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Zebrafish Serotonin N-Acetyltransferase-2: Marker for Development of Pineal Photoreceptors and Circadian Clock Function¹

Section on Neuroendocrinology, Laboratory of Developmental Neurobiology (Y.G., S.L.C., M.A.A.N, D.C.K), and Laboratory of Molecular Genetics (R.T., A.C.), National Institute of Child Health and Human Development, National Institutes of Health, Department of Physiology, Uniform Services University of Health Sciences (M.A.A.N.), Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Dr. David C. Klein, Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health 49/5A38, 49 Convent Drive, Bethesda, Maryland 20892-4480. E-mail: klein{at}helix.nih.gov

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 larvae indicated that zfAANAT-2 mRNA abundance exhibits a dramatic 24-h rhythm in a 14-h light, 10-h dark cycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock at this stage. The techniques described in this report were also used to determine that zfAANAT-2 expression is altered in two well characterized genetic mutants, mindbomb and floating head. The observations described here suggest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.

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