Characterizing the mechanistic basis for sex-dimorphic responses to retinoic acid signaling in the developing brain

项目摘要/摘要 性激素和性染色体基因指导性特有特征的发育 神经发育，包括躯体基因表达和灰质体积的差异。加在一起，这些 发育计划导致了分子、细胞和组织水平上的根本差异 雄性和雌性。虽然一些神经疾病与X染色体基因有明确的联系，如Rett 综合征和脆性X综合征，包括自闭症在内的其他疾病表现出性别偏见的外显，没有 明确遗传机制。这项提案旨在解决性别-二态转录的机制基础 发育中大脑对维甲酸信号的反应。维甲酸信号转导对于 调节协调神经系统正常发育的基因调控程序，以及最近的工作 研究表明，自闭症风险基因Foxp1在维甲酸信号转导中上调。初步 研究表明，视黄酸信号对女性大脑皮质中Foxp1的特异性上调有反应 有机化合物，而不是雄性。该项目将同样利用干细胞衍生的皮质有机体来模拟 神经体外发育以更好地了解性别二态表型的分子基础 暴露在维甲酸中。目标1将表征性别二态全基因组表达的变化作为响应 维甲酸。雄性和雌性器官将接受维生素A治疗，维生素A是维甲酸的前体， 用于scRNA-seq.这个数据集将发现女性器官中特异上调的基因 维甲酸。此外，我将鉴定雄性和雌性器官中表现出最大的细胞类型 基因表达变化对维甲酸的响应，有助于深入了解细胞类型特异性对 维甲酸在神经发育中的作用。在目标2中，X-连接赖氨酸的剂量与 去甲基酶KDM5C和维甲酸诱导的性别特异性基因调控程序将被描述。 女性干细胞衍生的有机体中KDM5C的敲除以及随后H3K4的全基因组变化 甲基化由KDM5C去甲基化，将通过Cut&Tag来确定。一位公认的增强剂 Foxp1基因座与H3K4me3标记重合，该基因组位置上依赖KDM5C的甲基化将是 特别重要。KDM5C基因敲除对维甲酸诱导的Foxp1表达的影响 然后用免疫组织化学方法进行询问。总之，这些实验将进一步加深我们对 神经发育过程中表观遗传学、基因表达和细胞信号通路的交集， 为性别二态发育计划提供了重要的机制基础。这将进一步推动我们的 了解神经发育障碍的病因学与性别偏见外显，同时发现 治疗干预的潜在候选者。

Project Summary/Abstract Sex hormones and sex chromosome genes instruct development of sex-specific features during neurodevelopment, including differences in somatic gene expression and gray matter volume. Together, these developmental programs lead to fundamental differences at the molecular, cellular, and tissue level between males and females. While some neurological disorders have clear links to X chromosome genes, such as Rett Syndrome and Fragile X Syndrome, other disorders including Autism show sex-biased penetrance with no clear genetic mechanism. This proposal aims to address the mechanistic basis of sex-dimorphic transcriptional responses to retinoic acid signaling in the developing brain. Retinoic acid signaling is indispensable for modulating gene regulatory programs that orchestrate proper nervous system development, and recent work has shown that the Autism risk gene FOXP1 is upregulated in response to retinoic acid signaling. Preliminary studies have shown specific upregulation of FOXP1 in response to retinoic acid signaling in female cortical organoids, and not in males. This project will similarly leverage stem cell-derived cortical organoids to model neurodevelopment in vitro to better understand the molecular basis of sex-dimorphic phenotypes upon exposure to retinoic acid. Aim 1 will characterize sex-dimorphic genome wide expression changes in response to retinoic acid. Male and female organoids will be treated with vitamin A, the precursor to retinoic acid, and used for scRNA-seq. This dataset will uncover genes upregulated specifically in female organoids in response to retinoic acid. Additionally, I will identify cell types in both male and female organoids that exhibit the greatest gene expression changes in response to retinoic acid, lending insight into cell type-specific sensitives to retinoic acid during neurodevelopment. In Aim 2, the relationship between dosage of X-linked lysine demethylase KDM5C and retinoic acid-induced sex-specific gene regulatory programs will be characterized. Knockdown of KDM5C in female stem cell-derived organoids and subsequent genome-wide changes in H3K4 methylation, which is demethylated by KDM5C, will be determined by CUT&Tag. A putative enhancer at the FOXP1 locus coincides with H3K4me3 marks, and KDM5C-dependent methylation at this genomic site will be of particular importance. The impact of KDM5C knockdown on retinoic acid-induced FOXP1 expression will then be interrogated by immunohistochemistry. Together, these experiments will further our understanding of the intersection of epigenetics, gene expression, and cell signaling pathways during neurodevelopment, providing an an important mechanistic basis for sex-dimorphic developmental programs. This will further our understanding of the etiology of neurodevelopmental disorders with sex-biased penetrance, while uncovering potential candidates for therapeutic interventions.