A human-specific enhancer fine-tunes radial glia potency and corticogenesis

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications1-3. Human accelerated regions (HARs) are highly conserved DNA sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to species-specific cortical development is largely unknown4,5. HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development6. Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices, which contain more excitatory neurons. By measuring neural dynamics in vivo we show these anatomical features result in increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using fixed and live imaging, lineage analysis, and single-cell RNA sequencing. We discover Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential later. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. Finally, we show that Hs-HARE5 promotes progenitor proliferation by increasing canonical WNT signaling. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways to modulate brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex. Comparative gene expression profiling analysis of RNA-seq data for wildtype and HARE5Hs/Hs knock-in mice cortices at two timepoint, E12.5 and E14.5. Three biological replicates have been collected for each condition. Conditions: E12.5 WT, E12.5 HARE5Hs/Hs, E14.5 WT, E14.5 HARE5Hs/Hs.人类演化出异常扩张且结构高度复杂的大脑皮层，该特征与发育及基因调控层面的修饰改变存在关联1-3。 人类加速区域（Human Accelerated Regions, HARs）是一类高度保守的DNA序列，携带有人类特异性的核苷酸替换变异。尽管目前已有数千个被注释的HARs，但它们对物种特异性大脑皮层发育的功能性贡献仍在很大程度上尚不明确4,5。 HARE5是一类靶向WNT信号通路受体Frizzled8（FZD8）的HARs类转录增强子，在大脑发育过程中发挥活性6。 本研究借助基因组编辑的小鼠与灵长类动物模型，证实人类（Hs）来源的HARE5可通过调控神经前体细胞（Neural Progenitor Cells, NPCs）的增殖与神经发生能力，对大脑皮层发育及皮层连接进行精细调控。 携带Hs-HARE5敲入的小鼠，其新皮层体积显著增大，且兴奋性神经元数量更多。 通过在体监测神经活动动态，本研究证实上述解剖学特征可导致大脑皮层区域间的功能独立性显著提升。 为解析背后的发育调控机制，本研究通过固定成像与活细胞成像、谱系追踪分析以及单细胞RNA测序等手段，对神经前体细胞的命运进行了评估。 研究发现，Hs-HARE5可改变放射状胶质前体细胞的行为模式：在发育早期阶段其自我更新能力增强，而在后续阶段则表现出更为广泛的神经发生潜能。 本研究利用基因组编辑的人类与黑猩猩（Pt）来源的神经前体细胞及大脑皮层类器官，对Hs-HARE5与Pt-HARE5的相对增强子活性及功能进行了评估。 通过这些正交实验策略，本研究证实HARE5中存在的4个人类特异性变异可增强其增强子活性，进而促进神经前体细胞的增殖。 最后，本研究证实Hs-HARE5可通过激活经典WNT信号通路，促进神经前体细胞的增殖。 上述研究结果阐明了调控DNA上的微小变异如何通过直接作用于关键信号通路，进而调控大脑发育过程。 本研究揭示了HARs作为关键调控元件的全新功能，其对于人类大脑皮层的扩张与结构复杂性维持至关重要。 本研究针对两个时间节点（E12.5与E14.5）的野生型及HARE5Hs/Hs敲入小鼠的大脑皮层组织的RNA测序数据开展了比较基因表达谱分析。每个实验条件均设置3次生物学重复，实验分组包括：E12.5野生型组、E12.5 HARE5Hs/Hs敲入组、E14.5野生型组以及E14.5 HARE5Hs/Hs敲入组。