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Title: Nr4a2在斑马鱼间脑多巴胺能神经元发育中的基因调控和分子学机制研究
Author: 罗光睿
Degree Level: 博士
Issued Date: 2009-04-30
Degree Grantor: 中国科学院上海生命科学研究院
Place of Degree Grantor: 上海生命科学研究院
Supervisor: 乐卫东
Keyword: 斑马鱼 ; 多巴胺能神经元 ; Nr4a2 ; Neurog1 ; 神经巢蛋白 ; BAC同源重组
Alternative Title: Gene Regulation and Molecular Mechanisms of Nr4a2 in Zebrafish Diencephalon Dopamine Neuron Development
Major: 神经生物学
Abstract: 斑马鱼作为一种新兴的模式动物,有着其独特的优势,包括体积小、体外发育、易于操作、胚胎透明、繁殖能力强、发育快速等。通过近二十年来对斑马鱼的研究,研究人员们已经建立了比较完善的基因组序列信息,基因操作方法以及各个系统的形态和解剖基础等。近十年来对斑马鱼多巴胺(DA)系统发育的研究取得了很多进展,具备了DA能神经元相关的基本知识,并且基本确定了在背侧结节(posterior tuberculum,PT)区域的DA神经系统与哺乳动物中脑DA神经系统的同源性。 在此基础上,我们对一个在DA能神经元发育过程中发挥重要作用的转录因子Nr4a2进行了深入研究。首先我们分析了斑马鱼中存在的两个人类NR4A2基因的同源基因nr4a2a和nr4a2b,发现在基因结构上nr4a2b和人类NR4A2的同源性更高,于是我们进一步研究了nr4a2b在斑马鱼胚胎发育时期的时空表达变化,以及它与DA能神经元的共定位情况。我们发现虽然nr4a2b和PT区域的DA能神经元并没有明显的共定位,但是却表达在紧靠在DA能神经元背侧的一群细胞中,并且这群细胞与表达在神经前体的基因neurog1有共定位。Nr4a2的功能缺失能够导致PT区域DA能神经元的减少,同时伴随着DA神经递质的减少,这一表型能够部分被外源性的小鼠Nr4a2蛋白所挽救。另外,通过检测没有降解的nr4a2b的mRNA,发现原来表达Nr4a2b的神经元在Nr4a2功能缺失之后非但没有减少而是有一定的增多[1],我们的实验说明了Nr4a2b的功能主要是表达在DA能神经前体上以促进PT区域的神经前体向终末神经元分化而并非是维持神经元的存活,而nr4a2b阳性细胞数目的增多很可能是由于分化阻滞造成的累积,Nr4a2b很可能是和Neurog1处在同一条调控通路来调控斑马鱼PT区域的DA能神经元的分化和成熟。 另外,在成年的斑马鱼中枢神经系统中,nr4a2b的表达和PT区域一群体积较小的纺锤形的TH阳性神经元有部分共定位,同时我们发现在前顶盖区域nr4a2b阳性细胞紧靠在TH阳性神经元的背侧,并且两群细胞交界处有少量的共定位细胞。而在间脑脑室周围区域我们发现了神经巢蛋白Nestin和nr4a2b的共定位,并且在这些共定位细胞的腹外侧分布有nr4a2b单阳性的细胞,而这两群细胞随年龄的增长会有明显减少。因此我们认为,Nr4a2b主要表达在神经前体里并且促进神经前体向终末神经元的分化,其表达随年龄的增加而减少提示了神经前体细胞分化能力的减弱,这可能与某些神经退行性疾病相关。 为了进一步研究Nr4a2b的功能及其阳性细胞的命运,我们需要建立nr4a2b::EGFP特异表达的转基因系。因为普通的基因克隆的方法没法得到足够长的调控EGFP在内源性Nr4a2b阳性细胞中特异表达的启动子,我们采用了BAC同源重组的方法,将EGFP重组到BAC中Nr4a2b翻译的起始位点下游。构建好之后通过将重组BAC在体进行瞬时显微注射,我们确定了EGFP与内源性的nr4a2b的共定位,从而获得了可特异性驱动EGFP在Nr4a2b阳性细胞中表达的重组BAC,进一步的稳定转基因的筛选正在进行中。 综上所述,我们通过在胚胎和成体斑马鱼中研究转录因子Nr4a2促进DA能神经元分化的功能,对DA能神经元分化的机制进行了进一步阐明,并且通过构建特异驱动EGFP在Nr4a2b阳性细胞中表达的重组BAC,为建立更好地研究DA能神经元发育的方法提供了线索,也为下一步DA系统相关神经药物的筛选奠定了基础。
English Abstract: Zebrafish, a small vertebrate model, has several advantages compared to other animal models: small volume, ectogenesis, easy to manipulate, transparent embryos, large scale of reproduction, and short developmental stage. In the past 20 years, numerous platforms such as genome sequencing, gene manipulation techniques and morphology and anatomy observation have been set up to study the biology of zebrafish in detail. Using these platforms, zebrafish dopamine (DA) systems have been widely investigated and DA neuron in the posterior tuberculum (PT area) has been identified as the homolog to the DA system in the midbrain of human brain. Taking these advantages of zebrafih for study of early development, we have investigated the detailed function of Nr4a2, a transcriptional factor believed to have important function in the DA neuron development. There are two homologues of human NR4A2 in zebrafish, nr4a2a and nr4a2b. Synteny analysis has shown that the genomic location of nr4a2b is more conserved with human NR4A2. To uncover the molecular mechanisms by which Nr4a2b contributes to the development of DA neurons, we have studied the topographic distribution of nr4a2b transcripts, and found that although nr4a2b transcripts do not co-localize with the transcripts of DA neuron markers (tyrosine hydroxylase, TH and DA transporter, DAT) in the PT area, however, it co-localizes with a neuronal progenitor marker (neurog1). Knockdown of Nr4a2 results in a significant decrease of DA neurons in the PT area, accompanied by a reduction of DA transmitter, which can partially be rescued by the injection of mouse Nr4a2 mRNA. Surprisingly, the number of nr4a2b-positive cells in Nr4a2-deficient embryos is increased by about 1.6 fold. These results suggest that Nr4a2 may play an important role in the differentiation and maturation rather than the survival of DA progenitors in the PT area during zebrafish embryogenesis. The increase of nr4a2b-positive cells might be caused by the blockage of the downstream differentiation pathway and the continuous incoming upstream differentiating Neurog1-positive cells. Neurog1 and Nr4a2b might work in the same pathway to regulate the differentiation of DA neurons in the PT area during zebrafish embryogenesis. In addition, we have investigated the correlation between TH and nr4a2b in adult brain and found that nr4a2b is co-localized with some spindle-shaped TH-positive cells in PT area. We have also documented some of the cells co-expressing TH and nr4a2 in the pretectum area at the boundary of the two groups of cells. Furthermore, we have demonstrated that the nr4a2/Nestin co-expressed cells, which are declined with aging, are localized beside the diencephalic ventricle. These findings indicate that Nr4a2 might be expressed in the neuronal progenitors to play a crucial role in the differentiation process in the adult brain, and the decrease of Nr4a2 expression with aging may suggest its possible association with neurodegenerative diseases. To further study the function of Nr4a2b in zebrafish and the fate of the Nr4a2b- positive cells, we need to set up the nr4a2b::EGFP line to specifically drive the EGFP to express in the Nr4a2b-positive cells. Since the promoter to drive the endogenous Nr4a2b expression is too long to clone by the conventional techniques, we make the construct by using the BAC homologous recombineering technology to insert the EGFP to the downstream of nr4a2b promoter region. With the novel recombinant construct, we are able to detect the co-localization of EGFP and endogenous nr4a2b. Our initial result shows that we have successfully constructed the recombinant BAC to drive the EGFP to specifically express in the Nr4a2b-positive cells. The screen of stable transgenic line is still undergoing. In summary, the results from our studies of Nr4a2’s role in both embryonic and adult zebrafish further clarify the detail mechanisms of DA neuron differentiation, and the novel construction of recombinant BAC will greatly help us understand the molecular mechanisms of Nr4a2 in the DA neuron development. These data will also shed new light on leading to uncover the association of these genes in DA neuron dysfunction relevant to Parkinson’s disease and find the new drugs targeting on these devastating diseases.
Language: 中文
Content Type: 学位论文
URI: http://ir.sibs.ac.cn/handle/331001/2392
Appears in Collections:神经所(总)_学位论文

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Recommended Citation:
Nr4a2在斑马鱼间脑多巴胺能神经元发育中的基因调控和分子学机制研究.罗光睿[d].中国科学院上海生命科学研究院,2009.20-25
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