Visualization of Estrogen Receptor Transcriptional Activation in Zebrafish

Abstract

Estrogens regulate a diverse range of physiological processes and affect multiple tissues. Estrogen receptors (ERs) regulate transcription by binding to DNA at conserved estrogen response elements, and such elements have been used to report ER activity in cultured cells and in transgenic mice. We generated stable, transgenic zebrafish containing five consecutive elements upstream of a c-fos minimal promoter and green fluorescent protein (GFP) to visualize and quantify transcriptional activation in live larvae. Transgenic larvae show robust, dose-dependent estrogen-dependent fluorescent labeling in the liver, consistent with er gene expression, whereas ER antagonists inhibit GFP expression. The nonestrogenic steroids dexamethasone and progesterone fail to activate GFP, confirming ER selectivity. Natural and synthetic estrogens activated the transgene with varying potency, and two chemicals, genistein and bisphenol A, preferentially induce GFP expression in the heart. In adult fish, fluorescence was observed in estrogenic tissues such as the liver, ovary, pituitary gland, and brain. Individual estrogen-responsive neurons and their projections were visualized in the adult brain, and GFP-positive neurons increased in number after 17β-estradiol exposure. The transgenic estrogen-responsive zebrafish allow ER signaling to be monitored visually and serve as in vivo sentinels for detection of estrogenic compounds.

Nuclear receptor research in zebrafish

“…In addition, two transgenic zebrafish lines have been generated in which GFP is used, enabling microscopic analysis of the reporter activity. In the first line, a promoter with 5 EREs was utilized (Gorelick & Halpern 2011). After estrogen exposure, GFP expression was observed in embryonic and larval zebrafish in the brain, liver and pancreas (Gorelick & Halpern 2011, Hao et al 2013, consistent with the location of erb2 expression during these developmental stages (Gorelick & Halpern 2011).…”

“…In the first line, a promoter with 5 EREs was utilized (Gorelick & Halpern 2011). After estrogen exposure, GFP expression was observed in embryonic and larval zebrafish in the brain, liver and pancreas (Gorelick & Halpern 2011, Hao et al 2013, consistent with the location of erb2 expression during these developmental stages (Gorelick & Halpern 2011). Estrogen treatment of larvae also induced expression of GFP in cells in the ventral fin adjacent to the cloaca, in the developing olfactory organ and in the heart.…”

“…This induction appeared to be dependent on ER (it could be inhibited by the ER antagonist ICI 182,780 (fulvestrant)), although ER expression in these regions was not detectable by in situ hybridization at these stages. Interestingly, GFP induction by EDCs like EE2, BPA and genistein showed marked differences in the tissue specificity of the response (Gorelick & Halpern 2011). In adult fish, males showed no GFP expression in the liver, whereas displayed dim expression, which could be increased upon estrogen exposure.…”

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

“…Transgenic zebrafish have been developed that allow for the direct visualization of activity in signaling pathways, such as fibroblast growth factors 1 , retinoic acid 2 and estrogens 3 , in live embryos. Such tools enable screening for chemicals that perturb signaling pathways (assayed as change in fluorescence intensity) or for chemicals that modulate signaling in a tissue-specific manner (change in fluorescence localization) 4 .…”

“…The transgenic line Tg(5xERE: GFP) c262/c262 contains 5 tandem estrogen response element DNA sequences (ERE) upstream of green fluorescent protein (GFP) 3 . In the absence of ligand, ERs are normally inactive.…”