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, 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 (per1) 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.

Keywords:

• Danio rerio
• lager and lime mutant
• period
• temperature compensation
• phase response curve
• per1

Acknowledgements

We thank Rhia Pascua and Ronit Spotts for technical assistance and Fanny S. Ng and Qin Zhao for help with the meiotic mapping. We also thank Keith Barrett (Northwestern University) and the Harvard and Oregon Zebrafish Facilities for zebrafish strains and Keith Barrett and Joseph Takahashi (Northwestern University) for the zebrafish per1 sequence, cDNA clone, and several sets of PCR primers. J.D. was supported in part by the Houston Area Vision Training Program institutional grant 5-T32-EY007024 (NIH). This research was supported by NIH grant MH60939 and Texas Advanced Research Program grant 3652–761 awarded to G.M.C.