Isolation and Phenogenetics of a Novel Circadian Rhythm Mutant in Zebrafish

Abstract

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag dg2 ) mutant is shortened by 0.7 h in heterozygotes, 403 and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1 ðper1Þ is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag dg2 mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.

Diversity of zebrafish peripheral oscillators revealed by luciferase reporting

“…Like in DD, rhythms damped gradually in LL, then recovered in subsequent LD cycles. The fact that per3-luc-mediated bioluminescence rhythms in some organs were not affected by LL was remarkable, because the other two zebrafish per genes are not rhythmic in LL at least in cell lines and some tissues; per2 mRNA is induced by light (32,36), whereas per1 transcription is suppressed by light [per1 is also called per4 (33,34)]. Although the exact elements responsible for per3 mRNA cycling are not known, three E-boxes have been found within 1-kb upstream sequences of this gene (38).…”

“…There are several possibilities to explain the results: (i) unlike in cell lines, per2 and per1 mRNA may cycle in LL in the organs studied here, although the light-dependent suppression of per1 was observed in the brain, and larval fish and the PAC-2 cell line (33), and induction of per2 has been observed in embryos (40); (ii) per2 and per1 mRNA may not cycle, but their protein products may do so by posttranscriptional mechanisms, although such mechanisms are unlikely to compensate for the very low level of per1 mRNA in LL; (iii) per2 and per1 may not be expressed in these organs, although per1 mRNA has been detected in the retina, heart and spleen (ref. 34 and M.K., unpublished data); and (iv) PER2 and PER1 are expressed, but do not affect the per3 promoter.…”

“…For example, there are six cry genes in zebrafish, whereas only two such genes are present in mammals (30). On the other hand, only three transcripts have been identified for zebrafish per genes, per1, per2, and per3 (per1 is also called per4) (31)(32)(33)(34). Like in Drosophila, molecular rhythms in peripheral tissues from zebrafish are directly resettable by light even in culture (32,35), although the photoreceptor molecule responsible for this resetting is not yet known.…”

The neurogenetic frontier--lessons from misbehaving zebrafish

“…Such mutants have the potential to shed new light on the function of the vast brain territory that lies between sensory reception and motor neuron firing, and illuminate fundamental aspects of brain function such as learning and memory, behavioral choice, attention, motivation, emotion and biological rhythms. Indeed, the first such screen to be carried out in zebrafish identified a semi-dominant mutant with a reduced circadian period [30].…”

Big ideas for small brains: what can psychiatry learn from worms, flies, bees and fish?

“…80 These are appealing behaviors to study, because the cellular architecture of the larval zebrafish retina and spinal cord are similar to those of higher vertebrates. Larvae have also been used to study circadian rhythms and sleep-like states, 81,82 addiction, [83][84][85][86][87] habituation 88 and a variety of human disease model behaviors. 89,90 Adult zebrafish have a wider array of complex behaviors than their larval counterparts, including reproductive behaviors such as courtship and preferential mating, social behaviors including shoaling, aggression, alarm and territoriality and learning and memory.…”