i>p</i> = 0.66, F-test). The LD cycle curve fits to a 6th-order polynomial regression model (R² = 0.96 and Runs test for deviation from model <i>p</i> = 0.99).</p> <p>In each case, zeitgeber time is plotted on the <i>x</i>-axis while the relative expression levels (percentage) are plotted on the <i>y</i>-axis. <i>β-actin</i> levels were used to standardize the results. The highest band intensity in each experiment was arbitrarily defined as 100%, and then all other values were expressed as a percentage of this value. All experiments were performed in triplicate, and error bars denote the standard deviation.</p></div

Temperature Influences CLK Protein Expression and Function

<div><p>(A) In vitro luciferase assays of transiently transfected PAC2 cells. The combinations of CLK (Clk) and BMAL (Bml) expression vectors cotransfected with the 4x Ebox (−7) reporter plasmid are indicated for each assay result. Control cells were transfected with the reporter plasmid or with the pGL3 Control plasmid alone. Values represent the mean fold difference between luciferase activities measured in 30 °C and 20 °C, 60 h after transfection. All assays were standardized for transfection efficiency using a β-galactosidase assay. The results are based on four independent experiments, and error bars indicate the standard deviation.</p> <p>(B) Electrophoretic mobility shift assay of nuclear extracts from PAC-2 cells cultured at 20 °C or 30 °C on a LD cycle, and harvested at ZT3, 9, 15, and 21 (lanes 1 to 8). Three specific complexes are indicated by A, B, and an asterisk. Supershift assays of a ZT15, 30 °C extract (+Ab), used either a dopamine transporter antibody (Control) or a mouse clk antibody (Clock) (lanes 9 and 10). Complexes indicated by A, B, and an asterisk are all efficiently competed by a 25-, 50-, and 100-fold excess of cold E-box probe (lanes 12, 13, and 14, respectively, and compare with lane 11), but not with a 100-fold excess of a CRE probe (compare lane 15 with lane 11).</p> <p>(C) Western blotting assay using the anti-mouse CLK antibody of the same nuclear extracts tested in the electrophoretic mobility shift assay analysis of panel B. The migration of a 100-kDa marker band is shown. Below are shown western blotting results for the same extracts using an anti-mouse CREB antibody as a loading control.</p> <p>(D) Western blot assay of CLK protein in 30 °C extracts prepared at ZT9 or ZT21 (time points representing the trough and peak, respectively, of the CLK protein rhythm). Samples were prepared with (+) or without (−) treatment with alkaline phosphatase prior to electrophoresis and transfer. In panels B, C, and D, data are representative of at least three independent experiments.</p></div

Temperature Compensation and the Amplitude of E-box-Directed Rhythmic Expression

<div><p>(A) Bioluminescence profile of 4xE-box (−7) reporter cells held at 20 °C under a LD cycle and then transferred to DD conditions. Plates were counted once per hour and maintained in robotic stacking units between assays, where they were illuminated.</p> <p>(B) Equivalent experiment to panel A, with cells maintained at 30 °C.</p> <p>(C) Bioluminescence traces from 1.7-kb WT <i>per4</i> reporter cells maintained at 20 °C under LD cycle and DD conditions.</p> <p>(D) Bioluminescence traces from 1.7-kb WT <i>per4</i> reporter cells maintained at 30 °C under LD cycle and DD conditions.</p> <p>(E) RPA analysis of <i>per4</i> expression in WT PAC-2 cells held at 20 °C and 30 °C under an LD cycle for 3 d. RNA extracts were prepared on the fourth day at 3-h intervals during one 24-h cycle. Time 0 represents ZT 0: the onset of the light period. A white and black bar above the autoradiograph indicates the duration of the light and dark periods. RPA results with a β-actin loading control are also shown. “t” represents a tRNA control sample.</p> <p>(F) A bar graph shows quantification of the peak (ZT3) and trough (ZT15) <i>per4</i> expression values at 20 °C and 30 °C plotted as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030351#pbio-0030351-g001" target="_blank">Figure 1</a>, with error bars representing the standard deviation of three independent experiments.</p> <p>All bioluminescence traces represent the mean values of 16 independent wells. Each panel is representative of at least three independent experiments.</p></div

Model for Temperature Regulation of the <i>per4</i> Promoter

<div><p>(A) Temperature steps entrain the phase of the clock by driving expression levels of <i>per4</i> and other clock genes via a hypothetical enhancer element X. Temperature decreases result in expression increases, and vice versa. Although E-boxes ultimately mediate regulation of the <i>per4</i> promoter by the entrained clock, they do not participate in the temperature-driven response.</p> <p>(B) Temperature influences the amplitude of rhythmic <i>per4</i> expression that has been entrained by LD cycles in two ways: (1) by determining the amplitude of E-box-directed rhythmic expression, via changes in CLK protein levels, phosphorylation, and E-box binding, and (2) by driving expression changes through element X (see panel A). The promoter integrates these two regulatory mechanisms. The temperature-dependent amplitude of E-box-directed rhythmic expression would be predicted to involve the core feedback loops of the clock itself and, according to mathematical models, might thereby underlie temperature compensation.</p></div

Differential effect of cycloheximide on light-induced gene expression.

<p>(A–F) Quantitative RT-PCR analysis (qRT-PCR) of light inducible genes in PAC-2 cells in the presence (red traces) or absence (black traces) of cycloheximide (CHX) during 8 hours of light exposure (left panels) or constant darkness conditions (right panels). Cells were maintained for 3 days in DD prior to the experiment. 1 h before sampling, cells were treated with CHX (10 µg/ml). Each gene is indicated above its respective panels. Yellow and black bars above each panel indicate the light and dark periods, respectively. Relative mRNA levels are plotted on the y-axes and were set arbitrarily as 1 at time-point 0 hrs for each gene. Endogenous <i>β-actin</i> mRNA levels were not influenced by light or cycloheximide treatment and so these were used to normalize the expression of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051278#pone.0051278.s001" target="_blank">Figure S1</a> B). Time (hrs) is plotted on the x-axes. In each panel, points are plotted as the means of three independent experiments +/− SD. All statistical analyses (t-test and two-way ANOVA) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051278#pone.0051278.s006" target="_blank">Table S2</a>. The blocking of protein synthesis by cycloheximide treatment of PAC-2 cells was confirmed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051278#pone.0051278.s001" target="_blank">Figure S1</a> A.</p

Regulation of <em>per</em> and <em>cry</em> Genes Reveals a Central Role for the D-Box Enhancer in Light-Dependent Gene Expression

<div><p>Light serves as a key environmental signal for synchronizing the circadian clock with the day night cycle. The zebrafish represents an attractive model for exploring how light influences the vertebrate clock mechanism. Direct illumination of most fish tissues and cell lines induces expression of a broad range of genes including DNA repair, stress response and key clock genes. We have previously identified D- and E-box elements within the promoter of the zebrafish <em>per2</em> gene that together direct light-induced gene expression. However, is the combined regulation by E- and D-boxes a general feature for all light-induced gene expression? We have tackled this question by examining the regulation of additional light-inducible genes. Our results demonstrate that with the exception of <em>per2</em>, all other genes tested are not induced by light upon blocking of <em>de novo</em> protein synthesis. We reveal that a single D-box serves as the principal light responsive element within the <em>cry1a</em> promoter. Furthermore, upon inhibition of protein synthesis D-box mediated gene expression is abolished while the E-box confers light driven activation as observed in the <em>per2</em> gene. Given the existence of different photoreceptors in fish cells, our results implicate the D-box enhancer as a general convergence point for light driven signaling.</p> </div

Contribution of <i>de novo</i> protein synthesis to light–induced clock gene expression.

<p>(A) Under normal conditions light exposure triggers expression of the gene encoding the PAR bZip factor, TEF-1. This in turn binds to D-boxes in the <i>cry1a</i> and <i>per2</i> promoters and trans-activates gene expression. In parallel, light also entrains the circadian clock. Via binding of the CLOCK–BMAL complex, the clock regulates the E-box in the <i>per2</i> promoter and thereby contributes to light induced gene expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051278#pone.0051278-Vatine2" target="_blank">[21]</a>. The clock also regulates expression of the additional PAR bZip factors (PAR) that contribute to D-box driven transcription. (B) Upon light exposure and coincident inhibition of <i>de novo</i> protein synthesis by treatment with cycloheximide (+CHX), translation of TEF-1 and the other PAR bZip factors is prevented. Therefore, light-driven transactivation via the D-box enhancer of the <i>cry1a</i> promoter is abolished. However, light-induced expression of the <i>per2</i> promoter persists due to regulation by the E-box. Specifically, upon cycloheximide treatment the core clock machinery directs increased activation via the E-box in a light dependent manner. We speculate that this up-regulation of E-box driven expression may also influence other clock-regulated genes including those encoding the PAR bZip factors.</p

Light induced D-box enhancer activity requires <i>de novo</i> protein synthesis.

<p>(A–C and E) qRT-PCR analysis of luciferase mRNA expression in PAC-2 cells transfected with different heterologous luciferase reporter constructs, in the presence (red traces) or absence (green traces) of CHX during 8 hours of light exposure or DD conditions (+CHX, blue traces, −CHX, black traces). (D) qRT-PCR analysis of endogenous <i>per1b</i> expression in PAC-2 cells in the presence or absence of CHX during 8 hours of light exposure or DD conditions (colour coded the same as in panels A–C and E). Each construct is indicated above its respective panel. Relative mRNA levels are plotted on the y-axis and were set arbitrarily as 1 at time-point 0 hrs. Time (hrs) is plotted on the x-axis. In each panel, points are plotted as means of three independent experiments +/− SD. All statistical analyses (two-way ANOVA) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051278#pone.0051278.s006" target="_blank">Table S2</a> B or in the results section.</p