The Establishment of Left-Right Asymmetry in Mammalian Development

我们的外部身体平面图是明显对称的，整个身体的器官和组织分布是高度不对称的：心脏的左侧位置和肠环的方向。虽然这种器官的不对称位置和解剖结构的进化原因尚不完全清楚，但影响L-R不对称正常发育的基因突变导致许多病理和出生缺陷，表明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，对这些机制有一个清晰的认识具有重要的临床意义。淋巴结是发育早期形成的一种结构，对侧性发育至关重要。结细胞含有运动纤毛，这对LR不对称至关重要：它们的运动产生细胞外液流动（结流），产生形态梯度，携带信号因子囊泡，或对结周围的其他非运动纤毛产生向左的压力：结果是胚胎左侧Nodal、Lefty2（其抑制剂）和Pitx2等基因的不对称表达，并建立左侧身份。然而，由于产生LR不对称的确切机制尚不清楚，并且由于侧侧的正确产生具有明确的临床意义，因此更好地了解LR不对称是如何建立的是有益的。研究这一现象的唯一实验模型涉及在这些早期发育阶段成本高昂且难以操作的动物，因此需要一个更易于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，其中包括在悬浮中培养mESCs，使其聚集，随后在适当的刺激应用后，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，它们产生了一小部分细胞（淋巴结样结构；Nd-LS），表达淋巴结的标记物Nodal，它们能够打破双边对称性，表现为基因的单侧表达。因此，我建议使用这种胚胎类器官系统来研究哺乳动物发育过程中导致LR不对称建立的机制。首先，我将对Gastruloid Nd-LS的结构和功能进行详细分析，重点分析其分子和细胞成分，通过定量分析Gastruloid中节点特异性荧光报告基因的表达，并通过原位杂交链式反应测量节点相关基因的转录，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，确定体内类似淋巴结的纤毛的存在。通过结合微流体、化学和遗传的功能增益和损失实验，我将能够精确地对胃原体进行治疗，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，我将通过生成表达荧光融合蛋白的敲入系和对建立侧边性很重要的两个基因（Nodal和Lefty）的转录报告基因，实时定量测量和记录LR对称性破坏事件的动态。单细胞追踪可以让我将不对称的出现与报告基因表达联系起来。最后，通过显微外科技术切除Nd-LS，我将评估Nd-LS在物理消融后对LR不对称产生的重要性。我将能够使用该系统对LR模式所涉及的机制有一个重要的见解，并进一步证明胃原体是研究体内发育的一个很好的替代品。

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.