The knockdown of the maternal estrogen receptor 2a (esr2a) mRNA affects embryo transcript contents and larval development in zebrafish
The knockdown of the maternal estrogen receptor 2a (esr2a) mRNA affects embryo transcript contents and larval development in zebrafish
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DOI：
10.1016/j.ygcen.2010.12.020
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发表时间：
2011-05-15
影响因子：
2.7
通讯作者：
Dalla Valle, Luisa
中科院分区：
文献类型：
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作者：
Celeghin, Andrea;Benato, Francesca;Dalla Valle, Luisa
在斑马鱼中，排卵的卵母细胞装载有母体雌激素受体2a（esr 2a）mRNA，其作为颗粒状和丝状结构遍布中央卵质，并在1细胞阶段（受精后0.2小时，hpf）迅速重新定位在胚盘区域内，如原位杂交所示。该转录本可用于翻译，直到其从4至8 hpf急剧下降，被主要位于头部区域和卵黄囊周围的低水平合子esr 2a mRNA所取代，从24 hpf直到48 hpf孵化。为了测试母体esr 2a mRNA的功能作用，将1-或2-细胞胚胎注射10.3ng的吗啉代（MO）以敲低母体和合子esr 2a转录物的翻译（MO 2-esr 2a），其中错剪接MO（MO 3-esr 2a）以有效地阻断单独的合子转录物的转录后作用，以及非特异性MO对照。用MO 2-esr 2a处理增加了胚胎中的细胞凋亡，特别是在脑中，并在63%的15 dpf幼虫中引起严重畸形，而用MO 3-esr 2a和MO-对照处理的幼虫中为10-11%。缺陷包括身体生长延迟伴弯曲形状、卵黄囊持续存在伴肠下静脉减少和卵黄延伸肿胀、脑和脏颅发育异常、眼睛和耳泡变小、心包水肿、鱼鳔未充气和尾鳍退化伴异常循环游泳。受影响的幼虫只能存活12-14天。MO 2-esr 2a表型通过共注射30 pg/胚胎的突变的斑马鱼esr 2a mRNA来挽救，所述突变的斑马鱼esr 2a mRNA编码全长的Esr 2a，但在MO 2-esr 2a识别的区域中含有8个沉默突变。较低剂量（15 μ g）未能恢复死亡率和异常。将剂量提高到60和90 μ g会增加异常，但不会增加死亡率，而120 μ g时死亡率和异常均恶化，表明对Esr 2a有严格的定量要求。共注射抗p53 MO未能挽救MO 2-esr 2a表型，消除了脱靶效应的可能性。Pangenomic微阵列分析显示，240和219显着表达的转录本上调和下调，分别由8-hpf MO 2-esr 2a胚胎中的母体Esr 2a蛋白缺乏。此外，在48 hpf，162和120大概合子转录上调和下调，分别，但只有18个是共同的，每个8-hpf集。总共有705个基因的转录本受到Esr 2a敲低的影响。这些研究结果表明，在斑马鱼发育的表观遗传编程中，母体esr 2a mRNA可能被储存在卵母细胞中的母体17 β-雌二醇反式激活。(C)2010年爱思唯尔公司All rights reserved.
In zebrafish, ovulated oocytes are loaded with maternal estrogen receptor 2a (esr2a) mRNA which is spread as granular and filamentous structures throughout the central ooplasm and is promptly relocated inside the blastodisc area at the 1-cell stage (0.2 h post-fertilization, hpf), as shown by in situ hybridization. This transcript is available for translation until its sharp decline from 4 to 8 hpf, being replaced by low levels of zygotic esr2a mRNA mainly localized in the head region and around the yolk sac from 24 hpf until hatching at 48 hpf. To test the functional role of the maternal esr2a mRNA, 1- or 2-cell embryos were injected with 10.3 ng each of morpholino (MO) to knockdown translation (MO2-esr2a) of both maternal and zygotic esr2a transcripts, with a missplicing MO (MO3-esr2a) to effectively block post-transcriptionally the zygotic transcript alone, and with a non-specific MO-control. Treatment with MO2-esr2a increased apoptosis in embryos, especially in the brain, and caused severe malformations in 63% of 15 dpf larvae, as compared to 10-11% in those treated with MO3-esr2a and MO-control. Defects included body growth delay with curved shape, persistent yolk sac with reduced sub-intestinal veins and swollen yolk extension, abnormal brain and splanchnocranium development, smaller eyes and otic vesicles, pericardial oedema, uninflated swim bladder and rudimentary caudal fin with aberrant circular swimming. Affected larvae could survive for only 12-14 days. The MO2-esr2a phenotype was rescued with co-injection of 30 pg/embryo of mutated zebrafish esr2a mRNA encoding the full length of Esr2a, but containing eight silent mutations in the region recognised by MO2-esr2a. A lower dosage (15 pg) failed to recover mortality and abnormality. Raising the dosage to 60 and 90 pg increased abnormality, but not mortality, whereas with 120 pg both mortality and abnormality worsened, indicating a strict quantitative requirement of Esr2a. Co-injection of an anti-p53 MO failed to rescue the MO2-esr2a phenotype, eliminating the possibility of off-target effects. Pangenomic microarray analysis revealed that 240 and 219 significantly expressed transcripts were up- and down-regulated, respectively, by maternal Esr2a protein deficiency in 8-hpf MO2-esr2a embryos. Also at 48 hpf, 162 and 120 presumably zygotic transcripts were up- and down-regulated, respectively, but only 18 were in common with each of the 8-hpf sets. In total, the transcripts from 705 genes were affected by Esr2a knockdown. These findings suggest the involvement of maternal esr2a mRNA, presumably transactivated by maternal 17 beta-estradiol stored in the oocyte from enveloping granulosa cells, in the epigenetic programming of zebrafish development. (C) 2010 Elsevier Inc. All rights reserved.