Genetic association studies have identified many factors associated with neurodevelopmental disorders such as autism
spectrum disorder (ASD). However, the way these genes shape neuroanatomical structure and connectivity is poorly
understood. Recent research has focused on proteins that act as points of convergence for multiple factors, as these may
provide greater insight into understanding the biology of neurodevelopmental disorders. USP9X, a deubiquitylating enzyme
that regulates the stability of many ASD-related proteins, is one such point of convergence. Loss of function variants in
human USP9X lead to brain malformations, which manifest as a neurodevelopmental syndrome that frequently includes
ASD, but the underlying structural and connectomic abnormalities giving rise to patient symptoms is unknown. Here, we
analyzed forebrain-specific Usp9x knockout mice (Usp9x−/y) to address this knowledge gap. Usp9x−/y mice displayed
abnormal communication and social interaction behaviors. Moreover, the absence of Usp9x culminated in reductions to the
size of multiple brain regions. Diffusion tensor magnetic resonance imaging revealed deficits in all three major forebrain
commissures, as well as long-range hypoconnectivity between cortical and subcortical regions. These data identify USP9X
as a key regulator of brain formation and function, and provide insights into the neurodevelopmental syndrome arising as a
consequence of USP9X mutations in patients.