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

项目摘要 越来越有说服力的证据表明，基因组变异驱动了人类的衍生特征， 灵长类动物富含调节元件，但绝大多数这些进化相关的变异 还没有被发现或被描述。这是不幸的，因为在大约3500万的单身汉中， 核苷酸取代（SNP），500万个插入或缺失（indel），以及90兆碱基的结构性突变。 人类和黑猩猩基因组不同的变异是无数的变异， 发育、功能或疾病。识别进化相关的遗传变异，以及那些 涉及疾病或功能，可以通过分析中间体的物种差异来指导 分子表型（例如，转录组和表观基因组签名），这是最有可能的主要 由基因组变异决定的效应。在这个建议中，我们建议进行灵长类比较 功能基因组学揭示遗传变异，解释影响人类的谱系特异性表型 和非人类灵长类动物（NHP）的大脑，一个器官表现出明显的分子和功能差异 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研究中期胎儿和 死后人脑和NHP脑以及脑的新生儿（胎儿晚期和婴儿早期）发育 含有从多种灵长类动物干细胞和成纤维细胞分化的细胞的类器官共培养物， 淋巴母细胞样细胞系。在我们的第二个目标中，我们将补充这一基因差异的物种图谱， 通过对多个基因组中的SNP、插入缺失和大的复杂结构变体进行编目， 灵长类动物这将使我们能够区分血统特异性（即，人类和黑猩猩的对抗 猕猴）和人科特有的（即，人类和黑猩猩对猕猴）基因组变异。最后在 我们的第三个目标是，使用大规模平行报告分析， CRISPR/Cas9基因组编辑、人诱导多能干细胞和神经系统疾病小鼠模型 通过这些实验鉴定了发育、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， 染色质可能驱动人类和非人类大脑中的衍生表型， 可能与人类认知、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.