Pineal‐specific expression of green fluorescent protein under the control of the serotonin‐N‐acetyltransferase gene regulatory regions in transgenic zebrafish

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Abstract
Zebrafish serotonin‐N‐acetyltransferase‐2 (zfAANAT‐2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue‐specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT‐2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5′‐flanking region, a regulatory sequence in the 3′‐flanking region is required for pineal‐specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5′ and 3′ regulatory regions of the zfAANAT‐2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT‐2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal‐specific gene expression in the zebrafish. Published 2002 Wiley‐Liss, Inc.