Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

Abstract

Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post fertilization (dpf), harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP)). Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database). The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

Steroid modulation of neurogenesis: Focus on radial glial cells in zebrafish

“…Similarly, inhibition of estrogen synthesis by an aromatase inhibitor (ATD) results in a significant reduction of pgr expression in the brain of adult zebrafish [89]. Such an estrogenic regulation of pgr expression is further reinforced by a recent transcriptomic analysis showing that pgr is an estrogen target gene in zebrafish [90]. Concerning membrane progestin receptors, their roles and expression are poorly documented in the central nervous system [88].…”

Nuclear receptor research in zebrafish

“…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.…”

“…Finally, a microarray was performed during early developmental stages (1, 2, 3 and 4 days post fertilization (dpf) (Hao et al 2013)). In addition to well-known ER target genes like vtg1 (the only gene induced by estrogens at all stages studied (Hao et al 2013)), vtg3, vtg4 and esr1, these studies also revealed many metabolic genes (Martyniuk et al 2007, Hoffmann et al 2008 and genes involved in cell cycle and DNA repair (Lam et al 2011) as being regulated by ER.…”

The First Fifteen Years of Steroid Receptor Research in Zebrafish; Characterization and Functional Analysis of the Receptors

“…Microarray analysis has also been performed during early developmental stages (1, 2, 3 and 4 dpf). In addition to well-known Er target genes like vtg1 (the only gene induced by estrogens at all stages studied [32]), vtg3, vtg4 and er , these studies also revealed Er regulation of many metabolic genes [36,37] and genes involved in cell cycle and DNA repair [38].…”

“…In addition, two transgenic zebrafish lines have been generated in which GFP is used, which makes it possible to locate the reporter activity using fluorescence microscopy. In the first line, a promoter with 5 EREs was utilized, and GFP expression was observed in embryonic and larval zebrafish in the brain, liver and pancreas after estrogen exposure [31,32]. 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.…”