| 题名 | 文昌鱼Dkk和Kremen基因家族表达图谱和功能的研究 |
| 作者 | 张煜珺 |
| 学位类别 | 硕士 |
| 答辩日期 | 2008-12 |
| 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 | 文昌鱼 白氏文昌鱼 Dkk Kremen GATA factors Otx |
| 其他题名 | Expression and Functional Analysis of Dkk and Kremen Genes in Amphioxus (Branchiostoma belcheri) |
| 学位专业 | 发育生物学 |
| 中文摘要 | 文昌鱼长期作为脊索动物的祖先模型被研究。它与脊椎动物发育机制的比较为后者的发生和进化提供了大量证据。Wnt信号通路在动物胚胎发育中行使着多样而重要的功能：如胚胎轴系的建立，胚层分化，神经图式形成等。在腔肠动物胚胎发育的早期，Wnt/β-catenin主要参与动植物半球极性的形成和胚层分化——很可能是Wnt通路的祖先功能。而在高等脊椎动物胚胎发育的早期，Wnt/β-catenin通路对于背腹轴、前后轴和左右轴极性的建立发挥着至关重要的作用。我们的研究主要集中在文昌鱼Wnt/β-catenin信号通路的两种调节因子Dickkopf（Dkk）和Kremen，以探明这些Wnt信号调节因子的祖先功能，以及在脊椎动物中获得新功能的进化历程。 分泌性蛋白Dkk是Wnt信号通路的抑制因子，协同它的高亲和性受体Kremen，在两栖类胚胎头部的发育中起着关键作用。基于脊椎动物Dkk和kremen的报道以及佛罗里达文昌鱼基因组序列信息，我们运用分子克隆的方法，得到白氏文昌鱼Dkk家族的两个基因：BbDkk124和BbDkk3，以及Kremen家族的5个基因：BbKremen-a，BbKremen-c，BbKremen-d，BbKremen-e，BbKremen-g，用整体胚胎原位杂交的方法研究了它们的表达图谱，并在293T细胞和非洲爪蟾胚胎这两个系统中检测了它们对Wnt信号活性的影响和胚胎发育表型的影响。结果表明文昌鱼Dkk和Kremen的表达区域与脊椎动物的同源基因并不相同，出现了较大分歧，但BbDkk124作为Wnt信号抑制因子的功能是保守的。Kremen家族的两个基因BbKremen-e和BbKremen-g在293T细胞内对Wnt通路的影响不显著，而在非洲爪蟾系统中，引起胚胎不同的畸形表型。我们的实验结果为脊椎动物Dkk和Kremen 基因家族的进化提供了一些资料。 此外我们还研究了文昌鱼GATA家族的基因，这个家族在脊椎动物和非脊椎动物的发育中行使重要的动能，在进化上也是非常保守的。脊椎动物的GATA基因分为两个亚群：GATA1/2/3和GATA4/5/6。通过生物信息分析，我们在文昌鱼的基因组中找到了三个GATA基因：一个GATA1/2/3亚家族基因，两个GATA4/5/6亚家族基因，另外还找到一个类GATA基因。我们克隆了白氏文昌鱼GATA123的一段序列并研究了它在早期胚胎发育中的特异性表达。结果表明GATA123在原肠胚的中内胚层表达，而在神经胚晚期和幼体早期，GATA123在脑泡和消化道中部区域表达。这种表达模式与头部发育的重要基因Otx相类似。我们的研究结果提示在文昌鱼脑泡的发育过程中GATA123和Otx很可能协同发挥着重要的作用。 |
| 英文摘要 | The Cephalochordate amphioxus, widely considered as a model of vertebrate ancestor, is a key organism for understanding of the origin and evolution of vertebrate developmental mechanisms. Wnt signaling pathway is well conserved from cnidarians to vertebrates and play versatile roles during embryonic development, such as axis formation, germ layer specification and neural system development. Its ancestral function is suggested to be regulation of animal-vegetal axial polarity and germ layer segregation, while in vertebrates, it plays key roles in the dorsal-ventral axis formation at early stages and later ones in the anterior-posterior patterning and left-right asymmetry of the body. Our researches focus on the analysis of two families of Wnt regulators, Dickkopf (Dkk), Kremen in amphioxus. Secreted protein Dkk and its receptor Kremen are Wnt signaling inhibitors, which are expressed in anterior region in vertebrates and regulate anterior embryonic development. Based on vertebrate Dkk and Kremen sequences and analysis of amphioxus (Branchiostoma floridae) genome, we have cloned two Dkk family genes: BbDkk124, BbDkk3 and five Kremen genes: BbKremen-a，BbKremen-c，BbKremen-d，BbKremen-e, BbKremen-g in amphioxus (Branchiostoma belcheri). We studied their expression patterns and tested their ability to inhibit Wnt signaling in reporter gene analysis in 293T cells as well as phenotypic analysis in Xenopus embryos. Our results suggest their expression patterns are not conserved compared with vertebrate homologs. However, the function of BbDkk124 as a Wnt signaling inhibitor is conserved and when injected in Xenopus embryos, it can induce anteriorized phenotypes with big heads and short trunks. BbKremen-e and BbKremen-g are functionally different in Xenopus embryos. These results provide important clue on the evolution of Dkk and Kremen functions. We also studied the amphioxus GATA factors, which are evolutionarily conserved and play crucial roles during embryonic development in both vertebrates and invertebrates. Vertebrate GATAs can be divided into two subgroups, the GATA1/2/3 and the GATA4/5/6 classes. Through genomic analysis, we have identified three GATA factors, representing the GATA1/2/3 and GATA4/5/6 subfamilies respectively, and one GATA like protein in the genome of amphioxus (Branchiostoma floridae, cephalochordata), the most basal chordate subphylum. We have cloned partial sequence of GATA123 in the amphioxus Branchiostoma belcheri (BbGATA123) and studied its expression pattern during early embryonic development. Expression of BbGATA123 is first detected in the mesendoderm during gastrulation. Interestingly, in the late neurula and early larva stages, it is expressed strongly in the cerebral vesicle and the mid gut region. Its expression is compared to Otx, a gene known crucial for the development of anterior structures. Our data suggest that GATA123, together with Otx, might play an important role in the development of amphioxus cerebral vesicle, the counterpart of the vertebrate brain. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6084]  |
| 专题 | 昆明动物研究所_发育生物学 |
| 推荐引用方式 GB/T 7714 | 张煜珺. 文昌鱼Dkk和Kremen基因家族表达图谱和功能的研究[D]. 北京. 中国科学院研究生院. 2008. |
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