Background: The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish cyclops (cyc) mutants lack ventral brain, including the region that gives rise to the SCN. We have used cyc embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression.Results: We find that pineal rhythms are present in cyc mutants despite the absence of an SCN. The arginine vasopressin-like protein (Avpl, formerly called Vasotocin) is a peptide hormone expressed in and around the SCN. We find avpl mRNA is absent in cyc mutants, supporting previous work suggesting the SCN is missing. In contrast, expression of the putative circadian clock genes, cryptochrome 1b (cry1b) and cryptochrome 3 (cry3), in the brain of the developing fish is unaltered. Expression of two pineal rhythmic genes, exo-rhodopsin (exorh) and serotonin-N-acetyltransferase (aanat2), involved in photoreception and melatonin synthesis, respectively, is also similar between cyc embryos and their wildtype (WT) siblings. The timing of the peaks and troughs of expression are the same, although the amplitude of expression is slightly decreased in the mutants. Cyclic gene expression persists for two days in cyc embryos transferred to constant light or constant dark, suggesting a circadian clock is driving the rhythms. However, the amplitude of rhythms in cyc mutants kept in constant conditions decreased more quickly than in their WT siblings.Conclusion: Our data suggests that circadian rhythms can be initiated and maintained in the absence of SCN and other tissues in the ventral brain. However, the SCN may have a role in regulating the amplitude of rhythms when environmental cues are absent. This provides some of the first evidence that the SCN of teleosts is not essential for establishing circadian rhythms during development. Several SCN-independent circadian rhythms have also been found in mammalian species. Thus, zebrafish may serve as a model system for understanding how vertebrate embryos coordinate rhythms that are controlled by different circadian clocks.
Bibliographical noteFunding Information:
We would like to thank Drs. Josh Gamse (Vanderbilt University, Nashville, TN), Eric Mintz (Kent State University, Kent, OH), and Neal Peachey (Cleveland Clinic, OH) for their helpful comments on this manuscript, Dr. Yoshitaka Fukada (University of Tokyo, Japan) for providing the full-length exorh cDNA, Dr. Michael Romero (Mayo Clinic, Rochester, MN) for sharing the pGEMHE plasmid and sequencing primers and advice on subcloning, Dr. Robert Molday (University of British Columbia, Canada) for providing the 4D2 antibody, Sandra Albro (Case Western Reserve University) for assistance with statistical analysis, and Drs. David Klein (National Institutes of Health), Yoav Gothilf (Tel-Aviv University, Israel), and Bernard and Christine Thisse (University of Virginia) for plasmids. Allisan Aquilina-Beck, Adelle Schuman, Jessica Clay, Stephan Brannan, Kristine DiMonte, Lain Pierce, Olga Ponomareva, Perry Hwang, Gerry Babcock, Andy Schreiner, and Brian Chen provided expert technical assistance. Supported in part by research grant No. 5-FY02-259 from the March of Dimes Birth Defect Foundation (to JOL), an Edward Mallinckrodt, Jr. Foundation grant (to JOL), 1 RO1 HD054523 (to JOL and Joshua Gamse), the Hathaway Brown Student Research Program (to LGK), and a Phi Beta Kappa Student Research Grant Award (to RRN).