斑马鱼Dapper2的表达、调控及其对中胚层发育的作用机理

作者姓名：张丽霞
论文题目：斑马鱼Dapper2的表达、调控及其对中胚层发育的作用机理
作者简介：张丽霞，女， 1974年10月出生，2001年9月师从于清华大学孟安明教授，于2005年1月获博士学位。

中文摘要
发育生物学是当今生命科学的一个重要学科，其目的在于探索生物个体在发育过程中基因的表达、调控、功能及基因间的相互作用网络。

脊椎动物由单细胞受精卵发育到由多细胞组成的中期囊胚阶段时，通过细胞分化产生内胚层、中胚层和外胚层，不同胚层主要沿背－腹轴线、前－后轴线以及左－右轴线生成不同的组织、器官。

这些胚层的形成和分化涉及细胞与细胞之间的信号交流和相互作用，转化生长因子（TGF ）、Wnt、成纤维生长因子（FGF）等信号通路在胚层的形成和分化中起至关重要的作用，胚胎的正常发育必需对这些信号的强度进行精确的时空调控。

Nodal是TGFβ超家族成员，已知其是脊椎动物中、内胚层诱导的关键因子，也参与了胚胎的背部发育。

此外，Nodal信号还在胚胎的左右不对称发育及神经外胚层的前后轴线分化中起重要的作用。

然而，在胚胎发育中Nodal信号的强度是如何受到精确调控的，目前尚知道的不多。

斑马鱼因其高繁殖力、胚胎体外发育、胚胎透明、较完善的遗传操作技术等优点，已成为发育生物学研究的一种重要模式脊椎动物。

Nodal信号在斑马鱼和其它脊椎动物胚胎发育中的作用相当保守的，因此在斑马鱼上的研究结果也可以帮助理解其它物种上胚胎发育的相关机理。

本研究通过胚胎原位杂交的方法，从斑马鱼早期胚胎的cDNA文库中筛选到组织特异性表达基因dapper2（dpr2）。

一些证据表明，它是人的DAPPER2的同源基因,而其功能未见报导。

斑马鱼dpr2的表达开始于晚囊胚的背部胚盘边缘；至原肠作用开始时，其表达逐渐扩展到整个胚盘边缘，在背部组织中心（胚盾）中的表达最强，这些表达dpr2的细胞具有发育为中胚层组织的命运；在体节形成期，dpr2在神经管背部、侧板中胚层和尾芽中表达；胚胎发育到24小时时，其表达局限在血液前体细胞和尾芽中。

给单细胞斑马鱼胚胎注射squint(sqt)基因的mRNA,可以诱导dpr2的异位表达；在包括cyclops;squint、MZoep、MZsur在内的缺失Nodal 信号的斑马鱼突变体中，dpr2在原肠期没有表达。

这些结果表明，dpr2的表达受Nodal信号的激活。

当用吗啉环修饰的dpr2反义寡核苷酸（dpr2-MOs）抑制内源dpr2mRNA的翻译时，Nodal
信号通路的基因cyclops、sqt、oep、lefty1的表达得到增强，dpr2基因本身的转录量也显著增加，说明dpr2可以反馈负调控Nodal信号通路上的基因和其自身的表达。

本论文采用基因敲降和过量表达的方法研究了dpr2在斑马鱼胚胎发育中的作用。

在斑马鱼胚胎中注射dpr2-MOs，可以使中胚层组织增加，chordin、gsc、ntl、snail、shh等中胚层标记基因的表达增强；dpr2-MOs与斑马鱼Nodal基因sqt mRNA或Nodal抑制因子lefty1的反义寡核苷酸（lft1-MO）共注射，可以进一步加强Nodal信号的作用；用dpr2-MOs注射缺失Nodal 信号的斑马鱼oep突变体胚胎，原来不在腹侧脑组织和神经管基板表达的shh基因可以恢复表达。

与此相反，在胚胎中过量表达dpr2，可以导致胚胎中线中胚层组织发育缺陷，引起类似oep的表型。

这些结果表明，Dpr2在胚胎发育中的正常功能是抑制Nodal信号的中胚层诱导作用。

本论文还研究了dpr2与其它控制胚胎发育的重要信号通路Bmp、Fgf、Wnt的关系。

抑制dpr2的表达，Bmp信号的下游基因gata2在神经外胚层的表达不受影响，dpr2和bmp7mRNA共注射可以增强bmp7过量表达导致的胚胎腹部化，表明Dpr2在胚胎发育中可能不抑制Bmp信号的作用。

fgf8的过量表达与抑制dpr2表达时对胚胎发育的影响有所不同，而且fgf8的过量表达也不能像注射dpr2-MOs那样恢复shh在oep突变体的神经基板的表达，说明Dpr2在对中胚层发育的抑制作用方面可能与Fgf信号无关。

同样地，wnt8的过量表达与抑制dpr2表达时对胚胎发育的影响不同，而且wnt8或disheveled2的过量表达也不能像注射dpr2-MOs那样恢复shh在oep突变体的神经基板的表达，表明Dpr2对中胚层发育的抑制作用可能与Wnt信号无关。

鉴于TGFβ/Nodal信号的转导机制在不同物种之间是保守的，因而本研究利用哺乳动物细胞系进一步研究了斑马鱼Dpr2在TGFβ/Nodal信号转导中的作用及其机理。

利用响应TGFβ信号的ARE-luciferase报告系统的研究发现，在哺乳动物Hep3B和NIH3T3细胞中，Dpr2可以抑制TGFβ或表达持续激活的受体ALK4和ALK5刺激的ARE-luciferase报告系统的表达。

为了探讨Dpr2抑制TGFβ/Nodal信号转导的分子机理，通过免疫共沉淀实验发现，Dpr2能够与TGFβ/Nodal 的I型受体ALK4或ALK5结合，它与激活形式的ALK4和ALK5的结合更强；另一方面，Dpr2的过量表达导致ALK4或ALK5蛋白量的减少，说明Dpr2可能具有促进TGFβ/Nodal 的I型受体降解的功能。

在加入蛋白酶体抑制剂MG132时，Dpr2的过量表达仍然可以引起ALK4或ALK5蛋白量的减少；但加入氯化铵、Chloroquin或Bafilomycin等溶酶体抑制剂时，Dpr2的过量表达未导致ALK4和ALK5蛋白量的下降。

这些结果表明，Dpr2促进了TGFβ受体通过溶酶体途径而非蛋白酶体途径的降解。

通过免疫荧光亚细胞定位实验发现，Dpr2蛋白主要定位在晚期内吞体中，而不存在于早期内吞体、质膜微囊或溶酶体中。

由此推测，Dpr2参与了通
过胞吞作用进入细胞质的TGF /Nodal受体的分选，或者帮助将晚期内吞体中的受体带到溶酶体中降解。

由此本研究提出Nodal信号的中胚层诱导活性的一种新的调控机制，即胚胎中有Nodal 活性的区域表达其抑制因子Dpr2，后者与激活、内吞的Nodal受体结合，促进含Nodal受体的晚期内吞体向溶酶体转运，这些受体进入溶酶体后降解，通过这种途径使Nodal信号的强度受到调控。

关键词：斑马鱼，胚胎发育，dapper2，Nodal信号，TGFβ受体，内吞
Expression,Regulation and Functional Mechanism on Mesoderm
Development of Zebrafish Dapper2
Zhang Li Xia
ABSTRACT
Developmental biology is currently an important discipline in life sciences, which aims to investigate gene’s expression pattern, function and regulatory networks during development of organisms. When a fertilized egg of vertebrates develops into a multicellular embryo at midblastula stage, three germ layers, endoderm, mesoderm and ectoderm layers, are induced following cell differentiation. Subsequently, different germ layers will give rise to different tissues and organs along the dorsal-ventral, anterior-posterior and left-right axes. The formation and differentiation of the three germ layers involves intercellular communications and interactions. TGFβ, Wnt and FGF signals play important roles in the formation and differentiation of the germ layers and their activities have to be precisely regulated in a spatiotemporal fashion during normal embryonic development. It is known that Nodal and Nodal-related proteins, members of TGFβ superfamily, are key endogenous inducers for mesoderm and endoderm in vertebrates, and that they are involved in dorsal development of embryos. Nodal signals also play pivotal roles in left-right asymmetrical development and neuroectodermal anterioposterior patterning. However, it remains unclear how the Nodal signaling is controlled precisely during embryonic development. Zebrafish has become an important vertebrate model in developmental biology, because of its high reproductivity, external embryonic development, transparency and well-established genetic manipulation technologies. It has been found that functions of Nodal signals are well conserved between zebrafish and other vertebrates. Thus, knowledge obtained in zebrafish will also help understanding the mechanisms for embryonic development of other vertebrate animals.
In this study, a tissue-specific gene, dapper2 (dpr2), is identified during screening for genes with restricted expression patterns from an embryonic cDNA library of zebrafish using whole-mount in situ hybridization. Several lines of evidence indicate that this gene is an ortholog of
human DAPPER2, a factor with unknown functions. Zebrafish dpr2starts to express in the dorsal blastoderm of late blastulas. At the onset of gastrulation, dpr2is expressed in the entire blastodermal margin, with the highest level in the dorsal organizer (embryonic shield), which commit to mesodermal fate. It is expressed in dorsal neural tube, lateral mesoderm and the tail bud during segmentation, and it is restricted to blood progenitors and the tail bud at 24 h of development. Injecting of squint(sqt) mRNA into one-cell stage embryos induces ectopic expression of dpr2. In zebrafish mutant embryos that are deficient in Nodal signaling pathway, including cyclops;squint, MZoep and MZsur, dpr2 expression during gastrulation disappears. These results suggest that dpr2expression is activated by Nodal signaling. Furthermore, the expression of the Nodal pathway components cyclops, sqt, oep and lefty1as well as that of dpr2itself is enhanced when translation of endogenous dpr2mRNA is blocked with dpr2 antisense morpholinos (dpr2-MOs), implying that dpr2negatively regulates the expression of Nodal pathway components and dpr2 itself in a feedback fashion.
This study investigated the roles of dpr2in development of zebrafish embryos using both knockdown and overexpression approaches. Injection of dpr2-MOs in one-cell embryos results in increase of mesodermal tissues and enhances expression of mesodermal marker genes chordin, gsc, ntl, snail and shh. Injection of dpr2-MOs is able to enhance effect of overexpression of sqt, a zebrafish Nodal gene, and that of morpholino-mediated knockdown of lft1, an antagonist of Nodal signals. Knockdown of dpr2 with dpr2-MOs in oep mutant embryos that are zygotically deficient in Nodal signals can rescue shh expression in the ventral brain and the floor plate of the neural tube. In contrast, overexpression of dpr2 causes developmental defects in the midline mesodermal tissues and results in a phenotype resembling oep. These data altogether suggest that Dpr2 functions to inhibit mesoderm induction mediated by Nodal signaling. In addition, dpr2 expression increases in the embryos injected with dpr2-MO, suggesting a negative feedback regulation mechanism.
This study also investigated the potential involvement of dpr2in Bmp, Fgf and Wnt signaling pathways that play important roles in embryonic development. The fact that knockdown of dpr2 has no effect on the expression of the Bmp downstream gene gata2in the neuroectoderm and that coinjection of dpr2and bmp7mRNAs is unable to severe ventralized phenotypes by bmp7 overexpression suggests that Dpr2 is unlikely to antagonize Bmp signaling during embryogenesis. fgf8 overexpression in zebrafish embryos has an effect different from that of dpr2 knockdown and
fails to resume shh expression in the floor plate of oep mutant, excluding a possibility that Dpr2 is an antagonist of Fgf signals in development of zebrafish embryos. Similarly, effect of wnt8 overexpression results in an effect different from that of dpr2knockdown, and overexpression of wnt8or disheveled2overexpression is unable to rescue shh expression in the floor plate of oep mutant, indicating that inhibitory effect of dpr2on mesodermal development is irrelevant to Wnt signaling.
Considering that TGFβ/Nodal signal transduction is conserved among different species, roles of Dpr2 in this pathway and its underlying mechanism is investigated in mammalian cell lines. In mammalian Hep3B and NIH3T3 cells, Dpr2 overexpression inhibits expression of the ARE-luciferase reporter that is stimulated by TGFβ ligand or overexpression of the constitutively active TGFβ/Nodal type I receptor ALK4 or ALK5. Co-immunoprecipitation experiments reveal that Dpr2 physically binds to ALK4 and ALK5 with higher affinity with their activated form. The amount of ALK4 or ALK5 decreases when Dpr2 is co-expressed, implying that Dpr2 may promote degradation of these receptors. Decrease in ALK4 or ALK5 level caused by Dpr2 overexpression is not affected by addition of the proteasomal inhibitor MG132, but inhibited in the presence of the lysosomal inhibitor NH4Cl, chloroquin or Bafilomycin, suggesting that Dpr2 promotes degradation of the TGFβ/Nodal receptors via lysosomal pathway. Subcellular localization analyses by immunostaining indicate that Dpr2 primarily localizes in the late endosomes, but not in the early-endosomes, lipid-rafts or lysosomes. Thus, Dpr2 may be involved in sorting of endocytosed TGFβ/Nodal receptors in the cytoplasm, or it may help to transport of the receptors into lysosomes for degradation.
In summary, this study proposes a novel mechanism underlying regulation of mesoderm induction activity of Nodal signals. Embryos express the Nodal antagonist Dpr2 in domains with Nodal activity. Dpr2 binds to the endocytosed, activated Nodal receptors, and facilitates their transport to lysosomes for degradation. Thus, Nodal activity is regulated in this way.
Key borad:zabrafish, embryo development, dapper2, Nodal signal, TGFβreceptor, Endocytosis。