ERK Signaling Regulates Light-Induced Gene Expression via D-Box Enhancers in a Differential, Wavelength-Dependent Manner

The day-night and seasonal cycles are dominated by regular changes in the intensity as well as spectral composition of sunlight. In aquatic environments the spectrum of sunlight is also strongly affected by the depth and quality of water. During evolution, organisms have adopted various key strategies in order to adapt to these changes, including the development of clocks and photoreceptor mechanisms. These mechanisms enable the detection and anticipation of regular changes in lighting conditions and thereby direct an appropriate physiological response. In teleosts, a growing body of evidence points to most cell types possessing complex photoreceptive systems. However, our understanding of precisely how these systems are regulated and in turn dictate changes in gene expression remains incomplete. In this manuscript we attempt to unravel this complexity by comparing the effects of two specific wavelengths of light upon signal transduction and gene expression regulatory mechanisms in zebrafish cells. We reveal a significant difference in the kinetics of light-induced gene expression upon blue and red light exposure. Importantly, both red and blue light-induced gene expression relies upon D-box enhancer promoter elements. Using pharmacological and genetic approaches we demonstrate that the ERK/MAPK pathway acts as a negative regulator of blue but not red light activated transcription. Thus, we reveal that D-box-driven gene expression is regulated via ERK/MAPK signaling in a strongly wavelength-dependent manner

A Blind Circadian Clock in Cavefish Reveals that Opsins Mediate Peripheral Clock Photoreception

Evolution during millions of years in perpetual darkness leads to mutations in non-visual opsin genes (Melanopsin and TMT opsin) and an aberrant, blind circadian clock in cavefish

Evolutionary implications of size-selective mortality on the ontogenetic development of shoal cohesion : a neurochemical approach using a zebrafish, Danio rerio, harvest selection experiment

Size-selective mortality may evolutionarily alter life-history as well as individual behavioral and physiological traits. Moreover, size-selective mortality can affect group behavioral traits, such as shoaling and collective properties (e.g., shoal cohesion), which are relevant for finding food and reducing risk of predation. Here, we present experimental evidence using selection lines of zebrafish (Danio rerio) that were exposed to positive ( large-harvested), negative (small-harvested), and random (control) size-selective mortality for five generations, followed by eight generations during which harvesting was halted to remove maternal effects and to study evolutionarily fixed outcomes. We investigated changes in shoal cohesion and turnover in monoamines in zebrafish through ontogeny. To that end, we repeatedly measured inter-individual distance in groups of eight fish and the turnovers of dopamine and serotonin in brains of fish from juvenile to the adult stage at 40-day intervals. We, firstly, found that shoal cohesion was overall consistent through ontogeny at group levels suggesting the presence of collective personality. Secondly, we found a decrease in shoal cohesion through ontogeny in the small-harvested and control lines, while the large-harvested line did not show any ontogenetic change. Thirdly, the selection lines did not differ among each other in shoal cohesion at any ontogenetic stage. Fourthly, dopamine turnover increased through ontogeny in a similar way for all lines while the serotonin turnover decreased in the large-harvested and control lines, but not in the small-harvested line. The large-harvested line also had higher serotonin turnover than controls at specific time periods. In conclusion, intensive size-selective mortality left an evolutionary legacy of asymmetric selection responses in the ontogeny of shoal cohesion and the underlying physiological mechanisms in experimentally harvested zebrafish in the laboratory. Significant statement The evolution of animal behavior can be affected by human activities both at behavioral and physiological levels, but causal evidence is scarce and mostly focusing on single life-stages. We studied whether and to what extent size-selective harvesting, a common selection pattern in fisheries, can be an evolutionary driver of the development of shoal cohesion during ontogeny. We used a multi-generation experiment with zebrafish to study cause-and-effects of opposing size-selection patterns. We quantified shoal cohesion, and serotonin and dopamine turnover in the brain. We found that shoal cohesion emerged as a collective personality trait and that behavioral and physiological responses were asymmetrical with respect to the opposing selection patterns

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

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

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