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 glan d and retina. Large changes in the activity of this enzyme drive the circad ian 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 isolate d and used for in situ hybridization. In the adult, zfAANAT-2 mRNA is expre ssed exclusively in pineal cells and retinal photoreceptors, Developmental analysis, using whole mount in, situ hybridization, indicated that pineal z fAANAT-2 mRNA expression is first detected at 22 h post fertilization. Reti nal zfAANAT-2 mRNA was first detected on day 3 post fertilization and appea rs 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 c ycle, with high levels at night. This rhythm persists in constant darkness, indicating that the zfAANAT-2 mRNA rhythm is driven by a circadian clock a t this stage. The techniques described in this report were also used to det ermine that zfAANAT-2 expression is altered in two well characterized genet ic mutants, mindbomb and floating head. The observations described here sug gest that zfAANAT-2 mRNA may be a useful marker to study development of the pineal gland and of circadian clock mechanisms in zebrafish.