Identification of Genetic and Molecular Bases of Derived Phenotypes in Primate Brain Development

项目总结 越来越有说服力的证据表明，基因组变异驱动人类和 灵长类富含调控元素，但绝大多数这些与进化相关的变体 尚未被发现或表征。这是不幸的，因为在大约3500万单身人士中 核苷酸替换(SNP)、500万个插入或缺失(INDELs)和9000万个结构碱基 人类和黑猩猩基因组不同的变体有无数与 疾病发育、功能或疾病识别进化上相关的遗传变异，以及 与疾病或功能有关的，可以通过对中间体物种差异的分析来指导 分子表型(例如，转录和表观基因组特征)，这很可能是主要的 由基因组变异决定的效应。在这项建议中，我们建议进行灵长类比较 功能基因组学揭示解释影响人类的谱系特异性表型的遗传变异 以及非人灵长类(NHP)脑，这是一种显示出明显的分子和功能差异的器官 在物种之间。为此，我们的第一个目标是建立一个基因表达和开放染色质的分类学。 在灵长类动物中，应用单核RNA-seq和单核atac-seq研究胎儿中期和 新生儿(胎儿晚期和婴儿期早期)死后人和NHP脑以及脑的发育 含有从多个灵长类干细胞和成纤维细胞分化的细胞的有机共培养 淋巴母细胞系。在我们的第二个目标中，我们将补充这份物种基因差异图谱。 通过编目SNPs、Indels和多个大的、复杂的结构变体来表达和打开染色质 灵长类物种。这将使我们能够区分特定血统(即，人类与黑猩猩和 猕猴)和人科特有的(即，人类和黑猩猩与猕猴)基因组变异。最后，在 我们的第三个目标是整合和功能验证，使用大规模并行的记者分析， CRISPR/Cas9基因组编辑、人类诱导的多能干细胞和小鼠神经模型 通过这些实验确定了发育、关键调控元件和从头基因。通过这些 目的，我们将识别和功能验证基因组变异和基因表达模式，并开放 染色质可能驱动人类和非人类大脑的衍生表型，因此 似乎与人类认知、社会行为和神经精神疾病有关。

PROJECT SUMMARY Increasingly persuasive evidence suggests genomic variants driving derived features in humans and among primates are enriched in regulatory elements, but the vast majority of these evolutionarily relevant variants have yet to be discovered or characterized. This is unfortunate, as among the approximately 35 million single nucleotide substitutions (SNPs), 5 million insertions or deletions (indels), and 90 megabases of structural variants where the human and chimpanzee genomes differ are countless variants associated with development, function, or disease. Identifying evolutionarily relevant genetic variants, as well as those implicated in disease or function, can be guided by the analysis of species differences in intermediate molecular phenotypes (e.g., transcriptomic and epigenomic signatures), which are most likely the primary effects determined by genomic variation. In this proposal, we propose to perform primate comparative functional genomics to uncover genetic variants explaining lineage-specific phenotypes affecting the human and non-human primate (NHP) brain, an organ exhibiting pronounced molecular and functional differences between species. To do so, in our first aim we will develop a taxonomy of gene expression and open chromatin across primates, applying single nucleus RNA-seq and single nucleus ATAC-seq to study the mid- fetal and neonatal (late fetal and early infancy) development of the post-mortem human and NHP brain, as well as brain organoid co-cultures containing cells differentiated from multiple primate stem cells and fibroblasts and lymphoblastoid cell lines. In our second aim, we will complement this atlas of species differences in gene expression and open chromatin by cataloguing SNPs, indels, and large, complex structural variants in multiple primate species. This will allow us to differentiate between lineage-specific (i.e., human versus chimpanzee and macaque) and Hominidae-specific (i.e., human and chimpanzee versus macaque) genomic variants. Finally, in our third aim we will integrate and functionally validate, using the Massively Parallel Reporter Assay, CRISPR/Cas9 genome editing, human induced pluripotent stem cells, and mouse models of neural development, key regulatory elements and de novo genes identified through these experiments. Through these aims, we will identify and functionally validate genomic variants and patterns of gene expression and open chromatin potentially driving derived phenotypes in the human and non-human brain and consequently plausibly associated with human cognition, social behaviour, and neuropsychiatric disease.