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

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

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

Circadian Timing of Injury-Induced Cell Proliferation in Zebrafish

<div><p>In certain vertebrates such as the zebrafish, most tissues and organs including the heart and central nervous system possess the remarkable ability to regenerate following severe injury. Both spatial and temporal control of cell proliferation and differentiation is essential for the successful repair and re-growth of damaged tissues. Here, using the regenerating adult zebrafish caudal fin as a model, we have demonstrated an involvement of the circadian clock in timing cell proliferation following injury. Using a BrdU incorporation assay with a short labeling period, we reveal high amplitude daily rhythms in S-phase in the epidermal cell layer of the fin under normal conditions. Peak numbers of S-phase cells occur at the end of the light period while lowest levels are observed at the end of the dark period. Remarkably, immediately following amputation the basal level of epidermal cell proliferation increases significantly with kinetics, depending upon the time of day when the amputation is performed. In sharp contrast, we failed to detect circadian rhythms of S-phase in the highly proliferative mesenchymal cells of the blastema. Subsequently, during the entire period of outgrowth of the new fin, elevated, cycling levels of epidermal cell proliferation persist. Thus, our results point to a preferential role for the circadian clock in the timing of epidermal cell proliferation in response to injury.</p> </div

Early proliferating cells contribute to the formation of the new epidermis.

<p>(A) Left section: Schematic cartoon of an adult zebrafish caudal fin where the amputation site is indicated (Amp.) and the location of the stump and blastema (b) regions is defined. Right section: Schematic diagram of a transverse section through the zebrafish adult caudal fin. The identity of the principal structures is indicated. (B) Transverse sections of fins that 24 hours following amputation were labeled for 15 minutes with BrdU and then sampled at 24, 72 and 144 hpa. Histological sections through the tip of the new regenerating fin tissue (regenerated) and through the “original” portion of the fin (stump) are represented. Representative blue stained BrdU positive nuclei are indicated by black arrows and are predominantly restricted to the epidermal layers of the stump at all time points and in the regenerated epidermis at 72–144 hpa. (C) Sections from a comparable experiment to that presented in panel B, except that the 15 minutes BrdU labeling period was performed 72 hours after amputation. BrdU positive nuclei are visible in both epidermis (black arrows) and in the blastema region (red arrows) at all time points in the regenerating tissue.</p

Time of amputation defines kinetics of increased epithelial cell proliferation.

<p>(A, B) BrdU incorporation in caudal fins from fish maintained under LD cycles and amputated at the end of the light period (A, ZT12, dark blue bars) or at the end of the dark period (B, ZT0, light blue bars). Results from non-amputated control fish are plotted in both panels (black bars, A and B). In both the panels, on the Y-axis is plotted the % of BrdU positive nuclei with respect to the largest value (A, 48 hpa; B, 36 hpa). A significant increase in cell proliferation is evident sooner in fish amputated at ZT12 (A, 10–12 hpa) compared with fish amputated at ZT0 (B, 22 hpa). Each time point represents the mean value +/− SEM calculated for a minimum of n = 6 fish. In both panels, the first time point showing a significant difference from the control is indicated by the symbol “#” and a bracket. Black and white bars indicate dark and light periods. (C) Levels of <i>zfcyclin B1</i> mRNA expression following amputation either at ZT0 (red trace) or ZT12 (blue trace).</p

Mechanical abrasion increases circadian cell proliferation.

<p>(A), Upper section: Representative image of BrdU-stained caudal fin 48 hours after half of its surface was abraded. The remaining, non-treated half of the fin served as an internal control. Lower section: The results of quantification of the number of BrdU positive nuclei measured each 6 hours during one 24 hours period between 24 and 48 hours following abrasion performed at ZT3. On the Y-axis is plotted the % of BrdU positive nuclei with respect to the largest value (ZT15, abraded). (B) Western blot analysis using P-H3 Ser 10 and H3 antibodies and its quantification (below) of whole protein extracts prepared from the abraded and non-abraded (control) sections of fins. On the Y-axis is plotted the % of grey scale with respect to the highest value (ZT17, abraded). The precise times of sample preparation are indicated by ZT times. Each time point represents the mean value +/− SEM calculated for a minimum of n = 6 fish. The results of statistical analysis of the peak and trough values for the abraded fins are indicated by asterisks (Bonferroni's <i>post hoc</i> test p<0.0001) and horizontal “brackets” above the graphs (A and B). Furthermore, statistically significant differences observed at each time point between the abraded and non-abraded control fins are indicated for simplicity, by the symbol “#” and a bracket above only the first time point (panel A, Bonferroni's <i>post hoc</i> test p<0.001 and panel B, Bonferroni's <i>post hoc</i> test p<0.0001). Black and white bars represent the dark and light periods. All the quantitative data were subjected to Cosinor analysis to test for the presence or absence of 24-h rhythmicity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034203#pone.0034203.s005" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034203#pone.0034203.s002" target="_blank">Figure S2</a>).</p

Circadian rhythms of S-phase in zebrafish fins.

<p>(A) Numbers of BrdU positive nuclei in adult caudal fins oscillate under LD cycle conditions and (B) during the first day of DD following transfer from LD. On the Y-axis is plotted the % of the BrdU positive nuclei with respect to the peak points. (C, D) Quantitative RT-PCR analysis of <i>zfp21</i> and <i>zfcyclin A2</i> expression during 2 days of exposure to LD cycles. In each panel, the time of each sample is indicated either as zeitgeber time (ZT) (A, B, C, D) or circadian time (CT) (B). In each panel, each point is plotted as the mean +/− SEM of three independent experiments, each including a minimum of n = 4 fins per point. The results of statistical analysis are indicated above each graph by asterisks (Bonferroni's <i>post hoc</i> test p<0.0001) and horizontal “brackets” drawn between the peak and trough values analyzed. White and black bars below indicate the light and dark periods. All the data were subjected to Cosinor analysis to test for the presence or absence of 24-h rhythmicity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034203#pone.0034203.s005" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034203#pone.0034203.s002" target="_blank">Figure S2</a>).</p