The Establishment of Left-Right Asymmetry in Mammalian Development

哺乳动物发育中左右不对称性的建立

• 批准号: NC/P001467/1
• 负责人: David Turner
• 金额: $ 27.44万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FP001467%2F1
• 关键词: Establishment; Left; Right; Asymmetry; Mammalian

Our external body plan is overtly symmetrical the distribution of organs & tissues throughout our body, is highly asymmetrical: left placement of the heart & the direction of intestine looping. Although the evolutionary reasons for this asymmetrical placement & anatomy of organs are not fully understood, mutations in genes that impact the proper development of L-R Asymmetry leads to a number of pathologies & birth defects demonstrating its central importance during the early embryo development. It is therefore of great clinical importance to have a clear understanding of these mechanisms if one is to understand these disease states. The Node, a structure that forms during early development is central to the development of laterality. The cells of the Node contain motile cilia, shown to be critical to LR asymmetry: where either their motion generates a flow of extracellular fluid (nodal-flow) create morphogen gradients, carry vesicles of signalling factors or generate leftwards pressure on other, non-motile cilia surrounding the node: the outcome is the asymmetric expression of genes such as Nodal, Lefty2 (its inhibitor) & Pitx2 in the left of the embryo & the establishment of left identity. However, as the exact mechanisms generating LR asymmetry are still unclear and as the proper generation of laterality has clear clinical implications, it is beneficial to garner a better understanding of how LR asymmetry is established. The only experimental models that exist to study this phenomena involve animals which are costly & difficult to manipulate at these early developmental stages, therefore a more tractable model to study the acquisition of laterality is therefore required. I have recently developed an experimental approach which involves growing mESCs in suspension where they aggregate, following which & after application of appropriate stimulation, they display many of the characteristics of the embryo including polarised gene expression & axial elongation. Significantly the generate a small region of cells (the node-like structure; Nd-LS) which express Nodal, a marker of the node, & they are able to break bilateral symmetry, manifested by one-sided expression of genes. I therefore propose to use this embryonic organoid (Gastruloid) system to study the mechanisms that result in the establishment of LR asymmetry during mammalian development. Firstly, I will undertake a detailed analysis of the structure & function of the Gastruloid Nd-LS, focusing on its molecular & cellular components, comparing it to the embryo node by quantitatively analysing the expression node-specific fluorescent reporter genes in Gastruloids & measuring the transcription of genes associated with the node by in situ hybridisation chain reaction. Electron microscopy & immunostaining will allow an assessment of the structure & topographical features of the Nd-LS, determining the presence of cilia similar to the Node in vivo. Through a combination of microfluidics, chemical & genetic gain and loss of function experiments, I will be able to precisely apply treatments to the Gastruloids & quantitatively establish which signals are important in the establishment of the Nd-LS and the midline. I will then quantitatively measure & record the dynamics of the LR symmetry-breaking event in real time by generating knock-in lines expressing fluorescent fusion proteins & transcriptional reporters for two genes important in establishing laterality: Nodal & Lefty. Single-cell tracking will allow me to correlate the emergence of asymmetries with reporter expression. Finally, by removing this Nd-LS through microsurgical techniques, I will assess the importance of the Nd-LS on the generation of LR asymmetry following its physical ablation.I will be able to use the system to gain a significant insight into the mechanisms involved in LR patterning as well as further demonstrating how Gastruloids are an excellent replacement for studying in vivo development.

我们的外部身体计划是明显对称的，器官和组织在整个身体的分布是高度不对称的：心脏的左侧位置和肠道循环的方向。尽管器官这种不对称放置和解剖结构的进化原因尚不完全清楚，但影响左右不对称正常发育的基因突变会导致许多病理和出生缺陷，证明其在早期胚胎发育过程中的核心重要性。因此，如果要了解这些疾病状态，清楚地了解这些机制具有重要的临床意义。节点是早期发育过程中形成的结构，是侧向性发展的核心。节点的细胞含有运动纤毛，这对 LR 不对称性至关重要：它们的运动产生细胞外液流（节点流），产生形态发生素梯度，携带信号因子囊泡或对其他非对称性产生向左压力。节点周围的运动纤毛：结果是诸如Nodal、Lefty2（其抑制剂）和Pitx2等基因在胚胎左侧的不对称表达以及左侧身份的建立。然而，由于产生 LR 不对称性的确切机制仍不清楚，并且正确产生偏侧性具有明确的临床意义，因此更好地理解 LR 不对称性是如何建立的是有益的。研究这种现象的唯一实验模型涉及在这些早期发育阶段昂贵且难以操作的动物，因此需要一个更容易处理的模型来研究侧向性的获得。我最近开发了一种实验方法，其中涉及在悬浮液中生长 mESC，并在其中聚集，随后并在应用适当的刺激后，它们表现出胚胎的许多特征，包括极化基因表达和轴向伸长。值得注意的是，它们会生成一小部分细胞（节点状结构；Nd-LS），表达节点标记Nodal，并且它们能够打破双边对称性，这通过基因的单侧表达来体现。因此，我建议使用这种胚胎类器官（原肠胚）系统来研究导致哺乳动物发育过程中 LR 不对称性建立的机制。首先，我将详细分析原肠胚 Nd-LS 的结构和功能，重点关注其分子和细胞成分，通过定量分析原肠胚中表达节点特异性荧光报告基因并测量其与胚胎节点的比较，通过原位杂交链式反应转录与节点相关的基因。电子显微镜和免疫染色将允许评估 Nd-LS 的结构和拓扑特征，确定与体内节点相似的纤毛的存在。通过微流体、化学和遗传功能获得和丧失实验的结合，我将能够精确地对原肠胚进行治疗，并定量确定哪些信号在 Nd-LS 和中线的建立中很重要。然后，我将通过生成表达荧光融合蛋白和转录报告基因的敲入线，实时定量测量和记录 LR 对称性破坏事件的动态，这两个基因对于建立偏侧性很重要：Nodal 和 Lefty。单细胞追踪将使我能够将不对称的出现与记者的表达联系起来。最后，通过显微外科技术去除这种 Nd-LS，我将评估 Nd-LS 对物理消融后 LR 不对称性产生的重要性。我将能够使用该系统来深入了解所涉及的机制LR 模式，并进一步证明原肠胚如何成为研究体内发育的绝佳替代品。