SMPD4 deficiency disrupts indirect neurogenesis and neuronal migration in gyrencephalic cortex

Variants in SMPD4 cause severe neurodevelopmental disorders characterized by microcephaly, simplified gyral patterns, and cortical malformations in humans, yet Smpd4 knockout mice exhibit minimal cortical abnormalities, displaying phenotypes primarily restricted to cerebellar defects. This striking species-specific disparity has hindered understanding of the cellular and molecular mechanisms underlying SMPD4-related cortical pathology due to the lack of appropriate gyrencephalic animal models that accurately recapitulate human brain development. We utilized in utero electroporation combined with CRISPR-Cas9 genome editing technology to efficiently knock out Smpd4 in the developing gyrencephalic neocortex of ferrets at embryonic day 34. Ferrets were analyzed at postnatal days 1, 2, 6, and 16 using immunohistochemistry, live-cell imaging, and single-cell RNA sequencing to comprehensively characterize the cellular and molecular consequences of SMPD4 deficiency. SMPD4-deficient ferret cortices successfully recapitulated key human phenotypes, exhibiting markedly reduced progenitor cell proliferation with significantly prolonged mitotic duration. Quantitative analysis revealed a profound reduction in basal intermediate progenitors and decreased neuronal output. Mechanistically, loss of SMPD4 compromised nuclear envelope integrity, disrupted mitotic spindle orientation leading to aberrant cell division patterns, impaired primary cilia formation, and substantially reduced intermediate progenitor morphological complexity. Additionally, Smpd4 knockout induced severe migration defects in cortical neurons, with many neurons failing to reach their appropriate cortical layers. Single-cell RNA sequencing analysis of over 20,000 cells from control and Smpd4 knockout cortices revealed that SMPD4 deficiency significantly reduced intermediate progenitor cell numbers and caused widespread dysregulation of genes previously associated with human lissencephaly, simplified gyration, microcephaly, developmental delay, and epilepsy. Gene ontology enrichment analyses identified disrupted pathways involved in cell cycle regulation, neurogenesis, chromosome organization, apoptosis, and neuronal migration. These findings provide critical mechanistic insights into how SMPD4 deficiency disrupts cortical development through impaired intermediate progenitor generation and neuronal migration, establishing ferrets as an invaluable gyrencephalic model system for investigating SMPD4-related neurodevelopmental disorders. This model offers significant translational potential for understanding human cortical malformations and developing therapeutic interventions for affected patients.