PROJECT SUMMARY There are fundamental gaps in our understanding of DDX3X syndrome, a genetic condition accounting for up to 2% of intellectual disability (ID) in females and caused by mutations in the X-linked gene DDX3X. Most affected individuals are females with DDX3X haploinsufficiency. A recent study using cell models and in utero manipulations in mouse has shown that Ddx3x regulates cortical neurogenesis and mRNA translation in neuronal progenitors. Yet, the lack of a mouse model with construct validity for DDX3X syndrome has impeded to faithfully capture the molecular and cellular determinants of the cognitive, social and motor deficits observed in individuals with DDX3X syndrome. There is a critical need to fill these gaps because, until we do so, understanding DDX3X syndrome and developing effective therapeutics remain out of reach. To address this unmet need, a mouse modeling DDX3X haploinsufficiency (Ddx3x+/-) was generated in our laboratory. The long-term goal is to unravel the pathophysiology of ID and identify targets for therapeutics. The overall objective is to capture the molecular and cellular mechanisms underlying DDX3X syndrome. The central hypothesis is that Ddx3x dictates the formation of cortical projection neurons subserving cognitive, social, and motor functions, and does so by regulating mRNA translation in the developing cortex. The rationale is that, once we identify the mechanisms of DDX3X syndrome and reliable phenotypes in the mouse model, therapeutics can be developed and tested. The hypothesis will be tested by pursuing three Specific Aims: 1) Assess the neurodevelopmental defects in a mouse model of DDX3X syndrome; 2) Capture the molecular defects in a mouse model of DDX3X syndrome; and, 3) Correlate cellular function to behavioral outcomes in Ddx3x mutant mice. Under Aim 1, the developmental and adult cognitive, social, and motor behavior of Ddx3x+/- mice will be measured with a standardized behavioral battery. Cortical projection neurons will be studied in Ddx3x+/- mice by combining cellular and in vivo (e.g., in utero electroporation) approaches. Under Aim 2, Ddx3x targets in projection neurons will be mapped using the translating ribosome affinity purification (TRAP) method for discovery and its viral-based development (vTRAP) for independent validation. Under Aim 3, conditional Ddx3x mice with the gene ablated in the forebrain or in specific cortical layers will be tested for development and adult behavior. The proposal is innovative because it addresses the neurobiology of a largely unknown ID gene and characterizes the first mouse model of DDX3X syndrome. It is also innovative because it bridges genetics, biochemistry, molecular and cellular neuroscience, developmental biology, and behavioral neuroscience. The application is significant because it will advance our understanding of ID pathophysiology, while shedding new light on corticogenesis, a fundamental process for brain functioning. These results are e...