CK1δ inhibition disrupts clock-controlled rhythmic locomotor activity in zebrafish larvae.

<p>Locomotor activity of zebrafish larvae was detected by the DanioVision observation chamber. The PF-670462 inhibitor was added to the water at 5 dpf at different concentrations, A-0.5 μM, B-1 μM and C-5 μM (red lines). Control larvae were treated with DMSO at the same concentrations (black lines). The larvae were kept under constant conditions (dim light) and the distance moved (cm, y-axis) was recorded over time (hours, x-axis). The horizontal bars represent the light conditions before and during the experiment. White boxes represent light, black boxes represent dark and grey boxes represent dim light. The rhythm of locomotor activity was completely abolished by the CK1δ inhibitor.</p

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

The effect of CK1δ inhibition on rhythmic pineal <i>aanat2</i> mRNA expression is reversible.

<p>Reversibility was determined after two (A) or three (B) LD cycles for re-entrainment. <b>Top panels</b>: Experimental design. The horizontal bars represent the light conditions before and during sampling; white boxes represent light, grey boxes represent subjective day and black boxes represent dark. Middle panels: Whole-mount ISH signals (dorsal views of the heads) of representative specimens treated with DMSO (control, a) or with PF-670462 (b). ZT 2-10b refers to the second 24 h cycle of the sampling. Bottom panels: Signal intensities in the pineal gland. Values represent the mean ± SE optical density of the pineal signals. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test for each treatment. Following removal of the inhibitor and re-entrainment, for two (A) or three (B) LD cycles, the rhythm of aanat2 mRNA re-appeared but seemed to be shifted.</p

CK1ε inhibition does not affect clock-controlled rhythmic locomotor activity in zebrafish larvae.

<p>Locomotor activity of zebrafish larvae was detected by the DanioVision observation chamber. Distance moved (cm) is plotted on the y-axis and time (hours) on the x-axis. The horizontal bars represent the lighting conditions before and during the experiment. White boxes represent light, black boxes represent dark and grey boxes represent dim light. The PF-480567 inhibitor was added to the water at two different concentrations, A-5 μM and B-10 μM (blue lines). Control larvae were treated with DMSO at the same concentrations (black lines). CK1ε inhibition did not affect the locomotor activity rhythm, but did have a slight phase shifting effect.</p

The effect of CK1δ -inhibition on peripheral circadian clocks is reversible.

<p>Bioluminescence assay of cells transfected with per1b:Luc (A) and Ebox:Luc (B). Cells were maintained under LD cycles, and then the inhibitor, PF-670462 (5 μM), was added to the cell culture 1.5 h before lights on (black arrows). Cells were maintained in LD conditions for 2 days after which the inhibitor was washed away 3.5 h before lights on (red arrows). After one LD cycle for re-entrainment, the cells were transferred to constant darkness for 24 h. Control cells were treated with DMSO. Bioluminescence is plotted on the y-axis and time (hours) on the x-axis. White/black bars show the light and dark periods, respectively. The clock-controlled rhythmic promoter activity reappeared immediately following removal of the inhibitor. Thus the effects of CK1δ inhibition are reversible.</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

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

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

Rhythmic clock gene expression in the blastema region following amputation.

<p>(A) Schematic representation of the experimental plan showing the amputation site (left section), the times of sampling (ZT and hpa) with respect to the lighting conditions (central section) and the location of the stump and blastema (b) regions analyzed (right section). Amputation was performed at the dark-light transition (ZT 0, red arrow). (B) Quantitative RT-PCR analysis of a blastema marker (<i>zfmsxb</i>) in the stump and blastema regions of amputated fins used as a control for the enrichment of blastema cells in the blastema samples. (C–G) Quantitative RT-PCR analysis of clock genes expression in the stump (black bars) and blastema (blue bars) regions of amputated fins. Each experiment was performed in triplicate with a minimum of 6 fins (n = 6) pooled together for each timepoint. Cosinor analysis of the clock gene expression in the stump as well in the blastema region shows 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>). Black and white bars beneath each panel indicate the dark and light periods of the lighting regimes.</p