The Establishment of Left-Right Asymmetry in Mammalian Development

我们的外部身体计划是公开对称的器官和组织分布在我们的身体，是高度不对称的：左放置的心脏和方向的肠循环。虽然这种器官的不对称放置和解剖结构的进化原因尚未完全了解，但影响L-R不对称性正常发育的基因突变导致了许多病理和出生缺陷，证明了其在早期胚胎发育中的核心重要性。因此，如果要了解这些疾病状态，明确了解这些机制具有重要的临床意义。节点，在早期发育过程中形成的结构是侧化发展的核心。淋巴结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的流动（逆向流动）产生形态梯度，携带信号因子的囊泡或对节点周围的其他非运动纤毛产生向左的压力：结果是基因如Nodal、Lefty 2（其抑制剂）和Pitx 2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不对称的确切机制仍不清楚，并且由于正确产生侧化具有明确的临床意义，因此更好地理解LR不对称是如何建立的是有益的。存在的唯一的实验模型来研究这种现象涉及动物是昂贵的和难以操纵在这些早期发育阶段，因此，一个更容易处理的模型来研究的收购偏侧化，因此需要。我最近开发了一种实验方法，该方法涉及在悬浮液中培养mESC，在悬浮液中它们聚集，然后在施加适当的刺激后，它们显示出胚胎的许多特征，包括极化基因表达和轴向伸长。值得注意的是，它们产生了一个小区域的细胞（节点样结构; Nd-LS），其表达节点的标志物Nodal，并且它们能够打破双边对称性，表现为基因的单侧表达。因此，我建议使用这个胚胎类器官（Gastruloid）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，我将对Gastruloid Nd-LS的结构和功能进行详细的分析，重点是其分子和细胞组成，通过定量分析Gastruloid中节点特异性荧光报告基因的表达并通过原位杂交链反应测量与节点相关的基因的转录，将其与胚胎节点进行比较。电子显微镜和免疫染色将允许评估Nd-LS的结构和地形特征，确定与体内淋巴结相似的纤毛的存在。通过微流体、化学和遗传获得以及功能丧失实验的结合，我将能够精确地对Gastruloids进行治疗，并定量地确定哪些信号在Nd-LS和中线的建立中是重要的。然后，我将通过产生表达荧光融合蛋白和转录报告基因的敲入系，真实的定量测量和记录LR破坏事件的动态，这两个基因在建立偏侧性方面很重要：Nodal和Lefty。单细胞跟踪将使我能够将不对称的出现与报告基因表达联系起来。最后，通过显微外科技术去除Nd-LS，我将评估Nd-LS在物理消融后对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LR模式化的机制，并进一步证明Gastruloids是研究体内发育的绝佳替代品。

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.