The Circadian Clock System

All organisms demonstrate a wide variety of daily rhythms of behavioral, physiological, biochemical, and molecular processes, which are driven by an endogenous biological circadian clock.

Research in our lab focuses on the development and mechanisms of the clock using zebrafish as a model. We combine a variety of molecular-genetic tools in order to understand how the circadian clock system develops, how it functions and how it is synchronized with the environment.

Mechanisms underlying rhythmic and pineal-specific gene expression

A central component of the biological clock in vertebrates is the melatonin-rhythm generating system in the pineal gland. This system translates photoperiodic information into many daily and annual physiological rhythms, such as sleep/wake cycles and seasonal reproduction. In non-mammalian vertebrates the pineal gland is photoreceptive and contains an endogenous circadian oscillator that drives the melatonin rhythms.

These functions require an accurate spatio-temporal expression of an array of specific genes/proteins. Among these is the arylalkylamine-N-acetyltransferase (AANAT), a key enzyme in the melatonin production pathway. In zebrafish, aanat2 is a clock-controlled gene; exhibiting rhythmic expression in the pineal gland as early as two days post-fertilization (Gothilf et al., 1999). Studies aimed at understanding the basis for this pineal-specific and rhythmic expression have lead to the identification of a new regulatory mechanism that is based on the interaction of clock and homebox proteins and simultaneously controls tissue-specificity and rhythmicity (Appelbaum et al., 2004; Appelbaum et al., 2005). This line of research is being extended by identifying and investigating additional genes that exhibit pineal-specific and rhythmic expression pattern (Alon et al., 2009).

Functional development and entrainment of the circadian oscillator

The circadian clock in the zebrafish pineal appears to be functional as early as the second day of development. We have been studying the functional development of this system using the pineal aanat2 mRNA rhythm as a marker, and discovered that light is mandatory for the development of a functional circadian clock (Ziv et al., 2005; Ziv and Gothilf, 2006a). Light induces the expression of period2 (per2), mainly in the pineal gland, and PER2 knockdown abolishes Aanat2 rhythms, suggesting that light-induced per2 expression is an important event in the development of a functional circadian clock (Ziv et al., 2005; Ziv and Gothilf, 2006a).

Light induces per2 expression even prior to pineal gland formation (mid-blastula to early segmentation; 4-16 hr post-fertilization). Interestingly, light exposure and light-induced expression of per2 at these early developmental stages are required for setting the phase of the circadian clock. The 24-h rhythm is then maintained throughout development as evident by clock-controlled rhythms of aanat2 expression during the third and fourth day of development (Ziv and Gothilf, 2006b). The implication of these findings is that the circadian oscillator is maintained during the rapid proliferation and differentiation that occur during development.

Regulatory mechanism of GnRH system development

Hypothalamic Gonadotropin-releasing hormone (GnRH) is a key peptide in the control of reproduction in all vertebrates. The embryonic origin of hypothalamic GnRH neurons is the olfactory placode. During development, they migrate backwards and settle in the hypothalamus. Correct course of migration and positioning of these neurons are crucial for reproductive fitness. The ontogeny of this system was investigated in zebrafish by in situ hybridization and expression assays of promoter-reporter constructs in live fish (Palevitch et al., 2007). A transgenic line that express EGFP under the control of the GnRH3 promoters was developed (Abraham et al., 2008) and is being utilized to investigate the effect of endogenous and exogenous factors on the development of this system (Abraham et al., 2009; Palevitch et al., 2009).

The Zebrafish Model

Over the past few years, the zebrafish has become a valuable vertebrate model system for developmental genetics. Its fast development, short generation time, large number of embryos, external fertilization and transparency, and accessibility of the embryos in early developmental stages, are just a few of the advantages the zebrafish offers as a model. As a result, techniques for mutagenesis, gene knockdown, transgenesis, and accumulation of genomic and proteomic knowledge, including the complete sequencing of the zebrafish genome, are now available.

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Chow MM, Kight KE, Gothilf Y, Alok D, Stubblefield J, Zohar Y (1998). Multiple GnRHs present in a teleost species are encoded by separate genes: analysis of the sbGnRH and cGnRH-II genes from the striped bass, Morone saxatilis. J. Mol. Endocrinol. 21:277-289.

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Falcon J, Gothilf Y, Coon SL, Boeuf G, Klein DC (2003). Genetic, temporal and developmental differences between melatonin rhythm generating systems in the fish pineal organ and retina. J Neuroendocrinol. 15:1-5.

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Appelbaum L, Toyama R, Dawid IB, Klein DC, Baler R, Gothilf Y (2004). Zebrafish serotonin-N-acetyltransferase-2 gene regulation: Pineal-restrictive downstream module (PRDM) contains a functional E-box and three photoreceptor conserved elements. Mol. Endocrinol. 18:1210-1221.

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Abraham E, Palevitch O, Ijiri S, Du SJ, Gothilf Y, Zohar Y (2008). Early development of forebrain gonadotrophin-releasing hormone (GnRH) neurones and the role of GnRH as an autocrine migration factor. J. Neuroendocrinol. 20(3):394-405.

Palevitch O, Abraham E, Borodovsky N, Levkowitz G, Zohar Y, Gothilf Y (2009). Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin-releasing hormone 3 neurons in zebrafish. Dev Dyn. 238(1):66-75.

Alon S, Eisenberg E, Jacob-Hirsch J, Rechavi G, Vatine G, Toyama R, Coon SL, Klein DC, Gothilf Y (2009). A new cis-acting regulatory element driving gene expression in the zebrafish pineal gland. Bioinformatics.