【作者】 王红岩；

【导师】 李玉新； 黄艳新；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2014， 博士

【摘要】 体节（Somite）是所有脊椎动物胚胎发育早期在准体节中胚层（PSM）内沿从头到尾的方向按固定时间间隔不断重复形成的上皮细胞团。体节发生（Somitogenesis）是指体节形成的生物学过程，这一过程在时间和空间上都受到严格调控，从而保证体节以固定的时间间隔在正确的位置生成。如果体节发生过程受到干扰，形成的体节就会出现缺陷，从而导致身体发育异常，产生各种先天性疾病。因此，作为脊椎动物身体发育的重要基础，对体节发生过程进行深入研究进而揭示其内在调控机制，对于保证动物身体正常发育以及相关疾病的诊疗具有重要的理论和现实意义。体节发生在时间上具有节律性，这种节律性被PSM中一个呈周期性振荡的分节时钟（Segmentation Clock）调控。分节时钟由Notch、Wnt和成纤维细胞生长因子（FGF）三个通路交互构成，这些通路靶基因的振荡表达对体节的正常形成至关重要。大量的分子生物学实验证明，分节时钟内存在多个呈周期性表达的基因，同时这些基因之间存在调控关系。单纯通过生物学实验方法很难从系统层面上阐述分节时钟基因振荡表达的分子机制，近年来系统生物学建模方法被广泛应用于分节时钟的研究，并且，有一些系统生物学模型提出。但是，迄今为止，这些模型大多仅包含信号通路的某一部分，而且模型结构和描述生化反应的公式过于简单抽象，建模的目标也只是停留在模拟基因振荡表达这一简单应用层面上。应用微分方程对通路信号转导过程进行模拟，整合多种调控关系对多个通路进行整合建模，以及通过模型分析对靶基因振荡表达机制进行深入探索等方面的研究成果还较少。本文首先通过系统生物学方法对体节发生相关的Notch和Wnt单个通路信号转导过程进行建模，利用常微分方程表示通路中从受体激活到靶基因转录的各个生化反应过程，并在此基础上，通过引入三层调控关系，构建了整合的Notch和Wnt通路交互模型。模型可以准确再现分节时钟靶基因的振荡表达，并且与生物实验结果吻合。模拟结果表明，Notch通路中以Hes7为中心的负反馈环和Wnt通路中以Axin2为核心的负反馈环是单个通路靶基因振荡表达的关键。Wnt信号对Notch配体Dll1的转录调控，以及Notch靶基因Hes7对Wnt抑制基因Nkd1的转录抑制，是交互模型中两个通路靶基因保持振荡表达的关键。通路中存在多个负反馈环是靶基因的振荡表达的关键，但是其作用机制各不相同，其中，Notch通路中以Lfng基因为中心的负反馈环对于分节时钟振荡周期的调控具有重要影响。在前述研究的基础上，通过引入FGF通路以及Notch、Wnt和FGF通路之间的调控关系，构建了一个包括全部Wnt、Notch和FGF等分节时钟相关通路的交互模型。该模型首次模拟了Wnt和FGF信号对Notch靶基因Hes7的转录激活，Wnt信号对Notch激活子Msgn1的转录激活，以及Notch靶基因Hes7对FGF抑制基因Dusp4的转录抑制等多种调控关系。模型分析结果表明，PSM中Wnt和FGF信号浓度由高到低的变化对应于分节时钟振荡由启动到停止的全过程。分节时钟的稳定振荡不仅依赖通路中反馈环，而且依赖于通路间交互调控相关的反应参数。并且，通路中存在多种互补机制，保证了分节时钟在扰动下仍能够保持稳定振荡。本文提出了多个不同层次的体节发生相关通路信号转导模型，并利用模型模拟对分节时钟靶基因稳定振荡表达的分子机制进行研究。这些模型可以为相关的实验研究提供指导，对揭示体节发生相关疾病的发病机理提供参考。更多还原

【Abstract】 Somites are epithelial cell group budding off periodically from the rostral end of thepresocratic mesoderm (PSM). Somitogenesis is the process by which somites form.Somitogenesis is strictly regulated in time and space by which somites can be formed atregular intervals in the right position. If the somitogenesis is disturbed, normal somiteformation will be destroyed which results in abnormal physical growth thereby bringingabout various congenital diseases. Consequently, it is important for ensuring the normaldevelopment of vertebrate bodies as well as the diagnosis and treatment to related diseases toresearch into somitogenesis and then to reveal its regulation mechanisms.Somitogenesis has rhythm in time and it is regulated by a periodic oscillatorysegmentation clock in the PSM. The segmentation clock is formed by the crosstlak of theNotch, Wnt and fibroblast growth factors (FGF) pathways and the oscillatory expression oftheir target genes is essential to normal somite formation. A lot of experiments of molecularbiology proved that there are many periodic expressing genes in the segmentation clock andthere are regulations among these genes. However, it is very difficult to uncover themolecular mechanisms of the oscillatory expression of genes in the segmentation clock insystematic level only through the methods of the biological experiment. In recent years,systems biology modeling methods are widely used in the research on the segmentation clockand some models have been proposed. Unfortunately, most of these models contained onlypart of the signaling pathways in the segmentation clock and the model structures as well asformulae representing biochemical reactions are too simple and abstract. Moreover, the goalof modeling is only to simulate the oscillatory expression of genes. The research findingsabout simulating the signal transduction process of pathways through differential equations,modeling multiple pathways by integrating multi-levels of crosstalk and exploring themechanisms of the oscillatory expression of genes through model analyses are still lacking.In this thesis, first of all, we constructed the mathematical models of the signaltransduction process of the Notch and Wnt pathways in isolation using systems biologymethods. Every biochemical reaction in the signaling pathway, from the activation ofreceptors to the transcription of target genes, is modeled by ordinary differential equations.On that basis, we constructed a crosstalk model by integrating the Notch and Wnt pathwaysthrough introducing three levels of crosstalk between the pathways. These models canaccurately reproduce the oscillatory expression of the target genes in the segmentation clockand the simulation results are in accord with biological experimental findings. The simulationresults indicated that the negative feedback loop centered on Hes7in the Notch pathway and the negative feedback loop centered on Axin2in the Wnt pathway are essential to theoscillation of the target genes in the single pathway. The transcriptional regulation of Wntsignals to the Notch ligand Dll1and the transcriptional repression of the Notch target geneHes7to the Wnt inhibitor Nkd1gene are essential to the oscillation of the target genes in thecrosstalk model. In addition, multiple negative feedback loops in the pathways are alsoessential to the oscillation of the target genes but the mechanisms are different. Among them,the negative feedback loop centered on Lfng gene in the Notch pathway is very important toregulate the oscillatory period of the segmentation clock.On the basis of our previous research, we constructed a comprehensive model of thesegmentation clock that contains the Notch, Wnt and FGF pathways through adding the FGFpathway as well as the regulations among the Notch, Wnt and FGF pathways. The modelsimulated the transcriptional activation of the Wnt and FGF signals to the Hes7gene, thetranscriptional activation of the Wnt signal to the Notch activator Msgn1and thetranscriptional repression of the Notch target gene Hes7to the FGF inhibitor Dusp4for thefirst time. The results of model analyses indicated that the concentration of Wnt and FGFsignals changing from high level to low level along PSM corresponds to the process fromstart to stop of the oscillation of the segmentation clock. Moreover, the stable oscillation ofthe segmentation clock depends not only on the negative feedback loops in the pathways butalso on the reaction parameters related to the regulations among pathways. In addition, thereare many complementary mechanisms in the pathways that ensure the segmentation clock canmaintain stable oscillation under perturbations.In this thesis, we proposed a number of models of the signal transduction pathwaysinvolved in somitogenesis in different level and studied the molecular mechanisms by whichthe target genes in the segmentation clock maintain stable oscillation. These models canprovide guidance for the experimental research on somitogenesis and provide reference forexploring the pathogenesis of the diseases related to somitogenesis.更多还原