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

Rhythmic clock gene expression in zebrafish caudal fins.

<p>(A–D) Quantitative RT-PCR analysis of clock gene expression in the adult caudal fin of zebrafish. (A–C) All genes show statistically significant differences between peak and trough values (Bonferroni's <i>post hoc</i> test p<0.0001) under light-dark (LD) conditions. (C) <i>zfper1b and zfclock1</i> rhythmic expression persists on the first day in constant darkness DD. (D) Lack of oscillation of <i>zfper1b</i> after 15 days in DD, free running conditions, compared with the rhythmic expression still observed after 1 day under DD conditions. The time of each sample is indicated either as zeitgeber time (ZT, where ZT0 is defined as lights on and ZT12, lights off) under LD cycle conditions (A–C) or circadian time (CT) under constant darkness (C–D). The results of statistical analysis are indicated above each graph by asterisks and colour-coded horizontal “brackets” drawn between the peak and trough values analysed. Black and white bars beneath each panel indicate the dark and light periods of the lighting regimes. Data for all genes 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>). For each time point a pool with a minimum of n = 5 fins were used. In each panel, points are plotted as means of three independent experiments +/− SEM. (E) Mean levels of bioluminescence measured from an <i>in vivo</i> luciferase assay of primary zebrafish caudal fin cell cultures. Cells were transiently transfected with the clock regulated reporter construct <i>zfper1b-luc </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034203#pone.0034203-Vallone1" target="_blank">[25]</a> and then assayed in real time while being exposed to various lighting regimes. On the X-axis is plotted the assay time (hours) from the start of the experiment. Blue arrows indicate the daily peaks of bioluminescence while a red arrow denotes the point where the phase of the LD cycle was reversed (LD to DL). Bioluminescence levels were plotted as means +/− SEM from three independent fin primary cultures.</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

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

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

Circadian rhythms of M-phase in zebrafish fins.

<p>(A) Immunofluorescence of representative adult caudal fins stained using a phospho Histone H3 Ser 10 (P-H3 ser10) primary antibody at different zeitgeber times. (B) Western blot analysis and quantification of fin whole protein extracts in LD (left panel) and after 1 day in DD (right panel) using Histone H3 (H3) and P-H3 ser10 antibodies. On the Y-axis is plotted the values of grey-scale quantification using Scion Image software and calculated as % relative to the peak timepoint (ZT16 and CT15 respectively). (C,D) Quantitative RT-PCR analysis of two mitotic markers: <i>zfcyclin B1</i> (C) and <i>zfwee1</i> (D). In all experiments, the time points are indicated either as zeitgeber time (ZT) or circadian time (CT). White and black bars below each panel indicate the corresponding lighting conditions. Each experiment was performed in triplicate with a minimum of 4 fins (n = 4) for each timepoint. Data are plotted as means +/− SEM of three independent experiments. 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. 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

Amputation differentially induces cell proliferation.

<p>(A) Schematic representation of the experimental plan showing the amputation site (left panel), the times of sampling (ZT, right panel,,above) and the corresponding hours post amputation (hpa, right panel, below). Amputation was performed before the light-dark transition (ZT 9, red arrow) and fins were subsequently harvested every 6 hours (starting from 18 hpa). Each time point was collected following a 15 minutes incubation with BrdU. (B) Representative fins stained for BrdU incorporation at the time points indicated in panel A. (C) Quantification of the number of S-phase nuclei in amputated fins (blue bars) and non-amputated controls (black bars). On the Y-axis is plotted the % of BrdU positive nuclei with respect to the sample with the largest value (amputated, 48 hpa). The result of statistical analysis of the peak and trough values for the amputated fins is indicated by asterisks (Bonferroni's <i>post hoc</i> test p<0.0001) and horizontal “brackets” above the graph. Furthermore, statistically significant differences observed at each time point between the amputated and control non-amputated fins are indicated for simplicity, by the symbol “#” and a bracket above only the first time point (18 hpa) (Bonferroni's <i>post hoc</i> test p<0.0001, ###). (D) Quantification of the level of BrdU staining at the ray tips in the blastema region using Scion Image software. Inset panel (D): magnified view of the fin ray tips region stained for BrdU incorporation. A red square delimits the area that has been quantified. White and black bars below each panel indicate the light and dark 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>). Each time point represents the mean values calculated for each fin ray in a total of n = 4 to 6 fins and expressed as % of the grey scale value.</p