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.