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Light is the key entraining stimulus for the circadian clock, but several features of the signaling pathways that convert the photic signal to clock entrainment remain to be deciphered. Here, we show that light induces the production of hydrogen peroxide (H 2 O 2 ) that acts as the second messenger coupling photoreception to the zebrafish circadian clock. Treatment of light-responsive Z3 cells with H 2 O 2 triggers the induction of zCry1a and zPer2 genes and the subsequent circadian oscillation of zPer1 . Remarkably, the induction kinetics and oscillation profile in response to H 2 O 2 are identical to those initiated by light. Catalase ( Cat ), an antioxidant enzyme degrading H 2 O 2 , shows an oscillating pattern of expression and activity, antiphasic to zCry1a and zPer2 . Interestingly, overexpression of zCAT results in a reduced light-dependent zCry1a and zPer2 gene induction. In contrast, inhibition of zCAT function enhances light-mediated inducibility of these clock genes. These findings implicate the enzymatic function of zCAT enzyme in the negative regulation of light-dependent clock gene transcriptional activation. Our findings provide an attractive link between the regulation of the cellular reduction/oxidation (redox) state and the photic signaling pathways implicated in circadian control.

MAP kinase phosphatase 1 (MKP1) is a negative regulator for the mitogen-activated protein kinase (MAPK)—mediated signal transduction, a key pathway that leads to the regulated expression of circadian clock genes. Here the authors analyzed mkp1 expression by in situ hybridization and found that mkp1 is a light-inducible and clock-controlled gene expressed in the central pacemaker neurons of the hypothalamic SCN. Interestingly, mkp1 presents a marked similarity to the clock core gene per1 in terms of the gene expression profiles as well as the gene promoter organization. Both mkp1 and per1 are subject to bimodal regulation in the SCN: the external light-dependent acute up-regulation and the functional clock-dependent circadian oscillation. Consistent with this, the authors show that mkp1 gene has a per1-like promoter that contains 2 functionally distinct elements: cAMP-responsive element (CRE) and E-box. CRE sites present in the mkp1 promoter constitute the functional binding sites for the CRE binding protein (CREB), which serves as an important regulator that mediates the light-induced signaling cascades in the SCN neurons. Furthermore, the authors show that the E-box present in the mkp1 promoter is necessary and sufficient for transcriptional control exerted by circadian clock core regulators that include a positive complex CLOCK/BMAL1 and a negative factor CRY1. The authors' studies on mkp1 have identified for the first time a gene encoding a phosphatase that functions in light-dependent and time-of-day-dependent manners in the mammalian central clock structure SCN.

The Aurora kinases are cell cycle-regulatory serine-threonine kinases that have been implicated in the function of the centrosomes, kinetechores, chromosome dynamics, and cytokinesis. In comparison with other tissues, there are high levels of expression of Aurora-B and -C in testis. What their respective roles in mammalian spermatogenesis are is an open question. Here we describe the expression and distribution patterns of the three kinases in mouse testis using in situ hybridization and immunohistochemistry. Importantly, the localization of Aurora-B is tightly regulated during spermatogenesis, whereas Aurora-C expression appears to be testis specific. To address the function of Aurora-B in spermatogenesis, we have generated transgenic mice using a pachytene-stage-specific promoter driving the expression of either wild-type Aurora-B or an inactive form of the kinase. Expression of the inactive Aurora-B results in abnormal spermatocytes, increased apoptosis, spermatogenic arrest, and subfertility defects. The function of Aurora-C may also be targeted in the Aurora-B transgenic mutants. To address the function of Aurora-C in testis, we generated Aurora-C knockout mice by homologous recombination. Remarkably, Aurora-C null mice were viable, yet the males had compromised fertility. Aurora-C mutant sperm display abnormalities that included heterogenous chromatin condensation, loose acrosomes, and blunted heads. These findings indicate that Aurora-B and Aurora-C serve specialized functions in mammalian spermatogenesis.

International audience; Disruption of overt circadian rhythms can occur without influencing the endogenous pacemaker, the so-called 'masking' effect classically elicited by light. As the physiological pathways involved in light masking remain elusive, we analyzed mice lacking the dopamine D2 receptor. Although circadian rhythmicity was normal, D2R-null mice showed a markedly deficient light masking response, indicating that D2R-mediated signaling is an essential component of the neuronal pathways leading to light masking of circadian rhythms.

Environmental cues modulate a variety of intracellular pathways whose signaling is integrated by the molecular mechanism that constitutes the circadian clock. Although the essential gears of the circadian machinery have been elucidated, very little is known about the signaling systems regulating it. Here, we report that signaling mediated by the dopamine D2 receptor (D2R) enhances the transcriptional capacity of the CLOCK:BMAL1 complex. This effect involves the mitogen-activated protein kinase transduction cascade and is associated with a D2R-induced increase in the recruiting and phosphorylation of the transcriptional coactivator cAMP-responsive element-binding protein (CREB) binding protein. Importantly, CLOCK:BMAL1-dependent activation and light-inducibility of mPer1 gene transcription is drastically dampened in retinas of D2R-null mice. Because dopamine is the major catecholamine in the retina, central for the neural adaptation to light, our findings establish a physiological link among photic input, dopamine signaling, and the molecular clock machinery.

International audience; Most organisms adapt the timing of their physiology to the cyclic changes of the environment with the use of intrinsic time-keeping systems called circadian clocks. Central features of the molecular clock mechanism are transcription- and translation-based negative feedback loops: clock genes and their products interact to generate oscillation of specific transcripts and proteins, and, ultimately, circadian rhythmicity and behavior. Various transcription factors constitute the molecular clock, and the signal transduction cascades governing their function appear to be crucial for the fine-tuning of the circadian cycle.

The molecular machinery that governs circadian rhythmicity is based on clock proteins organized in regulatory feedback loops. Although posttranslational modification of clock proteins is likely to finely control their circadian functions, only limited information is available to date. Here, we show that BMAL1, an essential transcription factor component of the clock mechanism, is SUMOylated on a highly conserved lysine residue (Lys 259 ) in vivo. BMAL1 shows a circadian pattern of SUMOylation that parallels its activation in the mouse liver. SUMOylation of BMAL1 requires and is induced by CLOCK, the heterodimerization partner of BMAL1. Ectopic expression of a SUMO-deficient BMAL1 demonstrates that SUMOylation plays an important role in BMAL1 circadian expression and clock rhythmicity. This reveals an additional level of regulation within the core mechanism of the circadian clock.

International audience; The zebrafish probably constitutes the best animal system to study the complexity of the circadian clock machinery and the influence that light has on it. The possibilities of producing transgenic fishes, to establish light-responsive cultured cells, and to directly explore light phototransduction on single clock cells are all remarkable features of this circadian system. This article describes some of the most useful methodologies to analyze the behavioral, cellular, and molecular aspects of the zebrafish circadian clock system.

The zebrafish probably constitutes the best animal system to study the complexity of the circadian clock machinery and the influence that light has on it. The possibilities of producing transgenic fishes, to establish light-responsive cultured cells, and to directly explore light phototransduction on single clock cells are all remarkable features of this circadian system. This article describes some of the most useful methodologies to analyze the behavioral, cellular, and molecular aspects of the zebrafish circadian clock system.

CLOCK and BMAL1 proteins, members of the basic helix-loop-helix PAS (PER-ARNT-SIM) superfamily of transcription factors which bind to the E-box DNA motif, are required for the high-level expression of the circadian clock genes period (per) and cryptochrome (cry). CRY inhibits transcriptional activity of the CLOCK-BMAL1 heterodimer, generating a negative-feedback loop that is the core element of the circadian oscillator.We show that zebrafish CRY (zCRY1a) neither disrupts the association between zfCLOCK and zfBMAL nor inhibits binding of the zfCLOCK-zfBMAL heterodimer to an E-box-bearing DNA fragment. Instead it binds to the heterodimer to form a stable zCRY1a-zfCLOCK-zfBMAL-E-box complex. Another zebrafish CRY protein, zCRY4, does not have transcriptional inhibitor activity, whereas zCRY1a has strong activity. zCRY4 does not associate with zfCLOCK and zfBMAL. We also show that the presence of a chemical reductant in the reaction mixture is crucial for efficient binding of the CLOCK-BMAL heterodimer to E-box bearing DNA, which is indicative of the reduction/oxidation (redox)-sensitive character of the heterodimer.Our findings suggest that CRY represses CLOCK-BMAL-mediated transcription by interacting directly with the zfCLOCK-zfBMAL-E-box complex.