A Cellular Resolution Census of the Developing Human Brain

项目摘要/摘要 我们的目标是创建发育中的人类新大脑皮层的时空单细胞分辨率图，以便 确定存在多少种不同的细胞类型，并解开它们复杂的发育历史。我们会 我们的分析建立在基于转录签名的细胞类型的多模式分类上，但 在可能的情况下，辅之以生理和表观遗传特征。我们还将检查瞬变细胞 种群只存在于发育阶段，我们将保留我们所有细胞的位置信息 用于创建发育细胞图谱的数据，该图谱根据细胞类型在 成长中的人脑。我们开发了用于大规模并行分析分子的创新策略 和使用基于液滴的捕获技术的原代人类皮质细胞的生理特性， 内容显微镜，以及对转录状态的成对生理反应。我们建议进行我们的 对发育中的人类大脑皮质特定区域以及纹状体进行的综合细胞观察， 丘脑、下丘脑和小脑，我们将使用单核测序来解锁发育 传统上难以研究的时间点。我们的项目将阐明细胞的起源 通过解决三个具体目标来实现人类皮质的多样性：1)我们将使用单细胞RNA测序来 询问神经发生和胶质形成是如何进行的，并在发育的大脑中产生关键的细胞类型。 我们假设，促进区域化和连通性的关键事件可以转录 从怀孕的前三个月到出生后的不同阶段，提供了对细胞身份如何确定的洞察。 2)我们对人脑细胞图谱的发育方法提供了一个描述瞬变的机会。 在边缘地带和亚板块区域出现的早期发育的细胞群，并在 新生儿期。这些细胞类型被认为在建立大脑结构和 功能，但它们在人类大脑发育中的特点仍然很差。我们假设 这些群体可以通过转录来识别，并可以解释这些临时群体的不同角色集 人口。3)转录状态是识别细胞类型的强大工具，但它们不能捕获 整个分子特征的复杂性。我们将描述细胞特异性激动剂的反应和染色质状态 在已定义的转录类中反映异质性。我们假设的交集 转录的生理状态和表观遗传状态将为细胞类型分类提供额外的细微差别。 我们的结果将为发育中的人脑提供一个细胞分类学框架，并创建一个 全面的细胞分辨率图，分子定义的细胞类型贯穿整个功能区域 发育中的人脑。这一独特的资源将成为人类大脑研究的蓝图 功能，疾病中细胞类型的选择性脆弱性，以及使我们独一无二的大脑进化特征。

Project Summary/Abstract We aim to create a spatiotemporal single cell resolution map of the developing human neocortex in order to establish how many distinct cell types are present and to unravel their complex developmental history. We will build our analysis on a multimodal classification of cells types based on transcriptomic signatures but complemented where possible by physiological and epigenetic features. We will also examine transient cell populations present only during developmental stages, and we will retain positional information for all our cell data to create a developmental cell atlas that plots the diversity of cell types according to their locations in the growing human brain. We have developed innovative strategies for massively parallel profiling of molecular and physiological properties of primary human cortical cells using droplet based capture technologies, high content microscopy, and paired physiological responses to transcriptional state. We propose to conduct our integrated cellular survey of developing human brain in specific regions of the cortex, as well as in the striatum, thalamus, hypothalamus and cerebellum, and we will use single nuclei sequencing to unlock developmental time points that have been traditionally difficult to study. Our project will shed light on the origins of cellular diversity in the human cortex by addressing three specific aims: 1) We will use single cell RNA-sequencing to interrogate how neurogenesis and gliogenesis proceed and give rise to key cell types in the developed brain. We hypothesize that key events promoting regionalization and connectivity can be transcriptionally distinguished from the first trimester to postnatal stages, providing insights into how cell identity is determined. 2) Our developmental approach to the human brain cell atlas provides an opportunity to characterize transient cell populations that appear early in development in the marginal zone and subplate regions, and disappear at neonatal stages. These cell types are presumed to play important roles in establishing brain architecture and function, but they remain poorly characterized in developing human brain. We hypothesize the heterogeneity of these populations can be identified transcriptionally and can explain a diverse set of roles for these transient populations. 3) Transcriptional states are a powerful tool for cell type identification, but they do not capture the entire complexity of molecular features. We will profile cell-specific agonist responses and chromatin state that reflect heterogeneity within defined transcriptional classes. We hypothesize that the intersection of physiological state and epigenetic state to transcription will provide additional nuance to cell type classification. Our results will provide a framework of cellular taxonomy in the developing human brain and create a comprehensive cellular resolution map of molecularly defined cell types throughout functional regions of the human brain during development. This unique resource will serve as a blueprint for studies of human brain function, selective vulnerability of cell types in disease, and the features of brain evolution that make us unique.