Understanding neuronal migration disorders using human tissue models

大脑皮层是许多使我们成为人类的高级认知功能的所在地，例如我们的高级学习和语言。我们知道皮层的正确组织对于这些功能至关重要，但在发育过程中如何实现这一点仍然是难以捉摸的。来自神经发育障碍研究的证据表明，神经元迁移对于确保正确数量的神经元在正确的时间到达正确的位置至关重要。这种疾病的一个关键例子是无脑回畸形，其中皮质的折叠（外表面上的皱纹）大大减少。这种折叠的缺乏与认知缺陷有关，并且被认为是由于神经元的过度迁移。本项目将使用尖端的人类细胞和组织培养系统来研究神经发育障碍中神经元迁移是如何失调的。它将利用两个实验室的专业知识，将Long实验室在人类胎儿新皮层发育和外植体模型方面的经验与Berninger实验室在人类诱导多能干细胞（iPSC）和类器官模型方面的经验相结合。我们将使用多学科的方法，包括实时成像，转录组分析，共聚焦成像和细胞生物学。第一年-人胎儿新皮层外植体模型中的神经元迁移;从iPSC建立人脑类器官在第一年，学生将学习胎儿组织外植体，iPSC和类器官培养系统。将使用慢病毒转染和GFP标记的iPSC建立内源性（对照）神经元迁移模式。到今年年底，他们还将把iPSC衍生的神经祖细胞/早期神经元/神经胶质细胞移植到胎儿组织外植体中。他们将学习关键的细胞/组织培养技术，显微镜（共聚焦和延时），免疫荧光和组织学，以及图像分析。第2年-神经元迁移障碍的iPSC/类器官和iPSC/外植体模型中的神经元迁移学生将使用第一年建立的技术，但使用来自神经元迁移障碍患者的iPSC。我们目前有几个线在使用，包括脑室周围异位，多脑回和小头畸形。将这些细胞的迁移缺陷与等基因对照（如果可用）或下一个合适的对照以及第一年观察到的内源性细胞行为进行比较。还将在所用模型、2D、类器官和胎儿组织外植体之间比较迁移。第3年-识别神经元迁移缺陷的机制;在iPSC/类器官和人类胎儿新皮质模型中拯救这些缺陷使用第2年收集的数据，学生现在将研究观察到的神经元迁移缺陷背后的机制，以及如何在不同的模型系统中拯救这些缺陷。这种拯救可以是遗传的（即CRISPR，质粒电穿孔，病毒转染）或通过药理学操作（即小分子，功能性抗体）。

The cerebral cortex is the seat of many of the higher cognitive functions that make us human, such as our advanced learning and speech. We know that the correct organisation of the cortex is vital for these functions, but how this is achieved during development remains elusive. Evidence from studies on neurodevelopmental disorders has indicated that neuronal migration is crucial to ensure that the right number of neurons end up in the right place at the right time. A key example of such a disorder is lissencephaly, where the folding of the cortex (the wrinkles on the outer surface) is greatly reduced. This lack of folding is associated with cognitive defects and has been suggested to be due to an over-migration of neurons.This project will use cutting-edge human cell and tissue culture systems to investigate how neuronal migration is dysregulated in neurodevelopmental disorders. It will take advantage of both laboratories' expertise, combining the Long lab's experience in human fetal neocortex development and explant models with the Berninger lab's experience in human induced pluripotent stem cells (iPSC) and organoid models. We will use a multidisciplinary approach, including live-imaging, transcriptome analysis, confocal-imaging and cell biology. Year 1 - Neuronal migration in human fetal neocortex explant models; establishment of human cerebral organoids from iPSCs In this first year, the student will learn the fetal tissue explant, iPSC and organoid culture systems. Endogenous (control) neuronal migration patterns will be established using lentiviral transfection and GFP labelled iPSCs. Towards the end of this year, they will also graft the iPSC derived neural progenitors/early neurons/glial cells into the fetal tissue explants. They will learn key cell/tissue culture techniques, microscopy (confocal and time-lapse), immunofluorescence and histology, as well as image analysis. Year 2 - Neuronal migration in iPSC/organoid and iPSC/explant models of neuronal migration disorders The student will use the techniques established in the first year, but with iPSCs from patients with neuronal migration disorders. We currently have several lines in use, including periventricular heterotopia, polymicrogyria and microcephaly. The migration defects of these cells will be compared to isogenic controls wherever available, or the next suitable control, as well as the endogenous cell behaviour observed in the first year. The migration will also be compared across the models used, 2D, organoids and fetal tissue explants. Year 3 - Identification of mechanisms underlying defects in neuronal migration; rescue of these defects in iPSC/organoid and human fetal neocortex models Using the data collected in year 2, the student will now study the mechanisms behind the neuronal migration defects observed and how these may be rescued in the different model systems. This rescue can be genetic (i.e. CRISPR, plasmid electroporation, viral transfection) or via pharmacological manipulation (i.e. small molecules, functional antibodies).