In vitro modeling of a mammalian embryonic neural tube

项目摘要 建立适合实验操作和药物的哺乳动物神经发育体外模型 筛选对于描绘癌症的分子基础和开发癌症的临床治疗是至关重要的， 神经系统疾病，影响全球超过10亿人。为了实现这一目标，三维 神经类器官已经发展并显示出神经组织的一些结构和功能特征。 然而，虽然这些类器官表现出与真实大脑相似的显微解剖学，但它们缺乏适当的模式 沿着前后（AP）和背侧（DV）轴以及与周围非神经元的相互作用 调节其发育和功能的组织。因此，迄今为止开发的神经类器官无法 从感觉器官获得输入并将信号传播到身体的其他部分。因此，它们无法模仿 一个功能齐全的神经系统，严重限制了它们的使用。我们力求通过建立一个 体外胚胎模拟系统，其包含完全图案化的神经管，能够接收信号，以及 系统地传递它们。我们的策略是在小鼠或人类的聚集体中启动胚胎发育， 胚胎干细胞（ESC），具有空间受限的形态发生活性中心， WNT和NODAL并充当组织者。我们对小鼠胚胎干细胞的初步研究表明，这些指示 聚集体发育成“胚状体”，形成原肠胚并形成所有三个胚层。其中，1/5是 沿沿着AP和DV轴形成图案，并且是两侧对称的。这些胚状体还含有神经板 由柱状神经上皮细胞组成，逐渐折叠成神经管。而后脑和脊髓 在胚胎发育过程中，由于胚胎发育过程中存在脊髓，胚胎缺少大脑的前部，即前脑和中脑。我们假设 前脑区域的缺乏是由于后验因素产生的过多的指导性信号， 纠正这个问题将优化这个模型，用于正常和病理神经系统的研究。 发展在目标1中，我们将进一步定义细胞组成和时空分子解剖学 利用高通量单细胞技术研究双侧对称胚状体发育中的神经系统 转录组分析以及组织特异性分子标记。在目标2中，我们将促进 通过抵消后向因子WNT和NODAL分泌的有效梯度， 组织中心。最后，在目标3中，我们将通过产生和分析胚状体来扩展我们的研究 含有可以折叠成神经管的神经板，来自人类ESC。出于道德原因，我们不会 试图制造前脑区，以避免在体外创造人类有机体。总之， 这些研究的成功完成将建立稳健的体外模型，以研究 哺乳动物的神经系统这也将为创造患者特异性的人类神经系统奠定基础。 系统模型，使用人类诱导多能干细胞。

Project Summary Creating in vitro models of mammalian neural development amenable to experimental manipulation and drug screening is critically important for delineating the molecular basis of, and developing clinical therapies for, neurological disorders that affect more than one billion people worldwide. Toward this goal, three-dimensional neural organoids have been developed and display some structural and functional features of neural tissues. However, while these organoids exhibit microanatomy similar to the authentic brain, they lack proper patterning along the Anterior-Posterior (AP) and Dorsal-Ventral (DV) axes and interactions with surrounding non-neural tissues that modulate their development and function. Thus, the neural organoids developed, to date, are unable to get input from sensory organs and disseminate signals to the rest of the body. Therefore, they cannot mimic a fully functional nervous system, strongly limiting their use. We seek to rectify this deficiency by building an in vitro embryo-mimetic system that contains a fully patterned neural tube, capable of receiving signals, and relaying them systemically. Our strategy is to initiate embryonic development in aggregates of mouse or human embryonic stem cells (ESCs), instructed with a spatially restricted morphogen activity center, which secretes WNT and NODAL and acts as an organizer. Our preliminary studies with murine ESCs show that these instructed aggregates develop into “embryoids” that gastrulate and form all three germ layers. Amongst them, 1/5 are patterned along AP and DV axes and are bilaterally symmetrical. These embryoids also contain a neural plate made of columnar neuroepithelial cells that progressively folds into a neural tube. While hindbrain and spinal cord are present, the embryoids lack the anterior most part of the brain, forebrain and midbrain. We hypothesize that the lack of anterior brain domains results from an excess of instructive signaling by the posteriorizing factors, and that correcting this problem will optimize this model for the study of normal and pathological neural development. In Aim 1, we will further define the cellular composition and spatio-temporal molecular anatomy of the developing nervous system of bilaterally symmetrical embryoids using high throughput single-cell transcriptome profiling, as well as tissue specific molecular markers. In Aim 2, we will promote formation of anterior brain domains by counteracting the potent gradient of posteriorizing factors WNT and NODAL secreted by the organizing center. Finally, in Aim 3, we will extend our study by producing and analyzing embryoids containing a neural plate that may fold into a neural tube, from human ESCs. For ethical reasons we will not attempt at producing the anterior brain domain to avoid the creation in vitro of a human organismal. Altogether, successful completion of these studies will establish robust in vitro models to study formation and function of the mammalian nervous system. It will also lay the groundwork for the creation of patient-specific human nervous system models, using human induced pluripotent stem cells.