QQ客服 官方微博 反馈留言

题名	非洲爪蟾Nkx6.3在原肠运动和神经嵴诱导调控中的功能及爪蟾神经管腹侧图式形成的研究
作者	赵树华
学位类别	博士
答辩日期	2009-06
授予单位	中国科学院研究生院
授予地点	北京
导师	毛炳宇
关键词	Nkx6 爪蟾 Nkx6.3 表达 原肠运动 细胞粘附 细胞运动 神经嵴 Wnt8 Fgf8 Bmp4 Slug Nkx6.2 启动子 转基因 Dbx 背腹图式 Shh
其他题名	The roles of Xenopus Nkx6.3 in gastrulation movement and neural crest induction and studies on Xenopus ventral neural tube patterning
中文摘要	神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发神经嵴（neural crest）是一类脊椎动物特有的多潜能迁移细胞。这一类细胞历经“表皮—间充质”转换（EMT），与神经管背侧的其它细胞分离，经由不同路线迁移，定位于胚胎外周各处，后分化为不同的细胞类型包括外周神经系统、颅面骨骼系统及色素细胞等。神经嵴的发育是一个多途径多步骤的过程，受多种信号通路及转录因子调控。这些调控因子相互调节形成精密网络，可被划分为三个主要层次类群：分泌性信号分子（BMP、Wnt、FGF、Delta）、神经板边界特异基因（Msx、Pax3/7、 Zic1、Dlx3/5）、神经嵴特异基因（Snail/Slug、AP-2、FoxD3、Twist、Id、cMyc、Sox9/10）。本文第一章主要概述不同组织来源的各种分泌信号在神经嵴诱导中的作用以及他们之间的整合调控。 Nkx6家族蛋白是一类进化上保守的转录因子，在脊椎动物中枢神经系统（CNS)的图式形成和胰腺的发育中有重要作用。在第二章，我们描述了非洲爪蟾中Nkx6家族基因的克隆及其表达图式。与小鼠和鸡中的同源基因类似，爪蟾的Nkx6家族基因在胚胎发育过程中主要表达于中枢神经系统和前部内胚层组织。其中Nkx6.1和Nkx6.2在神经胚期神经板表达重合，晚期都表达于后脑和脊髓的腹侧。Nkx6.3从卵裂期到神经胚早期都表达于非神经外胚层，而尾芽期表达于后脑后部和腮弓。在内胚层中，Nkx6.2在尾芽期表达于底索。在蝌蚪期，Nkx6家族的三个基因分别表达于前部内胚层的衍生物，包括胰腺、胃、食道和肺。 Nkx6.3是最近发现的Nkx6家族新成员，它在爪蟾中的表达与Nkx6.1和Nkx6.2有了较大分歧。在第三章，我们通过功能获得及功能缺失实验来探讨Nkx6.3在爪蟾早期发育中的功能。我们发现原肠期前过量或抑制Nkx6.3表达都会影响胚胎原肠运动的正常进行。我们通过动物帽延伸实验证明Nkx6.3参与了细胞运动。半定量RT-PCR结果显示，Nkx6.3可以调控一些粘附分子的表达。以上结果说明Nkx6.3通过调控粘附分子的转录而参与细胞运动的调控。我们还发现，在爪蟾胚胎中Nkx6.3的过表达或抑制表达都导致神经嵴标记基因表达降低。进一步研究发现，32细胞期在不同部位注射Nkx6.3 mRNA可以异位诱导或抑制Slug的表达。动物帽实验显示，Nkx6.3单独过表达可以诱导神经嵴发生，而迄今为止转录因子中只有Snail1具有这一单独诱导能力。在爪蟾胚胎及动物帽中，过表达Nkx6.3都可以诱导Fgf8、Wnt8而抑制BMP4的转录，而且Nkx6.3对这些分泌因子的调控方式是不同的。4细胞期过表达Nkx6.3的胚胎，在促进Fgf8和Wnt8而抑制BMP4的同时，却抑制神经板边界特异基因Msx1、Pax3和神经嵴特异基因Slug的表达，说明Nkx6.3对神经嵴的诱导调控在神经板边界基因层次还存在抑制作用。32细胞过表达Nkx6.3会细胞自主性抑制以及细胞非自主诱导Msx1、Pax3、Slug的表达。Nkx6.3异位诱导Dlx5却抑制Dlx3的表达，说明Dlx5可能是Nkx6.3负调控的直接靶基因。由此，我们提出Nkx6.3的神经嵴诱导调控分为两个层次：分泌信号分子水平的正调控和神经板边界决定水平的负调控。在脊椎动物的神经发生过程中，神经管背腹不同层次形成不同的神经元。这些神经元细胞的命运由背腹起源的多种形态发生素决定。形态发生素通过浓度梯度确定了一组转录因子在神经管背腹不同层次的特异表达，这些基因的组合调控决定了神经前体细胞的命运。然而，这些转录因子是如何解读形态发生素梯度信号的还不是很清楚。第四章，我们通过对神经管腹侧特异表达的转录因子的调控区进行预测，确定了可能调控这些基因表达的保守区段。此外，我们改进了爪蟾转基因操作，并用这一技术确证了Nkx6.2的调控区域。Dbx1、Nkx2.2及Pax6的转录调控区已在小鼠或爪蟾中报道过。由此我们得到了两对在神经管背腹图式中相互作用的转录因子的调控区域：Nkx6.2和Dbx1、Nkx2.2和Pax6。通过对Nkx6.2和Dbx1的调控保守区的转录因子结合位点的预测，我们发现这四个基因以及Wnt信号之间存在大量的相互调控。然而在这两个基因的调控区，我们没有发现Gli的调控位点，暗示这两个基因可能不受Shh的直接调控。我们还克隆了Dbx家族的两个基因，并检测了它们的时空特异性表达，发现Dbx2是母源性表达的，而Dbx1是合子型基因。这两个基因的表达图式相似，都在神经板中线两侧成线状表达，尾芽期在神经管中部表达。过表达Dbx2抑制神经元的初级分化，说明它可能与Dbx1一样具有维持神经板细胞未分化状态的功能。Dbx2的过表达还抑制Nkx6.2及Dbx1的表达，说明它们可能一起参与了神经管腹侧图式的调控。
英文摘要	Neural crest is a multipotent, migratory cell population, which exists only in vertebrate embryos. These cells undergo migration along distinct pathways to various sites in the periphery, and then differentiate into various cell types, ranging from the peripheral nervous system to the craniofacial skeleton and pigment cells. The neural crest development is guided by a carefully orchestrated gene regulatory network, which can be divided into several modules, including: secreted signal molecules (BMP, Wnt, FGF, Delta), neural plate specifiers (Msx, Pax3/7, Zic1, Dlx3/5) and neural crest specifiers (Snail/Slug, AP-2, FoxD3, Twist, Id, cMyc, Sox9/10). In the first part, we mainly focused on the secreted signal pathways, which arise from different tissues, play distinct roles and, induce neural crest formation by integrations at multi-levels. The evolutionarily conserved Nkx6 family transcription factors play important roles in the patterning of the central nervous system (CNS) and pancreas in vertebrates. In the second part, we described the cloning and expression patterns of the three Nkx6 family genes in Xenopus laevis. Like their mouse and chicken homologues, Xenopus Nkx6 family genes are mainly expressed in the CNS and anterior endodermal tissues during embryonic development. Nkx6.1 and Nkx6.2 share overlapping expression domains in the ventral neural tube at neurula stages and later in the ventral part of developing hindbrain and spinal cord. Nkx6.3 is detected in the non-neural ectoderm from cleavage to early neurula stages and in the caudal hindbrain and the mandibular arch at tail bud stages. In the endoderm, Nkx6.2 is expressed in the hypochord at tail bud stages. At tadpole stages, the three Nkx6 genes are differentially expressed in the anterior endoderm derivatives, including the pancreas, stomach, esophagus, and lung. Nkx6.3 is a recently reported member of Nkx6 family. Nkx6.3 shows distinct expression with the other two Nkx6 genes during early embryonic development in Xenopus laevis. In the third part, we reported the roles of Nkx6.3 in Xenopus by gain and loss of function studies. Both overexpression and knockdown of Nkx6.3 before gastrula stages led to gastrulation defects. Overepression of Nkx6.3 inhibited activin induced animal cap elongation, which could be rescued by co-injected dominant negative construct HDC. Further, we showed that Nkx6.3 regulated adhesion molecules expression by RT-PCR. These results suggest that Nkx6.3 plays roles in cell movements by regulating adhesion molecule expression levels. We also found that both gain and loss of function of Nkx6.3 inhibited the expression of neural crest marker Slug. Injection of Nkx6.3 mRNA at 32-cell stage led to inhibition or ectopic expression of Slug at different sites. In animal caps, overexpression of Nkx6.3 induced the expression of neural crest markers. These results indicate that Nkx6.3 is sufficient for neural crest induction. Further studies showed that Nkx6.3 up-regulated Wnt8 and Fgf8, but inhibited BMP4 in both animal caps and embryos. However, in the Nkx6.3 overexpressed embryos, the neural plate border specifiers, Msx1 and Pax3, as well as neural crest marker, Slug, were inhibited, indicating an existence of another regulating level of Nkx6.3 in the neural crest induction. Injection of Nkx6.3 at 32-cell stage inhibited Msx1, Pax3 and Slug in a cell autonomous manner while induced them in a cell unautonomous manner. In addition, we found that Nkx6.3 ectopicly induced Dlx5 while inhibited Dlx3, suggested that Dlx5 might be the direct target of Nkx6.3 at neural border specifier level. Thus, our data suggest that Nkx6.3 regulates neural crest induction at two levels: the positive contributions by regulating the secreted signaling molecules and the negative ones by inhibiting neural plate border specifiers. During the neural development of vertebrates, different types of neurons emerge along the dorso-ventral axis of the neural tube. These neurons are specified by different morphogens. A group of transcription factors are regulated by the gradient activities of these morphogens, and the cooperative regulation of these genes determines the fates of preneural cells. However, the way how these transcription factors interpret the gradient activities is not clear yet. In the fourth part of this thesis, we have tried to predict the potential regulatory sequences of these transcription factors by screening for conserved non-coding regions of these genes. In addition, we improved the transgenic method in Xenopus, by which we confirmed the regulatory region of Nkx6.2. The transcriptional regulatory regions of Dbx1, Nkx2.2 and Pax6 have been reported in mouse or Xenopus. Thus we got regulatory regions of two pairs of transcription factors which interact with each other during the neural tube pattern: Nkx6.2 and Dbx1, Nkx2.2 and Pax6. We showed that there were lots of cross talks between these four genes as well as Wnt signals by binding site predictions and TOP-flash assay. No binding site of Gli was predicted in the regulating regions of Nkx6.2 and Dbx1, indicating that these genes are not regulated by Shh signaling pathway directly. We also described the cloning and expression pattern of Dbx family genes, Dbx1 and Dbx2. These two genes are similarly expressed as two thin stripes flanking the midline in the neural plate and medially in the neural tube at the tailbud stages. Overexpression of Dbx2 inhibits N-tubulin, indicating a similar role of this gene in inhibiting neural differentiation as Dbx1. Dbx2 also inhibits expression of Nkx6.2 and Dbx1 but not Sox2, suggesting a patterning role of this gene in Xenopus neural differentiation.
语种	中文
公开日期	2010-10-22
内容类型	学位论文
源URL	[http://159.226.149.42:8088/handle/152453/6313]
专题	昆明动物研究所_发育生物学
推荐引用方式 GB/T 7714	赵树华. 非洲爪蟾Nkx6.3在原肠运动和神经嵴诱导调控中的功能及爪蟾神经管腹侧图式形成的研究[D]. 北京. 中国科学院研究生院. 2009.

个性服务

查看访问统计

相关权益政策

暂无数据

收藏/分享

除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。