Probing pineal-specific gene expression with transgenic zebrafish.
The pineal gland of zebrafish (Danio rerio) contains light-sensitive photoreceptor cells and plays an important role in the neuroendocrine system. The zebrafish exorhodopsin gene encodes a pineal-specific photoreceptive protein, whose promoter region harbors a cis-acting element, pineal expression-promoting element (PIPE), directing pineal-specific gene expression. For in vivo genetic studies on PIPE-binding proteins and their regulatory mechanisms, we generated a transgenic zebrafish line, Tg(P(20)-rh/P:gfp), that expresses green fluorescent protein (GFP) under the control of the zebrafish rhodopsin promoter fused with 20 PIPE repeats. In Tg(P(20)-rh/P:gfp) fish, PIPE-dependent gene expression is visualized by GFP fluorescence in the pineal gland along with PIPE-independent GFP signals in the retinal rod photoreceptors. The transgenic fish exhibit detectable and reproducible GFP fluorescence in the larval pineal gland by 5 days postfertilization. Antisense morpholino-mediated knock-down of a pineal transcription factor gene, otx5, suppresses pineal GFP expression in the transgenic line. In a pilot screen of N-ethyl-N-nitrosourea-treated fish of the GFP transgenic line, we isolated potential dominant mutations that cause attenuation of pineal GFP fluorescence with a marginal effect on the retinal GFP signal. The results suggest that the Tg(P(20)-rh/P:gfp) line will be useful for detecting deficits in PIPE-dependent gene expression in the pineal gland.
Kojima
Daisuke
D
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.
Dowling
John E
JE
Fukada
Yoshitaka
Y
D000818:Animals; D030801:Animals, Genetically Modified; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000502:physiology; D008027:Light; D010786:Photoreceptor Cells / Q000502:physiology / Q000528:radiation effects; D010870:Pineal Gland / Q000502:physiology*; D015027:Zebrafish / Q000235:genetics*
D049452:Green Fluorescent Proteins