【作者】 蔡召平；

【导师】 张红卫；

【作者基本信息】 山东大学 ， 发育生物学， 2009， 硕士

【摘要】 脊椎动物的胚胎图式形成需要多种保守的信号通——Nodal、Bmp、Wnt、Hedgehog和Fgf等,它们构成一个复杂的调控网络,参与调控胚胎发育过程中的各种生命活动。左-右轴是脊椎动物三个重要的体轴之一,它的形成同样需要多种信号通路的共同调节。本论文的第一部分利用氯化锂(LiCl)作为经典Wnt信号通路的特异激活剂,通过原位杂交技术,首次发现了经典Wnt信号参与体节发生的左右对称性调节。研究发现,在斑马鱼原肠胚早期用LiCl处理,能使胚胎在早体节期产生不对称性体节。体节发生中的两个关键调控分子:fgf8和her1,在LiCl处理过的胚胎中也出现两侧不同步表达的现象。并且,在胚胎发育后期,心脏和肝脏的左右位置特性也发生了改变。但是,Nodal信号通路的关键组分(southpaw,lefty1和pitx2a)的表达图式却没有发生相应的改变,这些结果表明,在斑马鱼中,LiCl造成的不对称性体节和器官水平上左右位置的缺陷并不是由于Nodal信号的改变引起的。另外,我们也发现,LiCl处理可能通过干扰中轴中胚层的发育间接引起southpaw(spaw)的异位表达。Hedgehog信号通路在胚胎发育中起着重要作用。它参与细胞增殖、分化和多种组织的模式形成。在脊椎动物中,Sonic hedgehog作为一种形态发生素,能形成浓度梯度,诱导周围的不同位置的细胞产生不同的分化命运。本论文的第二部分是以头索动物文昌鱼为实验材料,利用文昌鱼在生命演化过程中独特的进化地位,首次对Hedgehog信号通路的几个关键组分(hedgehog,patched,smoothened,gli,suppressor-of-fused)在系统进化学、基因组结构和表达图式上,进行了系统地研究。研究发现,文昌鱼Hedgehog通路几个主要分子在系统进化上都位于脊椎动物同源分子进化学分支的基部,符合文昌鱼在传统分类学中的进化地位。在基因组结构特征方面,文昌鱼与脊椎动物更相似。文昌鱼无论是对于无脊椎动物果蝇,还是对于脊椎动物斑马鱼和人而言,都倾向于拥有更多数量的外显子,甚至存在一些在其他物种中既没有相应大小也没有对应蛋白序列的外显子,说明文昌鱼Hedgehog通路几个主要基因,在基因表达产物的调节上具有一定的特殊性,还具有有头索动物的原始属性;在时空表达方面,Hedgehog通路几个主要基因的表达图式有很大的相似性,都在形成中和新形成的体节、分化中的原肠腔壁和头部中内胚层表达,这与在果蝇和脊椎动物同源基因的表达情况也很相似,说明Hedgehog信号通路在进化中十分保守。本论文的第三部分研究了Ecsit在斑马鱼早期胚胎发育中的功能。Ecsit是目前已知的BMP信号通路和Toll信号通路的唯一交叉点,在脊椎动物的胚胎发育和自身免疫调节中都具有重要的作用。通过在斑马鱼胚胎早期注射morpholino,抑制Ecsit mRNA的翻译,本研究首次发现,胚胎早期绝大部分Bmp信号,像bmp4,vox,eve1等,都受到了抑制,但vent的表达却没有明显的改变。这些结果说明Ecsit只介导部分的Bmp信号。此外,还发现巨噬细胞的早期marker基因lcp1的表达也受到了抑制,表明Ecsit可能同样参与巨噬细胞的成熟分化。更多还原

【Abstract】 Vertebrate embryonic patterning requires several conserved inductive signals, including Nodal,Bmp,Wnt,Hedgehog and Fgf signals,which have shown to constitute a complex regulatory network that are responsible for all the events in embryogenesis.Left-right(L-R) axis is one of the three major axes in vertebrates.The generation of L-R asymmetry also requires interaction of many signals.The first part of this study firstly discovers a second role of Wnt signaling in L-R regulation of somitogenesis,using LiCl chloride(LiCl) as a specific activator of canonical wnt signaling.,Our data show when embryos are treated by LiCl at early-gastrulation,asymmetric somites are found in early somite stage.The bilateral symmetric expressions of herl and fgf8a,two critical members in segmentation model, are desynchronized in LiCl-treated embryos.L-R asymmetries of the heart and liver are also defective in the subsequent development.However,the expressions of Nodal pathway components(southpaw,lefty1 and pitx2a) do not correspondingly changed. These results demonstrate defects in somitogenesis and organ asymmetries induced by LiCl are not due to the interruption of Nodal signaling.Moreover,aberrant spaw expressions after LiCl treatment probably result from the impairment of the notochord development in zebrafish.Hedgehog signaling plays a multiple role in many processes,including cell proliferation,cell-fate determination and differentiation of various tissues.In vertabrates,Sonic hedgehog acts as a morphogen that induces cells into different cell fates by different concentration.The second part of this study firstly focuses on the phylogenesis,genomics and express pattern of several important members of Hedgehog signaling in amphioxus.In this part,we have shown Hedgehog pathway genes(hedgehog,patched,smoothened, gli and suppressor-of-fused) are based on the vertebate branches.In genomic structure of these genes,amphioxus is more similar to vertebrates.Amphioxus tends to possess more exons than Drosophila and zebrafish and human.These results indicate the Hedgehog pathway genes in amphioxus still have primitive characteristics in gene regulatory.In expression pattern,these genes have many similarities to each other including somite,archenteron and head mesoendoderm and to homologes in Drosophila and vertebrates.This demonstrates Hedgehog signaling is evolutionary conserved.The third part of this study is mainly on Ecsit in zebrafish embryogenesis.Ecsit is so far the only known intermediate between Toll and Bmp signaling in vertebrates and plays a critical role in both embryogenesis and immunity regulation.By injection of Ecsit morpholino into 1-cell stage of zebrafish embyos,we find that most BMP signaling,including bmp4,vox and evel are inhibited.These results show Ecsit conveys part of Bmp signaling.Futhermore,early macrophage marker gene lcp1 is also suppressed,which show Ecsit may involve in maturation of macrophages.更多还原