<EM>SMPD4</EM> DEFICIENCY IMPAIRS CORTICAL EXPANSION IN A GYRIFIED MODEL: MECHANISMS OF DISRUPTED NEUROGENESIS AND NEURONAL MIGRATION

Mutations in the SMPD4 gene are associated with severe human neurodevelopmental disorders characterized by microcephaly, simplified cortical gyration, and developmental delays. The mechanistic understanding of these pathologies has been hindered by the lack of pronounced phenotypes in traditional rodent models, highlighting a critical need for gyrencephalic animal systems. To bridge this species-specific gap, we developed a novel in vivo model by combining in utero electroporation with CRISPR/Cas9-mediated genome editing to achieve efficient Smpd4 knockout in the developing neocortex of ferrets, a gyrencephalic mammal. Smpd4 deficiency in ferrets recapitulated key human cortical malformations. We observed a significant reduction in progenitor cell proliferation, accompanied by prolonged mitosis and a dramatic decrease in the basal intermediate progenitor (IP) pool, ultimately leading to reduced neuronal output. At the cellular level, Smpd4 loss compromised nuclear envelope integrity, disrupted mitotic spindle orientation, and impaired primary cilia formation. Furthermore, Smpd4-deficient IPs exhibited reduced morphological complexity, and postmitotic neurons displayed severe migration defects. Single-cell RNA sequencing analysis confirmed a significant depletion of the IP population and revealed dysregulation of gene networks implicated in lissencephaly, microcephaly, and epilepsy. Our findings establish the ferret as a powerful model for SMPD4-related corticogenesis disorders. We demonstrate that SMPD4 is essential for maintaining progenitor cell dynamics and neuronal migration in a gyrencephalic cortex, providing direct cellular and molecular insights into the etiologies of human cortical simplification and malformation syndromes.