【作者】 张立凤；

【导师】 桂永浩；

【作者基本信息】 复旦大学 ， 儿科学， 2007， 博士

【摘要】 Tbxl基因影响斑马鱼心脏发育机制的研究DiGeorge综合征是人类最常见的染色体缺失类型的先天性遗传病。DiGeorge综合征患者具有一些典型的临床特征，如先天性心脏病、中至重度的免疫功能缺陷、低钙血症、头面部的发育畸形等。许多研究表明22q11.2上的一段约112Mb范围的微小的杂合性缺失是这些疾病的病因，这一区段被命名为DiGeorge关键区段(DiGeorge critical region，DGCR)。人们应用小鼠的研究表明，Tbx1单倍体缺失常伴有第四咽弓动脉双侧或单侧发育不良，而纯合子缺失将更加严重的阻断咽弓动脉系统的发育，表现DiGeroge综合征的典型特征。小鼠Tbx1过表达也同样会导致一系列如异位锁骨下动脉，法勒氏四联征，肺动脉闭锁等异常。这些结果证实了Tbx1基因可能是DiGeorge综合征最重要的侯选基因。许多神经嵴细胞来源的结构，如头面部的骨骼，胸腺，甲状旁腺和心脏流出道在Tbx1-／-小鼠中均出现发育缺陷。因此，我们推测Tbx1基因作为DGS的重要候选基因，可能是在神经嵴细胞的发育中充当重要功能的基因。但是Tbx1基因只在咽弓、心脏和耳囊表达，并不在神经嵴细胞中表达，对于Tbx1基因影响神经嵴细胞发育的机制还不清楚。Tbx1基因通过与其它的转录因子的相互作用，形成一个转录调控的网络，并通过这个网络调控心脏的发育过程。那么，Tbx1是如何被调控的，它又与哪些转录因子有相互作用还不清楚。视黄酸(retinoic acid，RA)是维生素A在生物体内的活性代谢产物，在脊椎动物咽弓发育的过程中发挥重要的作用。视黄酸过量或缺乏的小鼠均表现出人类DGS综合征的典型特征，与Tbx1敲除和突变的表型非常相似。由于这种表型的高度相似性，促使人们提出这样的假设，在咽弓形成的过程中，视黄酸与Tbx1之间是否有相互作用关系。Tbx1与其它的转录因子如Nkx2-5、hand2、Bmp等之间的相互作用机制，以及与其它T-box基因家族成员之间的拮抗或协同的作用关系，也还不清楚。本研究将以Tbx1作为研究的突破口，对其上游调控因子以及与其他转录因子的相互作用和和其下游的靶基因进行深入研究。利用模式生物研究基因功能是一种行之有效的方法。斑马鱼作为脊椎动物的模式生物，其各系统的发育与人类相应系统有许多共同特点，2000年，Nasevicius等提出应用吗啡啉修饰性的寡核苷酸可下调斑马鱼基因的表达，而且可以多基因同时下调，因此这一快速有效的基因敲除方法，成为应用斑马鱼进行基因功能研究的重要手段。斑马鱼的心脏类似于人胚胎时的心脏，分为心房和心室，二者之间有辦膜。斑马鱼特别适合心脏发育的研究。这主要是因为：1．斑马鱼的心血管系统发育过程与人类有许多共同点，与人类的心脏发育具有相似的基因调控途径。2．斑马鱼胚胎体外受精，体外发育，完全透明，其心脏发育可以直接用眼睛观察，因此具有高度的可视性。3．斑马鱼的胚胎可以不依赖于有功能的心血管系统，甚至在整个心血管循环缺失的情况下，它都仍然能通过对氧的被动运输而生存，继续正常发育一段时间，这就为心血管系统严重缺陷的细节观察提供了可能。与鼠类模型相比，斑马鱼的主要优势是它提供了将经典遗传学与胚胎观察和操作结合起来研究的机会。斑马鱼的这些优势，使得对斑马鱼的研究异军突起，成为近几年使用最多的进行心脏发育生物学研究的模式动物，极大地推动了心血管疾病发病机制的研究。先天性心脏病是最常见的先天性遗传病，约1／3以上的先天性心脏病患者均合并流出道畸形。许多DiGeorge综合征患者只表现出心脏流出道畸形，不伴有DiGeorge综合征的其它典型特征。因此，可以说DiGeorge综合征与一些孤立型的先天性心脏病有相同的遗传学和分子机制，探讨DiGeorge综合征的发病机制的意义可能远远大于DiGeorge综合征本身。Tbx1在流出道的发育中发挥重要作用这一事实已经逐渐明朗，我们将进一步研究Tbx1与其他转录因子在心脏发育中的相互关系，深入探讨Tbx1基因在神经嵴细胞发育中的作用，以及Tbx1与视黄酸代谢通路的作用关系。第一部分斑马鱼Tbx1的表达模式和功能本部分拟利用斑马鱼在心血管发育遗传学优势，探讨Tbx1在斑马鱼胚胎发育中的功能及其调控机制。首先，利用多种手段探讨Tbx1在胚胎发育中的表达模式：其后，利用吗啡啉修饰的反义寡核苷酸技术在整体水平下调(knock-down)TbX1基因的表达，观察斑马鱼在发育过程中各个时期的表型变化，并进一步探讨这些变化的发生机制。首先，利用Real-time PCR和胚胎整体原位杂交技术动态分析了Tbx1在胚胎发育中的表达模式，整体原位杂交结果表明Tbx1在斑马鱼胚胎发育过程中表达在心脏、咽弓和耳囊，在心脏的表达，以流出道的表达最强。Tbx1的表达在36hpf-60hpf到达峰值，这一时期正是心脏和咽弓发育的关键时期，我们的结果说明Tbx1基因的功能可能和心脏，咽弓和耳囊的发育密切相关。其后，利用吗啡啉修饰的寡核苷酸技术在整体水平下调(knock-down)基因Tbx1的表达，探讨其异常表型及其机制。发现：1．Tbx1 Knock-down可导致斑马鱼耳囊发育畸形，耳囊体积减小，耳石减小，内部的听神经和半规管结构几乎完全缺失；2．Tbx1 Knock-down可导致咽弓表型异常，Tbx1 Knock-down胚胎前部的腭弓和角状弓存在，但是腭弓上的腭方软骨和角状弓上的舌颌软骨的体积明显减小，3-7咽弓的数目出现了不同程度的减少或完全消失；3．Tbx1 Knock-down可导致主动脉弓的异常表型，5条主动脉弓出现不同程度的缺失；4．Tbx1 Knock-down导致胸腺的异常表型，部分Tbx1 Knock-down胚胎胸腺缺如或体积缩小。第二部分咽弓和心脏发育相关基因在Tbx1 knock-down斑马鱼的表达胚胎发育受转录因子以及它们所构成的转录调控网络控制。在这一部分中，我们以Tbx1基因为切入点，研究Tbx1基因功能下调后，以其为中心的转录调控网络是如何发生变化的。首先我们观察了Tbx1 knock-down的情况下，另两个重要的T盒成员，即Tbx20和Tbx2的变化情况，我们观察到Tbx1 knock-down可以改变Tbx2在咽弓的表达模式；同时我们观察到Tbx1 knock-down后，Tbx20的表达出现下调。我们的结果说明T盒基因成员之间可以构成协同或拮抗的调控网络，对胚胎发育起着重要的调节作用。我们进一步对Tbx1与咽弓发育相关基因，Fgf8、Bmp2b和Hand2的关系进行了探讨。我们发现Tbx1 knock-down后，Bmp2b在咽囊和心脏的表达明显减低；Hand2在咽弓神经嵴细胞的表达明显减低；Tbx1 knock-down对Fgf8的表达没有影响。我们同时观察了Tbx1 knock-down会下调斑马鱼斑马鱼心脏结构基因Nppa的表达。第三部分Tbx1在神经嵴细胞迁移和分化中的作用Tbx1 knock-down将导致神经嵴细胞来源的结构，如甲状旁腺，胸腺，心脏流出道和头面部骨骼的发育异常，因此提示，Tbx1可能在神经嵴细胞的迁移、分化和增殖过程中发挥着重要作用，但是第一部分的研究中，我们发现Tbx1基因并不在神经嵴细胞表达，因此我们对Tbx1在神经嵴细胞发育中的作用进行了探讨。首先，我们选择神经嵴细胞迁移前、迁移中和迁移后等不同阶段的标志物进行原位杂交实验，dlx2α是咽弓神经嵴细胞特异性的标记物，我们发现Tbx1 knock-down胚胎，dlx2α在咽弓神经嵴细胞的表达出现明显下调，其它一些神经嵴细胞的标记物在Tbx1 knock-down胚胎的表达也表现为明显下调。说明，神经嵴细胞在Tbx1 knock-down后能够部分地出现迁移和分化，然而，神经嵴细胞的迁移过程部分地受到抑制，只有一部分神经嵴细胞能够正常地迁移至咽弓。我们进一步观察到，Tbx1 knock-down后，胚胎中脑，后脑和咽弓的凋亡细胞的数量明显增加，凋亡细胞的增加与这些部位神经嵴细胞在数量上的减少是符合的。在本部分中，我们直接观察到Tbx1参与调节斑马鱼胚胎心脏收缩功能的证据，斑马鱼胚胎Tbx1 knock down后心室收缩指数下降，心率减慢，可能Tbx1 knock-down减少了心神经嵴细胞的数量，进而导致斑马鱼心脏的收缩功能受到抑制。第四部分外源性视黄酸抑制斑马鱼Tbx1基因表达视黄酸过量或缺乏的小鼠均表现出与人类DGS综合征相似的的典型特征，与Tbx1突变或敲除的表型具有高度相似性。视黄酸可以控制众多转录因子在咽弓的表达。在本部分中，我们关注的是，在咽弓发育的过程中，视黄酸是否能够改变Tbx1的表达，继而影响咽弓的发育。首先我们观察到：1．外源性视黄酸处理可以导致斑马鱼胚胎死亡率和畸形率升高，且随处理浓度升高，死亡率和畸形率逐渐升高；2．视黄酸作用于斑马鱼心脏有特异的时间窗，适当浓度视黄酸时间窗内处理胚胎，将对心脏发育产生特异的影响；3．外源性视黄酸将导致斑马鱼胚胎心脏流出道和心室结构出现缺失。在以上研究的基础上，我们选择适当浓度视黄酸，时间窗内处理斑马鱼胚胎，观察了视黄酸对Tbx1基因表达的影响，我们观察到：1．外源性视黄酸可以下调斑马鱼Tbx1基因在心脏和咽弓的表达；2．Tbx1基因的表达下调主要表现在后部咽弓，前部咽弓Tbx1基因的表达与对照组相比没有明显差异；3．外源性视黄酸对Tbx1基因表达的抑制作用在36hpf最显著。更多还原

【Abstract】 The regulatory mechamisms of zebrafih Tbx1 in Cardiac DevelopmentTbx1, one of the genes mapped within the de122q11 locus in human, appears to be one of the earliest genes involved in pharyngeal arch development. Combined studies of heterozygous deletions within the DGCR and knockout experiments in mice have identified Tbx1 as a major genetic determinant of aortic arch malformations in DGS. In addition, homozygous for Tbx1 mutation in mice displayed a wide range of developmental anomalies encompassing almost all of the common features, including hypoplasia of the thymus and parathyroid glands, cardiac outflow tract abnormalities, abnormal facial structures, abnormal vertebrae and cleft palate. As a result of all these studies, Tbx1 currently represents the most promising candidate gene for DGS.DGS is the most common genetic deletion syndrome in humans, with an incidence of 1 in 4000 live births. DGS is characterized by congenital heart defects, thymus and parathyroid aplasia/hypoplasia, and craniofacial anomalies. These structures are derived from neural crest cells during embryogenesis. In common with mouse, chick, lamprey and zebrafish embryos, Tbx1 is expressed in the pharyngeal endoderm and mesoderm, not in neural crest cells. Thus, the expression pattern of Tbx1 raises a question which roles of Tbx1 played during the development of neural crest cells.The formation of pharyngeal arches is critically dependent on retinoic acid （RA）. Defects caused by excessive or insufficient RA in mouse recapitulate the typical phenotypes seen in human DGS/VCFS syndrome. Likewise, RALDH2 knockout and experimental manipulation of retinoid receptor in zebrafish also produce phenotypes of DGS. Over expression and haploinsufficieny of Tbx1 also produce similar DGS-like defects to those caused by RA treated models. The strong phenotypic reminiscence between RA treated DGS-like syndrome and Tbx1-/- mutants prompted us to investigate the effects of RA on the expression pattern of Tbx1.The zebrafish, Danio rerio, offers several distinct advantages as a genetic and embryological model system, including the external fertilization, rapid development and optical clarity of its embryos. Being a vertebrate, the zebrafish has a notochord, blood, heart and vasculature, kidney and optical systems that share many features with corresponding human systems. The zebrafish system bridges the gap between fruit fly/worm and mouse/human genetics, making it feasible to address issues of early development, organ formation, integrative physiology, pharmacology and complex disease.The zebrafish also have the outstanding feature for studying on cardiovascular development. 1. They are completely transparent during the first 3 days of development, which facilitates the experiment on cardiac development. 2. It is easy to trace the cardiac movement and circulation in living embryos. 3. Because of their small size, zebrafish embryos are not completely dependent on a functional cardiovascular system. Even in the total absence of blood circulation, they receive enough oxygen by passive diffusion to survive and continue to develop in a relatively normal fashion for several days, thereby allowing a detailed analysis of animals with severe cardiovascular defects. By contrast, avian and mammalian embryos die rapidly in the absence of a functional cardiovascular system. Forward genetics in zebrafish has led to the identification of several mutations affecting cardiac contractility.Tbx1 shows that both patterns of expression and gene function are generally conserved across vertebrate species. Morpholino （MO） antisense technology is a reliable gene knockdown method in zebrafish and can prevent translation of the target gene. Therefore, to investigate the developmental role of Tbxl, Tbxl specific morpholino antisense oligonucleotides were used in our study. However, knowledge remains limited concerning the upstream regulation of Tbx1 during pharyngeal arch development.Section 1 the expression patterns of Tbx1 and phenotypie analysis of Tbx1 knock-down zebrafish embryosIn this study, we describe that Tbx1 is highly expressed in heart as well as pharyngeal arches and otic vesicle during zebrafish embryogenesis. To determine the function of Tbx1 in zebrafish development, two different morpholino antisense oligonucleotides targeting the zebrafish Tbx1 were used. The cartilages in the mandibular and hyoid arches are drastically reduced in size. The cartilages in pharyngeal arches 3-7 are either seriously reduced in their number or completely absent in Tbx1 knock-down embryos. Tbx1 functionally knock-down produce smaller size in otic vesicle. We observed that the thymus are absent in nearly 60% Tbx1 morphant embryos. Counting the numbers of aortic arches reveals a significant reduction in their number after morpholinos injection when compared to control embryos. Conclusively, Tbx1 knock-down embryos are characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus.Section 2 Altered gene expression profiles in tbx1 morphant embryosT-box genes play important roles during embryogenesis in the vertebrate. Tbx2 is another member of T-box family. In this section, we found that Tbx1 knock-down can lead to altered expression of Tbx2 in the pharyngeal arches; this result raised a possibility that Tbx1 can regulate Tbx2 expression in the pharyngeal arches directly or indirectly. In addition, the expression of Tbx20 is also reduced in Tbx1 knock-down embryos.A large number of genes are expressed in the pharyngeal arches and implicated in pharyngeal arch development, such as Bmp2b, fgf8 and hand2. We showed that the expression of Hand2 and Bmp2b are reduced in the pharyngeal arches. We observed that there is no difference in the expression of Fgf8 between Tbx1 knock-down ad control embryo. The expression of Nppa is down-regulated in the ventricle in Tbx1 knock-down embryos. Taken together, these data indicate Tbx1 knock-down can lead to an altered gene expression profiles in zebrafish.Section 3 Tbx1 knock-down can cause reduced pharyngeal neural crest cells and cardiac dysfunctionWe have found that the structures derived from neural crest cells are seriously interrupted in Tbx1 knock-down embryos.Therefore these results raise a question whether Tbx1 plays an important roles in the control of neural crest cells fate. To understand the function of Tbx1 on neural crest development, we examined the expressions of some neural crest markers, such as foxd3, tfap-2, dlx2αand msx in Tbx1 knock-down embryos. We found that the expression ofdlx2αis seriously reduced by knockdown of Tbx1. The expressions of msx and trap-2 in pharyngeal neural crest cells are also decreased in Tbx1 knock-down embryos. In addition, Tbx1 Knock-down leads to an increase of cell apoptosis in the midbrain, hindbrain and pharyngeal arches. Therefore, we conclude that Tbx1 knock-down can produce the reduced amount of pharyngeal neural crest cells; in addition, at least in part, due to elevated level of apoptotic cells in this region.We also found defects in cardiac function in Tbx1 knock-down embryos. Depressed heart rate and reduced ventricular shortening fraction were observed in the Tbx1 knock-down embryos compared with control embryos. These results demonstrated that knock-down of Tbx1 can cause cardiac dysfunction due to reduced cardiac neural crest cells.Section 4 Exogenous RA down-regulates Tbx1 expression in zebrafishIn this part, we found that zebrafish embryo treated with 10^-7-10^-8mol/L RA at the end of gastrulation or the early segmentation, can be used as an experimental model to explore the mechanisms of cardiac development. Phenotypes of these embryos were analyzed, no detectable effect on the overall body plan was found. However these normal looking embryos, as the animals develop, exhibited selective defects of the heart tube.Time course analysis of zebrafish tbx1 expression by real-time quantitative PCR reveals that the expression level of Tbx1 was depressed due to RA treatment. The analysis of whole-mount in situ hybridization showed that exogenous RA can down-regulate Tbx1 expression in pharyngeal arches and out flow tract when compared to control embryos.更多还原