Yoav Gothilf

It’s time to swim! Zebrafish and the circadian clock

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research.

ZEBRAFISH SEROTONIN N-ACETYLTRANSFERASE-2 : MARKER FOR DEVELOPMENT OF PINEAL PHOTORECEPTORS AND CIRCADIAN CLOCK FUNCTION

Serotonin N-acetyltransferase (AANAT), the penultimate enzyme in melatonin synthesis, is typically found only at significant levels in the pineal gland and retina. Large changes in the activity of this enzyme drive the circadian rhythm in circulating melatonin seen in all vertebrates. In this study, we examined the utility of using AANAT messenger RNA (mRNA) as a marker to monitor the very early development of pineal photoreceptors and circadian clock function in zebrafish. Zebrafish AANAT-2 (zfAANAT-2) cDNA was isolated and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expressed exclusively in pineal cells and retinal photoreceptors. Developmental analysis, using whole mount in situ hybridization, indicated that pineal zfAANAT-2 mRNA expression is first detected at 22 h post fertilization. Retinal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appears to be associated with development of the retinal photoreceptors. Time-of-day analysis of 2- to 5-day-old zebrafish lar...

Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr−/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr−/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr−/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr−/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

Functional development of the zebrafish pineal gland: light-induced expression of period2 is required for onset of the circadian clock.

In zebrafish, the pineal gland is a photoreceptive organ that contains an intrinsic circadian oscillator and exhibits rhythmic arylalkylamine‐N‐acetyltransferase (zfaanat2) mRNA expression. In the present study, we investigated the role of light and of a clock gene, zperiod2 (zper2), in the development of this rhythm. Analysis of zfaanat2 mRNA expression in the pineal gland of 3‐day‐old zebrafish embryos after exposure to different photoperiodic regimes indicated that light is required for proper development of the circadian clock‐controlled rhythmic expression of zfaanat2, and that a 1‐h light pulse is sufficient to initiate this rhythm. Analysis of zper2 mRNA expression in zebrafish embryos exposed to different photoperiodic regimes indicated that zper2 expression is transiently up‐regulated by light but is not regulated by the circadian oscillator. To establish the association between light‐induced zper2 expression and light‐induced clock‐controlled zfaanat2 rhythm, zPer2 knock‐down experiments were performed. The zfaanat2 mRNA rhythm, induced by a 1‐h light pulse, was abolished in zPer2 knock‐down embryos. These experiments indicated that light‐induced zper2 expression is crucial for establishment of the clock‐controlled zfaanat2 rhythm in the zebrafish pineal gland.

Genetic, Temporal and Developmental Differences Between Melatonin Rhythm Generating Systems in the Teleost Fish Pineal Organ and Retina

Complete melatonin rhythm generating systems, including photodetector, circadian clock and melatonin synthesis machinery, are located within individual photoreceptor cells in two sites in Teleost fish: the pineal organ and retina. In both, light regulates daily variations in melatonin secretion by controlling the activity of arylalkylamine N‐acetyltransferase (AANAT). However, in each species examined to date, marked differences exist between the two organs which may involve the genes encoding the photopigments, genes encoding AANAT, the times of day at which AANAT activity and melatonin production peak and the developmental schedule. We review the fish pineal and retinal melatonin rhythm generating systems and consider the evolutional pressures and other factors which led to these differences.

Light directs zebrafish period2 expression via conserved D and E boxes.

A highly conserved promoter module in a vertebrate clock gene confers light-regulated gene expression.

Early Development of Forebrain Gonadotrophin-Releasing Hormone (GnRH) Neurones and the Role of GnRH as an Autocrine Migration Factor

Normal migration of the gonadotrophin‐releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio‐temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post‐fertilisation and by 4–5 days post‐fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino‐modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo, that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.

Seasonal variation of the three native gonadotropin-releasing hormone messenger ribonucleic acids levels in the brain of female red seabream

We studied the seasonal variation of the expression of genes encoding the three native gonadotropin-releasing hormones (GnRHs), namely salmon(s) GnRH, chicken(c) GnRH-II, and seabream(sb) GnRH in red seabream, Pagrus (Chrysophrys) major, in order to better understand the regulatory mechanisms of GnRH gene expression by environmental and endocrine factors. Female red seabream, reared under natural conditions, were collected monthly or bimonthly from October to June, and the levels of the three distinct GnRH messenger ribonucleic acids (mRNAs) in the brains of those fish (n = 4-6) were determined by ribonuclease (RNase) protection analysis. The levels of sbGnRH mRNA correlated well with the observed ovarian histology; the levels of sbGnRH mRNA of immature fish in October and December were low, and increased in February and March in conjunction with active vitellogenesis. The sbGnRH mRNA levels reached a maximum level in April (spawning season), after which they rapidly decreased together with the observed ovarian regression in June. In contrast, the levels of sGnRH mRNA showed no variation, while those of cGnRH-II mRNA were elevated only slightly in March and April. The increase in sbGnRH mRNA levels correlates with the increase in day length, water temperature and serum steroids levels, suggesting that these factors are candidates for regulators of sbGnRH synthesis.

Circadian clocks, rhythmic synaptic plasticity and the sleep-wake cycle in zebrafish

The circadian clock and homeostatic processes are fundamental mechanisms that regulate sleep. Surprisingly, despite decades of research, we still do not know why we sleep. Intriguing hypotheses suggest that sleep regulates synaptic plasticity and consequently has a beneficial role in learning and memory. However, direct evidence is still limited and the molecular regulatory mechanisms remain unclear. The zebrafish provides a powerful vertebrate model system that enables simple genetic manipulation, imaging of neuronal circuits and synapses in living animals, and the monitoring of behavioral performance during day and night. Thus, the zebrafish has become an attractive model to study circadian and homeostatic processes that regulate sleep. Zebrafish clock- and sleep-related genes have been cloned, neuronal circuits that exhibit circadian rhythms of activity and synaptic plasticity have been studied, and rhythmic behavioral outputs have been characterized. Integration of this data could lead to a better understanding of sleep regulation. Here, we review the progress of circadian clock and sleep studies in zebrafish with special emphasis on the genetic and neuroendocrine mechanisms that regulate rhythms of melatonin secretion, structural synaptic plasticity, locomotor activity and sleep.

Developmental Expression of Three Forms of Gonadotropin-Releasing Hormone and Ontogeny of the Hypothalamic-Pituitary-Gonadal Axis in Gilthead Seabream (Sparus aurata)

Abstract To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF. Salmon (s) GnRH mRNA-expressing cells were first seen in the olfactory placode at 3 DPF, started caudal migration at 14 DPF, and reached the preoptic areas at 59 DPF. Seabream (sb) GnRH mRNA-expressing cells were first detected in the terminal nerve ganglion cells (TNgc), ventral part of the ventral telencephalon, nucleus preopticus parvocellularis, and thalamus at 39 DPF, and extended to the nucleus preopticus magnocellularis at 43 DPF, ventrolateral hypothalamus at 51 DPF, and nucleus lateralis tuberis and posterior tuberculum at 59 DPF. Coexpression of sbGnRH and sGnRH transcripts was found in the TNgc. Using real-time fluorescence-based quantitative polymerase chain reaction, transcript levels of cGnRH-II and sGnRH were first detected at 1 and 1.5 DPF, respectively, and increased and remained high thereafter. Transcript levels of sbGnRH remained low after first detection at 1 DPF. Furthermore, these GnRH expression profiles were correlated with the expression profiles of reproduction-related genes in which at least four concomitant increases of GnRH, GnRH receptor, gonadotropin, gonadotropin receptor, and Vasa transcripts were found at 5, 8, 14, and 28 DPF. Our data provide an expanded view of the ontogeny of the GnRH system and reproductive axis in perciforms.