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题名	Nkx6.3调控神经嵴发育的机制和文昌鱼Hedgehog信号通路与纤毛关系的初步研究
作者	张祖明
学位类别	博士
答辩日期	2014-11
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	神经嵴 Nkx6.3 文昌鱼 Smoothened 纤毛
其他题名	Regulation of neural crest development by Nkx6.3 and the involvement of cilia in Hedgehog signaling in amphioxus
中文摘要	神经板边界是指位于脊椎动物胚胎神经板和表皮交界处的外胚层，将来分化成为神经嵴（neural crest）和基板（placode）。神经嵴与基板是脊椎动物所特有的结构，与脊椎动物的一些基本特征的发育密切相关。因此对神经板边界、神经嵴和基板的研究已成为探索脊椎动物进化、胚胎发育不可回避的重要内容。神经板边界的形成受到来自周围组织的BMP、Wnt、FGF等信号的调控，这些信号调节一组转录因子在神经板边界区域的特异性表达，包括Zic、Pax、Dlx和Msx家族的基因。这些基因被称为神经板边界的特化基因，它们进而调控神经嵴与基板的进一步分化。 在本文中，我们在非洲爪蛙中鉴定了一个新的神经板边界特化基因-Nkx6.3。我们发现Nkx6.3表达于爪蛙胚胎的表皮、基板与神经嵴，而在神经板中不表达。利用反义Morpholino抑制内源Nkx6.3蛋白的表达会抑制神经嵴的发育，并影响多种神经板边界基因的表达。在神经板边界区域过表达Nkx6.3会扩大部分神经板边界基因的表达范围（如Msx1, Zic1)，而抑制其它基因（如Pax3）和神经嵴特化基因的表达；而在神经板前部过表达Nkx6.3会诱导异位神经嵴的产生。同时我们发现Nkx6.3过表达会激活Wnt8的表达，这可能是其影响神经嵴形成的重要机制。通过进一步的研究我们发现，Msx1是Nkx6.3潜在的直接靶基因之一， Dlx3则会抑制Nkx6.3诱导神经嵴的活性。为了进一步寻找Nkx6.3和Dlx3的潜在靶基因，我们在爪蛙胚胎中共注射带有生物素化标签序列的Nkx6.3、Dlx3的mRNA与细菌来源的生物素化酶的mRNA，成功表达了生物素化的Nkx6.3和Dlx3蛋白，建立了bioChIP的方法，并通过克隆测序，初步获得了部分潜在的Nkx6.3靶位点序列，为进一步全面分析其靶基因奠定了基础。 Hedgehog(Hh)信号通路参与胚胎发育的诸多过程，包括脊椎动物肢芽极性的形成、神经管的形成等。成体中该信号通路的紊乱与一些癌症的发生相关，如髓母细胞瘤、基底细胞癌等。Hh信号通路的配体Hh通过与膜受体Patched（Ptch）结合激活该信号通路。这一通路首先发现于果蝇中。在没有Hh配体时，Ptch受体蛋白分布于细胞膜上，信号通路下游的Smoothened（Smo）蛋白分布于细胞质中。当配体Hh与Ptch结合后，Ptch被转运到细胞质中，解除对Smo蛋白的抑制，Smo被转运到细胞膜上，信号通路被激活。该信号通路在脊椎动物与无脊椎动物中的一个重要不同在于，在脊椎动物中，Hh信号通路的激活依赖于原纤毛。小鼠中的Ptch蛋白富集于原纤毛上，在其与配体结合时被清除出纤毛，Smo蛋白则被转移到原纤毛上，进而激活下游信号。然而，在进化过程中，Hh信号在何时、通过何种机制与纤毛相关联的，仍是一个未解之谜。 文昌鱼作为介于无脊椎动物和脊椎动物的中间物种，在进化中有着重要的地位。在本研究中，我们克隆了文昌鱼的Smo基因，初步研究了文昌鱼Smo是否定位于纤毛以及干扰纤毛形成是否会影响文昌鱼的Hh信号活性。在转染小鼠细胞中，文昌鱼的Smo蛋白可以转移到小鼠细胞的原纤毛上；在文昌鱼胚胎中敲低原纤毛的马达分子Kif3，Hh信号通路的活性受到抑制。这些数据提示，在文昌鱼中，Hh信号通路可能依赖于原纤毛，但仍有待于进一步的验证。 关键词：神经板边界，神经嵴，Nkx6.3，染色质免疫共沉淀，文昌鱼，Smoothened，纤毛
英文摘要	Neural plate border is generated in the junction of the neural plate and epidermis, and give rise to neural crest and placode，which contribute to many fundamental characters of vertebrate. Therefore, to understand the evolution and development of vertebrate，studies on neural plate border, neural crest and placode are unavoidable. The formation of neural plate border is regulated by the signals of BMP, Wnt and FGF from surrounding tissues. These signals induce a panel of transfactors expressing in neural plate border, including Zic, Pax, Dlx and Msx family genes. These genes are neural plate border specifiers and regulate the differentiation of neural crest and placode. In this paper, we identified a new neural plate border specifier - Nkx6.3 in X.laevis. Nkx6.3 is expressed in the epidermis, placode and neural crest, not in neural plate. Inhibiting the expression of Nkx6.3 by antisense Morpholino，the development of neural crest is repressed and some neural plate border specifiers are disturbed. The expression domain of some neural plate border specifiers(Msx1 and Zic1) are expanded, while other(Pax3) is inhibited. The neural crest marker genes are inhibited as well. However, when overexpressing Nkx6.3 in the anterior of neural plate, neural crest is induced around the anterior of neural plate. Beside, we found that when overexpressing Nkx6.3, the expression of Wnt8 is induced, which maybe a reason for the ability of Nkx6.3 to induce neural crest. Additionally, Msx1 is one of the direct candidate genes for Nkx6.3 and Dlx3 can inhibit the neural crest induction of Nkx6.3. Furthermore, Nkx6.3 and Dlx3 are biotinylated in the embryos of X.laevis when we injecting the mRNA of Nkx6.3 and Dlx3 fused with a short bio peptide tag and BirA, an enzyme from E.Coli. Hedgehog (Hh) signal pathway is involved in many development processes of embryo, such as the polarity of palm and the formation of neural tube. In adults, cancers, such as medulloblastoma and basal cell carcinoma, are happened when this signal pathway disrupted. Hedgehog signal pathway is activated by the binding of ligand Hh and receptor Patched(Ptch).This signal pathway is studied in fly first. Without Hh, Ptch is located to membrane and Smoothened(Smo) is located in cytoplasm. When Hh binding to Ptch, Ptch is translocated into cytoplasm and release the inhibition of Smo. Then the signal pathway is active. For Hh signal pathway, the most different between vertebrate and invertebrate is that in vertebrate this signal pathway is dependent on primary cilia. In mouse, Ptch is enriched in primary cilia, and removed when binding with ligand Hh. Then Smo is translocated to primary cilia and active downstream signal. However, the question, when and how Hh signal pathway related to primary cilia, puzzle researchers. Amphioxus is an important species for evolution, because of its transition position between vertebrate and invertebrate. In this study, we cloned the Smo gene of amphioxus and tested the possibility of primary cilia location of it. Moreover, the Hh activity was tested when disrupting the formation of cilia. The results show that amphioxus Smoothened protein can be located to the primary cilia when transfected into mouse cells. Additionally, the activity of Hh pathway is downregulated when knockdown Kif3, one of the dynein proteins in the primary cilia. These results suggested that Hh signal pathway maybe dependent on primary cilia in Amphioxus and this will be proved by more test. Keywords: neural plate border, neural crest, Nkx6.3, ChIP, Amphioxus, Smoothened, cilia
语种	中文
内容类型	学位论文
源URL	[http://159.226.149.26:8080/handle/152453/10179]
专题	昆明动物研究所_发育生物学
推荐引用方式 GB/T 7714	张祖明. Nkx6.3调控神经嵴发育的机制和文昌鱼Hedgehog信号通路与纤毛关系的初步研究[D]. 北京. 中国科学院研究生院. 2014.

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