Molecular recording to predict cell fate decisions and animal behavior

摘要 大脑是非常复杂的，我们对这个器官的理解仍然处于起步阶段。许多 关于大脑发育和功能的基本问题仍然存在。单细胞基因组学有望开辟新途径 但是几乎所有的单细胞方法都有一个缺点--细胞在被破坏时， 测量分子状态。这种"观察即破坏"使得不可能将分子 过去发生的事件的最终结果：如果你摧毁了一个中间祖先， 大脑来分析它的表观基因组，你无法知道祖先会采取什么样的细胞命运。 同样，在经历后对动物进行侧写会混淆动物的分子后果。 在接触病毒之前就有任何分子倾向如果你在 如果你没有经验，你就无法知道动物的结果-例如，如果动物是学习者， 没有这一缺点是几乎所有现有基因组方法所固有的，并且阻碍了各种各样的感兴趣的基因组学研究。 分析。我们最近开发了一种平台技术，即单小区"电话卡"（CC）， 转座子在一个瞬间捕获分子事件，然后在稍后的时间用单个转座子将它们读出。 细胞分辨率我们现在建议在此基础上开发一种强大、易于使用的技术 该平台以单细胞分辨率记录小鼠大脑中的分子事件，并将其与细胞命运联系起来 决策、行为结果、神经元活动概况和解剖位置。我们将开发一套广泛的 适用的试剂和方法，将其应用于一系列高影响力的"测试案例"，以证明其实用性， 并迅速将它们分发给更广泛的社区。

Abstract The brain is remarkably complex, and our understanding of this organ is still in its infancy. Many fundamental questions about brain development and function remain. Single cell genomics promises new ways to answer these questions, but nearly all single-cell methods share shortcoming – cells are destroyed when their molecular states are measured. This “destruction upon observation” makes it impossible to correlate molecular events that have occurred in the past with the final outcomes: if you destroy an intermediate progenitor in the brain to profile its epigenome, you have no way to know what cell fate the progenitor would have adopted. Likewise, profiling an animal after an experience is going to conflate the molecular consequences of the experience with any molecular predispositions that were there before exposure. And if you profile before the experience, you have no way of know of the animal's outcome – e.g. if the animal would have been a learner or not. This shortcoming is inherent to nearly all existing genomic methods and impedes a wide variety of interesting analyses. We have recently developed a platform technology, single-cell `Calling Cards' (CC) that uses transposons to capture molecular events at one instant in time and then read them out at a later time with single cell resolution. We now propose to build upon this foundation to develop a robust, easy to use technology platform to record molecular events at single cell resolution in the mouse brain and connect them with cell fate decision, behavioral outcomes, neuronal activity profiles, and anatomical location. We will develop a set of widely applicable reagents and methods, apply them to a series of high-impact “test cases” to demonstrate their utility, and rapidly distribute them to the broader community.