PROJECT SUMMARY Neurodevelopmental disorders (NDDs) encompass a broad constellation of highly prevalent and etiologically heterogeneous disorders that share several key diagnostic features. These include motor incoordination, abnormal sensory processing, intellectual and developmental disabilities, epilepsy, and neuropsychiatric co- morbidities such as autism, anxiety, and attention deficit and hyperactivity disorder (ADHD). Together, these findings suggest that NDDs result from the perturbations of brain regions critical for motor, non-motor, and sensorimotor information processing. One such region is the cerebellum, which forms multiple circuits with cortical and subcortical regions to modulate movement, language, cognition, sensory processing, executive function, and social behaviors. Intriguingly, cerebellar vermian hypoplasia (CVH) is a common neuroanatomical finding in NDDs and lesions or malformations of the vermis result in communication and behavioral deficits, suggesting that disrupted cerebellar development may perturb multi-modal information processing. In 2016, we and others discovered that a previously unrecognized NDD, the Hypotonia, Ataxia, and Delayed Development syndrome (HADDS, MIM#617330), results from either heterozygous loss-of-function (LOF) or single gene deletions of Early B-Cell Factor 3 (EBF3). EBF3-related NDDs are characterized by the namesake features, co-morbid autism, atypical cerebellar foliation, and CVH. EBF3 encodes a Collier/Olf/EBF3 (COE) transcription factor that regulates multiple neurodevelopmental processes, including GABAergic-inhibitory neuronal differentiation. We found that Ebf3 haploinsufficiency in mice recapitulated analogous neurobehavioral deficits and cerebellar dysmorphisms. Hence, elucidating the molecular and cellular mechanisms of EBF3-related NDDs affords a unique opportunity to study the impact of cerebellar dysfunction in NDDs. The primary goal of this proposal is to understand the molecular and cellular mechanisms of cerebellar inhibitory neuronal dysfunction in NDD pathogenesis through a detailed dissection of a well-defined monogenic disorder. Our unifying central hypothesis is that cerebellar inhibitory Purkinje cells and GABAergic interneurons are vulnerable to EBF3 LOF and lead to cerebellar dysfunction in EBF3-related NDDs. We will define the spectrum of cerebellar deficits, molecular alterations, and phenotype-genotype correlations in EBF3-related NDDs (Aim 1), identify Ebf3 expression in the cerebellum and associated gene regulatory networks for cerebellar inhibitory neuronal development (Aim 2), and determine the vulnerability of cerebellar inhibitory and excitatory neurons to Ebf3 LOF (Aim 3). This proposal focuses on incorporating in-depth human phenotyping with integrated mechanistic studies of Ebf3 haploinsufficiency at the cell-type-specific level in mouse models. The cross- species approaches in this project provide innovative opportunities to elucidate the interplay betwe...