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 筛查对于描述该病的分子基础和开发其临床治疗方法至关重要。 影响全球10多亿人的神经系统疾病。为了实现这一目标，三维 神经有机体已经被开发出来，并显示出神经组织的一些结构和功能特征。 沿前-后(AP)和背-腹侧(DV)轴及其与周围非神经细胞的相互作用 调节其发育和功能的组织。因此，到目前为止，开发出的神经有机体无法 从感觉器官获得输入，并将信号传递到身体的其他部位。因此，他们不能模仿 一个功能齐全的神经系统，强烈限制了它们的使用。我们试图通过建立一个In来纠正这一缺陷 体外胚胎模拟系统，包含一个完全有图案的神经管，能够接收信号，以及 有系统地转播它们。我们的战略是在老鼠或人类的聚合体中启动胚胎发育 胚胎干细胞(ESCs)，由空间受限的形态原活动中心指示，该中心分泌 WNT和Node，并充当组织者。我们对小鼠胚胎干细胞的初步研究表明，这些细胞 聚集体发育成原肠并形成所有三个胚层的“胚状体”。其中，五分之一是 沿AP和DV轴排列，两侧对称。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。而后脑和脊椎 脊髓存在，胚状体缺少大脑的最前部、前脑和中脑。我们假设 缺乏前脑区是后脑化因子过度传递指示信号的结果， 纠正这个问题将优化这个模型，用于正常和病理神经的研究 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胎神经系统发育 转录组图谱，以及组织特异性分子标记。在目标2中，我们将促进形成 通过抵消后化因子WNT和结节分泌的强大梯度来实现脑前区 由组织中心主办。最后，在目标3中，我们将通过生产和分析胚状体来扩展我们的研究 含有一个神经板，可以折叠成神经管，来自人类胚胎干细胞。出于道德原因，我们不会 试图产生前脑区域，以避免在体外创造人类有机体。总而言之， 这些研究的成功完成将建立强大的体外模型来研究血管形成和功能 哺乳动物的神经系统。它还将为创造特定于患者的人类神经奠定基础 系统模型，使用人类诱导的多能干细胞。

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.