The Establishment of Left-Right Asymmetry in Mammalian Development

我们的体外计划是公开对称的，器官和组织在我们全身的分布是高度不对称的：心脏的左侧位置和肠环的方向。虽然这种器官不对称放置和解剖的进化原因还不完全清楚，但影响L-R不对称正常发育的基因突变导致了许多病理和出生缺陷，表明了它在早期胚胎发育中的核心作用。因此，如果要了解这些疾病状态，对这些机制有一个清楚的了解是非常重要的临床意义。结点是在早期发育过程中形成的一种结构，是侧向发育的中心。结节的细胞含有运动纤毛，被证明是LR不对称的关键：它们的运动产生细胞外液的流动(节流)，产生形态原梯度，携带信号因子的小泡，或对结节周围的其他非运动纤毛产生向左压力：结果是Nodal、Lefty2(其抑制物)和Pitx2等基因在胚胎的左侧不对称表达&建立左侧身份。然而，由于LR不对称的确切产生机制尚不清楚，而且适当的偏侧性的产生具有明确的临床意义，因此对LR不对称的建立有更好的理解是有益的。现有的研究这种现象的唯一实验模型涉及到在早期发育阶段昂贵且难以操纵的动物，因此需要一个更容易处理的模型来研究偏侧性的获得。我最近开发了一种实验方法，涉及在悬浮中培养mESCs，在那里它们聚集在一起，随后&在应用适当的刺激后，它们显示出许多胚胎的特征，包括极化的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(结节样结构；ND-LS)，这些细胞表达节点的标志-节点，它们能够打破两侧的对称性，表现为基因的单方面表达。因此，我建议使用这个胚胎类器官系统来研究在哺乳动物发育过程中导致LR不对称性建立的机制。首先，我将对天花粉蛋白ND-LS的结构和功能进行详细的分析，重点分析其分子和细胞成分，通过定量分析天花粉蛋白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与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可以评估ND-LS的结构和形态特征，确定体内是否存在与Node相似的纤毛。通过结合微流体学、化学和遗传增益以及功能丧失实验，我将能够精确地对胃液进行治疗&定量地确定哪些信号在ND-LS和中线的建立中重要。然后，我将通过生成表达荧光融合蛋白的敲入线来实时定量测量和记录LR对称性破坏事件的动态，这是两个在建立侧向关系中至关重要的基因的转录报告：Nodeal和Lefty。单细胞跟踪将使我能够将不对称性的出现与记者的表达联系起来。最后，通过显微外科技术移除这种ND-LS，我将评估ND-LS在物理消融后LR不对称产生方面的重要性。我将能够使用该系统对LR图案化涉及的机制有一个重要的洞察，并进一步证明天麻素是研究体内发育的极佳替代品。

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.