斑马鱼神经系统早期发育阶段赖氨酸特异性去甲基化酶1的表达

doi:10.3969/j.issn.1673-5374.2012.34.010

中国神经再生研究(英文版) ›› 2012, Vol. 7 ›› Issue (34): 2719-2726.doi: 10.3969/j.issn.1673-5374.2012.34.010

• 原著:神经损伤修复保护与再生 • 上一篇下一篇

斑马鱼神经系统早期发育阶段赖氨酸特异性去甲基化酶1的表达

收稿日期:2012-08-15 修回日期:2012-08-15 出版日期:2012-12-05 发布日期:2012-11-06

Lysine-specific demethylase 1 expression in zebrafish during the early stages of neuronal development

Aihong Li¹, Yong Sun², Changming Dou³, Jixian Chen⁴, Jie Zhang⁵

1 Department of Internal Neurology, Affiliated Hospital of Nantong University, Nantong 226019, Jiangsu Province, China
2 Key Laboratory of Acupuncture Combined with Medication, Nanjing University of Chinese Medicine, Nanjing 210000, Jiangsu Province, China

3 Anhui Academy of Environmental Sciences, Hefei 230061, Anhui Province, China

4 Department of Cardiothoracic Surgery, Third Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, Zhejiang Province, China

5 Department of Immunology, School of Medicine, Nantong University, Nantong 226019, Jiangsu Province, China

Received:2012-08-15 Revised:2012-08-15 Online:2012-12-05 Published:2012-11-06
Contact: Jie Zhang, Ph.D., Associate professor, Department of Immunology, School of Medicine, Nantong University, Nantong 226019, Jiangsu Province, China zhangjie@ntu.edu.cn
About author:Aihong Li★, Master, Associate professor, Department of Internal Neurology, Affiliated Hospital of Nantong University, Nantong 226019, Jiangsu Province, China
Supported by:
The study was supported by the National Natural Science Foundation of China, No. 81102643; the Natural Science Foundation of the Higher Education Institutions of Jiangsu Province, No. 10KJB310010; the Science Foundation of Zhejiang Province, No. Y2100917; and the Science Foundation of Anhui Province, No. 1208085MB26.

摘要/Abstract

摘要：

赖氨酸特异性去甲基化酶1（LSD1）分子功能是作为转录调控因子改变组蛋白上甲基化修饰水平。但是，在模式生物斑马鱼中赖氨酸特异性去甲基化酶1的表达和分子功能还不清楚。实验揭示了斑马鱼赖氨酸特异性去甲基化酶1的不同发育阶段的表达谱。研究还发现在斑马鱼受精卵分裂期就有表达，这也提示赖氨酸特异性去甲基化酶1具有母系遗传的特性。斑马鱼发育早期（受精后0-48 h），前12h赖氨酸特异性去甲基化酶1在整个胚胎上表达，24 -72 h赖氨酸特异性去甲基化酶1表达区域逐渐向胚胎前部集中，尤其是大脑。抑制赖氨酸特异性去甲基化酶1活性（反环苯丙胺）或者下调赖氨酸特异性去甲基化酶1基因（吗啡啉反义寡核苷酸）表达均可以导致斑马鱼脑细胞缺损，以及神经活动相关基因gad65, gad75和reelin的大幅度下调（不包括hey1）。研究结果说明赖氨酸特异性去甲基化酶1在斑马鱼神经系统发育过程中的重要地位。

关键词: 神经再生, 神经发生, 斑马鱼, 赖氨酸特异性去甲基化酶, 吗啡啉, 反环苯丙胺, 神经细胞, 胚胎发育, 组蛋白甲基化, 组蛋白脱甲基酶, 脑

Abstract:

Lysine-specific demethylase 1 (Lsd1) is associated with transcriptional coregulation via the modulation of histone methylation. The expression pattern and function of zebrafish Lsd1 has not, however, been studied. Here, we describe the pattern of zebrafish Lsd1 expression during different development stages. In the zebrafish embryo, lsd1 mRNA was present during the early cleavage stage, indicating that maternally derived Lsd1 protein is involved in embryonic patterning. During embryogenesis from 0 to 48 hours post-fertilization (hpf), the expression of lsd1 mRNA in the embryo was ubiquitous before 12 hpf and then became restricted to the anterior of the embryo (particularly in the brain) from 24 hpf to 72 hpf. Inhibition of Lsd1 activity (by exposure to tranylcypromine) or knockdown of lsd1 expression (by morpholino antisense oligonucleotide injection) led to the loss of cells in the brain and to a dramatic downregulation of neural genes, including gad65, gad75, and reelin, but not hey1. These findings indicate an important role of Lsd1 during nervous system development in zebrafish.

Key words: zebrafish, lysine-specific demethylase, morpholino, tranylcypromine, nerve cells, embryonic development, histone methylation, histone demethylase, brain, neural regeneration

. 斑马鱼神经系统早期发育阶段赖氨酸特异性去甲基化酶1的表达[J]. 中国神经再生研究(英文版), 2012, 7(34): 2719-2726.

Aihong Li, Yong Sun, Changming Dou, Jixian Chen, Jie Zhang. Lysine-specific demethylase 1 expression in zebrafish during the early stages of neuronal development[J]. Neural Regeneration Research, 2012, 7(34): 2719-2726.

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引言

材料方法

Design

A controlled, observational, in vivo study.

Time and setting

Experiments were conducted at the Immunology Laboratory, Nantong University, China from January to July 2011.

Materials

Animals

Wild-type (AB* strain) zebrafish stocks were obtained from the International Zebrafish Research Center (WT group). Embryos were obtained from natural spawning of wild-type adults. Zebrafish were raised, maintained, and staged as previously described^[9].

Drugs

Lsd1 inhibitors, trans-2-phenylcyclopropylamine (referred to as PCPA hereafter; also known as tranylcypromine and parnate; Sigma, USA), were dissolved in ddH₂O at stock concentrations of 100 mM, and then diluted in embryo media to a final concentration of 40 μM. Control embryos were wild-type zebrafish. All embryos were incubated at 28.5°C^[15].

Methods

Whole-mount RNA in situ hybridization

Plasmids encoding zebrafish lsd1 (3’-UTR region) were kindly provided by Nantong University, China. Whole-mount RNA in situ hybridization using digoxigenin-labeled antisense RNA probes was performed using standard methods as previously described^[14]. Briefly, digoxigenin-labeled antisense RNA probes were produced using a digoxigenin-RNA labeling kit (Roche, Mannheim, Germany) following the manufacturers’ instructions. Hybridization and detection with an anti-digoxigenin antibody coupled to alkaline phosphatase was performed with fixed zebrafish embryos.

Western blot assay

Embryos (24 hpf) were completely deyolked in Ginzburg Fish Ringer buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO₃) and washed with PBS. The embryos were collected by centrifugation and lysed with 2 × Laemmli’s buffer for 30 minutes followed by centrifugation to remove the debris. After separation by 12.5% sodium dodecylsulfate polyacrylamide gel electrophoresis, the proteins were blotted onto a nitrocellulose filter and hybridized with rabbit polyclonal anti-H3K4 demethylation (H3K4 (2m)) antibody (1:1 000; Abcam, Cambridge, MA, USA).

Morpholino oligonucleotide injection

Morpholinos were obtained from Gene Tools (Philomath, OR, USA). For lsd1, the splice morpholino (exon 11) contained 25 bases. For control, a lsd1 mismatch morpholino (control MO, CON group) that included five mispaired bases was used. The sequences of lsd1 MO and CON (mis-match MO) were GGG ACC TTT GGC AAA GTT ACA AAC A and GGc ACC TTT cGC AAA cTT AgA AAg A, respectively. 0.05 μL lsd1 MO at 15.0 mg/mL (1 ng per embryo) was injected into the blastomere of one-cell or two-cell stage embryos (microinjector from Narishige, Japan). Control MO was injected at the same volume.

RT-PCR

Total RNA was extracted from 25–30 embryos using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. One microgram of total RNA was used as a template in a 20 mL RT-PCR reaction mixture using a one-step RT-PCR kit (Qiagen, Hilden, Germany). Quantitative analysis of RT-PCR expression prouction by REST soft (Stratagene, La Jolla, CA, USA). The RT-PCR conditions are as previously described^[15], except for a change in annealing temperature, which depended on the Tm value of the primers. PCR primers used to detect targeted exon 11 skipping are listed in Table 1. Product concentration was quantified using gapdh as an internal reference.

Histological examinations

At 6 dpf, 20 fish from each treatment were pooled and euthanized with Tricaine (Sigma). Bodies were fixed in Dietrich’s fixative^[40] and then embedded in paraffin and sectioned longitudinally along the entire dorsal-ventral axis at 4mm. Sections were stained with hematoxylin- eosin and examined under a light microscope (Olympus, Tokyo, Japan).

Photography

Stained embryos were examined with Olympus BX61 and SZX12 microscopes (Olympus, Japan), and photographed with a DP70 digital camera (Olympus). Images were processed using IMAGE PRO software (Media Cybernetics, Silver Spring, MD, USA).

讨论

文章快阅

延伸阅读

文章重点内容的中文介绍：
LSD1分子功能是作为转录调控因子改变组蛋白上甲基化修饰水平。但是，在模式生物斑马鱼中LSD1的表达和分子功能还不清楚。作者对4种生物的LSD1蛋白序列进行同源性比对发现斑马鱼与其它三物种的LSD1序列高度保守。研究还发现LSD1在斑马鱼受精卵分裂期就有表达，这也提示LSD1具有母系遗传的特性。斑马鱼发育早期（0至48 hpf），前12小时LSD1在整个胚胎上表达，24 至72 小时LSD1表达区域逐渐向胚胎前部集中，尤其是中枢神经系统。

研究通过吗啡啉修饰的反义寡核苷酸技术（morpholino）干扰目标mRNA翻译的方法下调lsd1表达，并观察斑马鱼发育受到的影响。作者设计了两个具有剪接功能的吗啡啉修饰反义寡核苷酸，下调了lsd1基因在斑马鱼中的表达。外显子11和17是LSD1功能蛋白必需的编码序列，失去其中任何一段，LSD1即丧失脱甲基酶活性。RT-PCR结果显示外显子11 和17在两种MO注射后分别被剪切掉，这也验证了吗啡啉修饰的反义寡核苷酸的有效性,说明LSD1 MO能够有效下调内源性的lsd1。此外，当斑马鱼暴露在一定浓度的PCPA(>20μM，反环苯丙胺)中，LSD1具有的组蛋白脱甲基酶活性H3-K4(2m)也被强烈抑制。

抑制LSD1酶活性（反环苯丙胺, PCPA）或者下调LSD1基因（siRNA吗啡啉）表达均可以导致斑马鱼脑细胞缺损，以及神经活动相关基因gad65, gad75和reelin的大幅度下调（不包括hey1）。

实验设计及文章构思：

LSD1与很多疾病有密切的关系。但是，lsd1敲除的小鼠在围产早期死亡，这也导致难以深入研究lsd1在发育过程中的作用及其机制。模式生物斑马鱼胚胎透明、体外发育、个体小、繁殖率高以及其心血管、血液、消化道、肝脏、肾脏以及视觉系统与人类相应系统有许多共同点，基因组序列与人的同源性更是达到65%以上。此外，斑马鱼所需部分的氧可以通过体表渗透，因此特别适用于心血管和血液系统早期发育的研究。课题就是利用斑马鱼作为模型来研究LSD1对于发育的影响并对其机制进行了初步的探索。

lsd1在斑马鱼的CNS部位持续高表达表明LSD1在神经系统发育上有一定的作用，但是具体分子机制不详。本研究将在斑马鱼基因中寻找与已知组蛋白脱甲基化酶（LSD1）同源的基因并分析在早期阶段的发育。重点研究LSD1在斑马鱼神经系统分化发育过程中所起的作用及其相关的分子机制。

与国内外同类研究的比较：

论文是利用斑马鱼作为模型来研究LSD1对于发育的影响并对其机制进行了初步的探索。在斑马鱼发育中， lsd1在胚胎上表达从受精后一直持续到整个发育早期，而且lsd1的表达量始终处于高水平，特别是在神经系统中。外显子11和17是LSD1功能蛋白必需的编码序列，失去其中任何一段，LSD1即丧失脱甲基酶活性。RT-PCR结果显示外显子11在MO注射后分别被剪切掉，这也验证了吗啡啉修饰的反义寡核苷酸的有效性,说明LSD1 MO能够有效下调内源性的lsd1。此外，当斑马鱼暴露在一定浓度的PCPA(>20μM，反环苯丙胺)中，LSD1具有的组蛋白脱甲基酶活性H3-K4(2m)也被强烈抑制。抑制LSD1酶活性表达均可以导致斑马鱼脑细胞缺损，以及神经活动相关基因gad65, gad75和reelin的大幅度下调（不包括hey1）。研究说明了LSD1在斑马鱼神经系统发育过程中的重要地位。

课题的创新性报告

目前，组蛋白的甲基化及脱甲基化机理的逐渐研究成为热点，并且有很多新的组蛋白脱甲基酶被发现。作为第一个发现的组蛋白脱甲基酶（2004），LSD1在模式生物斑马鱼中的研究尚不多，一些重要的信息上不清楚。本研究的特色之处就是选取斑马鱼作为模式生物，研究LSD1在起发育的早期阶段的表达情况；并进一步通过两种手段下调LSD1酶活性后，探讨斑马鱼的神经系统发育情况及涉及的关键分子。所以，本研究创新之处是利用斑马鱼这一优良的模式生物，揭示了LSD1表达情况和相关分子功能，进而为相关基因（特别是影响神经系统发育基因）的研究提供了新思路。

斑马鱼神经系统早期发育阶段赖氨酸特异性去甲基化酶1的表达

Lysine-specific demethylase 1 expression in zebrafish during the early stages of neuronal development

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