Organoids recapitulate brain development Gene expression changes and their control by accessible chromatin in the human brain during development is of great interest but limited accessibility. Trevino et al. avoided this problem by developing three-dimensional organoid models of human forebrain development and examining chromatin accessibility and gene expression at the single-cell level. From this analysis, they matched developmental profiles between the organoid and fetal samples, identified transcription factor binding profiles, and predicted how transcription factors are linked to cortical development. The researchers were able to correlate the expression of neurodevelopmental disease risk loci and genes with specific cell types during development. Science, this issue p. eaay1645 Organoids recapitulate the chromatin accessibility profiles of the developing human forebrain. INTRODUCTION The cerebral cortex is responsible for higher-order functions in the nervous system and has undergone substantial expansion in size in primates. The development of the forebrain, including the assembly of the expanded human cerebral cortex, is a lengthy process that involves the diversification and expansion of neural progenitors, the generation and positioning of layer-specific glutamatergic neurons, the cellular migration of γ-aminobutyric acid (GABA)–ergic neurons, and the formation and maturation of glial cells. Disruption of these cellular events by either genetic or environmental factors can lead to neurodevelopmental disease, including autism spectrum disorders and intellectual disability. RATIONALE Human forebrain development is, to a large extent, inaccessible for cellular-level study, direct functional investigation, or manipulation. The lack of availability of primary brain tissue samples—in particular, at later stages—as well as the limitations of conventional in vitro cellular models have precluded a detailed mechanistic understanding of corticogenesis in healthy and disease states. Therefore, tracking epigenetic changes in specific forebrain cell lineages over long time periods, has the potential to unravel the molecular programs that underlie cell specification in the human cerebral cortex and, by temporally mapping disease risk onto these changes, to identify cell types and periods of increased disease susceptibility. RESULTS We used three-dimensional (3D) directed differentiation of human pluripotent stem cells into dorsal and ventral forebrain domains and applied the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) in combination with RNA-sequencing (RNA-seq) to map the epigenetic and gene expression signatures of neuronal and glial cell lineages over 20 months in vitro. We show, through direct comparison with primary brain tissue from our study and several epigenetic datasets, that human stem cell–derived 3D forebrain organoids recapitulated in vivo chromatin accessibility patterns over time. We then integrated these data to discover putative enhancer-gene linkages and lineage-specific transcription factor regulators, including a diverse repertoire of factors that may control cortical specification. We validated protein expression of some of these transcription factors using immunofluorescence, confirming cellular and temporal dynamics in both primary tissue and forebrain organoids. Next, we used this resource to map genes and genetic variants associated with schizophrenia and autism spectrum disorders to distinct accessibility patterns to reveal cell types and periods of susceptibility. Last, we identified a wave of chromatin remodeling during cortical neurogenesis, during which a quarter of regulatory regions are active, and then highlighted transcription factors that may drive these developmental changes. CONCLUSION Using long-term 3D neural differentiation of stem cells as well as primary brain tissue samples, we found that organoids intrinsically undergo chromatin state transitions in vitro that are closely related to human forebrain development in vivo. Leveraging this platform, we identified epigenetic alterations putatively driven by specific transcription factors and discovered a dynamic period of chromatin remodeling during human cortical neurogenesis. Finally, we nominated several key transcription factors that may coordinate over time to drive these changes and mapped cell type–specific disease-associated variation over time and in specific cell types. Developing a human cellular model of forebrain development to study chromatin dynamics. ATAC-seq and RNA-seq studies over long-term differentiation of human pluripotent stem cells into forebrain organoids and in primary brain tissue samples reveal dynamic changes during human corticogenesis, including a wave associated with neurogenesis, and identify disease-susceptible cell types and stages. Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.

中文翻译：

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人类前脑发育模型中的染色质可及性动态

类器官概括大脑发育 基因表达变化及其在发育过程中通过人脑中可接近的染色质进行的控制是非常有趣的，但可及性有限。特雷维诺等人。通过开发人类前脑发育的三维类器官模型并在单细胞水平检查染色质可及性和基因表达，避免了这个问题。通过这项分析，他们匹配了类器官和胎儿样本之间的发育特征，确定了转录因子结合特征，并预测了转录因子如何与皮质发育相关联。研究人员能够将神经发育疾病风险位点和基因的表达与发育过程中的特定细胞类型相关联。科学，这个问题 p。eaay1645 类器官概括了发育中的人类前脑的染色质可及性概况。引言 大脑皮层负责神经系统中的高级功能，并且在灵长类动物中经历了显着的体积扩张。前脑的发育，包括扩张的人类大脑皮层的组装，是一个漫长的过程，涉及神经祖细胞的多样化和扩张，层特异性谷氨酸能神经元的产生和定位，γ-氨基丁酸的细胞迁移。 GABA)-能神经元，以及神经胶质细胞的形成和成熟。遗传或环境因素对这些细胞事件的破坏可导致神经发育疾病，包括自闭症谱系障碍和智力障碍。基本原理 人类前脑发育在很大程度上无法进行细胞水平研究、直接功能研究或操作。缺乏初级脑组织样本——尤其是在后期阶段——以及传统体外细胞模型的局限性，阻碍了对健康和疾病状态下皮质发生的详细机制理解。因此，长期跟踪特定前脑细胞谱系的表观遗传变化，有可能解开人类大脑皮层细胞特化背后的分子程序，并通过将疾病风险暂时映射到这些变化上，以确定细胞类型和时期增加疾病易感性。结果我们使用三维 (3D) 定向分化人类多能干细胞到背侧和腹侧前脑结构域，并应用高通量测序 (ATAC-seq) 结合 RNA 测序 (RNA- seq) 在体外绘制神经元和神经胶质细胞谱系超过 20 个月的表观遗传和基因表达特征。我们通过与我们研究中的原代脑组织和几个表观遗传数据集的直接比较表明，人类干细胞衍生的 3D 前脑类器官随着时间的推移重现了体内染色质可及性模式。然后，我们整合这些数据以发现推定的增强子-基因联系和谱系特异性转录因子调节因子，包括可能控制皮质规范的多种因素。我们使用免疫荧光验证了其中一些转录因子的蛋白质表达，证实了原代组织和前脑类器官中的细胞和时间动态。接下来，我们使用此资源将与精神分裂症和自闭症谱系障碍相关的基因和遗传变异映射到不同的可及性模式，以揭示细胞类型和易感期。最后，我们在皮质神经发生过程中发现了一波染色质重塑，在此期间四分之一的调节区域处于活动状态，然后突出显示了可能驱动这些发育变化的转录因子。结论 使用干细胞的长期 3D 神经分化以及原代脑组织样本，我们发现类器官在体外本质上会经历染色质状态转变，这与体内人类前脑发育密切相关。利用这个平台，我们确定了由特定转录因子驱动的表观遗传改变，并发现了人类皮质神经发生过程中染色质重塑的动态时期。最后，我们指定了几个关键的转录因子，它们可能会随着时间的推移而协调以驱动这些变化，并绘制出随着时间和特定细胞类型的细胞类型特异性疾病相关变异。开发人类前脑发育细胞模型以研究染色质动力学。ATAC-seq 和 RNA-seq 对人类多能干细胞长期分化为前脑类器官和初级脑组织样本的研究揭示了人类皮质发生过程中的动态变化，包括与神经发生相关的波，并确定疾病易感细胞类型和阶段。前脑发育的特点是高度同步的细胞过程，如果受到干扰，可能会导致疾病。为了绘制这些事件背后的调节活动，我们生成了人类三维前脑类器官中可接近的染色质图。为了捕捉皮质发生，我们在体外 20 个月内从背侧或腹侧前脑类器官中对神经胶质和神经元谱系进行了采样。在不同发育阶段的类器官中观察到在人类原代脑组织中鉴定的活性染色质区域。我们使用此资源来绘制疾病的遗传风险图并探索进化保护。而且，我们将染色质可及性与转录组学相结合，以确定调节人类皮质发生的假定增强子-基因联系和转录因子。总体而言，该平台提供了对人类前脑发育以前无法进入的阶段的基因调控动态的见解，包括神经精神疾病的特征。