Morpholino-Mediated Knockdown of ERα, ERβa, and ERβb mRNAs in Zebrafish (Danio rerio) Embryos Reveals Differential Regulation of Estrogen-Inducible Genes
Morpholino-Mediated Knockdown of ERα, ERβa, and ERβb mRNAs in Zebrafish (Danio rerio) Embryos Reveals Differential Regulation of Estrogen-Inducible Genes
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DOI：
10.1210/en.2013-1446
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发表时间：
2013-11-01
期刊：
影响因子：
4.8
通讯作者：
Callard, Gloria V.
中科院分区：
文献类型：
--
作者：
Griffin, Lucinda B.;January, Kathleen E.;Callard, Gloria V.
基因上不同的雌激素受体亚型(ERα和ERβ)在脊椎动物的雌激素作用中发挥着重要作用，但它们的独特和重叠功能尚不完全清楚。虽然哺乳动物每个亚型(ESR1和ESR2)都有一个基因，但硬骨鱼只有一个ESR1(ERα)和2个ESR2(ERβa和ERβb)基因。为了确定不同的ER亚型在调节雌激素诱导的转录靶标中的作用，将ESR特异的吗啉(MO)寡核苷酸显微注射到斑马鱼(Danio Rerio)胚胎中，以破坏DNA结合区外显子III/内含子III连接的剪接。每个MO都下调了各自的正常转录本，并增加了保留内含子III(ESR1 MO)或缺失或错接外显子III(esr2a和esr2b MOS)的变异体的产量。ESR1和esr2b MOS均可阻断雌激素对肝脏优势基因卵黄蛋白原和ERαmRNAs的诱导，但ESR2b是唯一能阻断脑优势基因细胞色素P450芳香酶BmRNA诱导的MOS。用esr2a MO敲除ERβa对雌激素诱导的3个mRNAs没有影响，但当与ESR1 MO联合注射时，减弱了ERα敲除的作用。结果表明，ERα和ERβb单独或协同作用于特定的基因靶点，是雌激素作用的正转录调节因子，但ERβa的作用尚不清楚。我们得出结论，斑马鱼胚胎中的分子轨道技术是在真实的生理背景下研究ER亚型相互作用的一种有利方法。
Genetically distinct estrogen receptor (ER) subtypes (ER alpha and ER beta) play a major role in mediating estrogen actions in vertebrates, but their unique and overlapping functions are not entirely clear. Although mammals have 1 gene of each subtype (ESR1 and ESR2), teleost fish have a single esr1 (ER alpha) and 2 esr2 (ER beta a and ER beta b) genes. To determine the in vivo role of different ER isoforms in regulating estrogen-inducible transcription targets, zebrafish (Danio rerio) embryos were micro-injected with esr-specific morpholino (MO) oligonucleotides to disrupt splicing of the exon III/intron III junction in the DNA-binding domain. Each MO knocked down its respective normal transcript and increased production of variants with a retained intron III (esr1 MO) or a deleted or mis-spliced exon III (esr2a and esr2b MOs). Both esr1 and esr2b MOs blocked estradiol induction of vitellogenin and ER alpha mRNAs, predominant hepatic genes, but esr2b was the only MO that blocked induction of cytochrome P450 aromatase B mRNA, a predominant brain gene. Knockdown of ER beta a with the esr2a MO had no effect on estrogen induction of the 3 mRNAs but, when coinjected with esr1 MO, attenuated the effect of ER alpha knockdown. Results indicate that ER alpha and ER beta b, acting separately or cooperatively on specific gene targets, are positive transcriptional regulators of estrogen action, but the role of ER beta a, if any, is unclear. We conclude that MO technology in zebrafish embryos is an advantageous approach for investigating the interplay of ER subtypes in a true physiological context.