Single‐Nucleus RNA Sequencing Reveals Mid‐Gestational Neurodevelopment Features in the Superior Temporal Plane from Fetuses with Nonsyndromic Cleft Lip and Palate

Nonsyndromic cleft lip and palate (NSCLP) is a common craniofacial malformation increasingly recognized to involve neurodevelopmental abnormalities, though the molecular basis remains unclear. Here, single-nucleus RNA sequencing of the superior temporal plane from mid-gestation NSCLP fetuses is performed, and profound alterations in cell-type composition, intercellular communication, and transcriptional programs are uncovered. Integrative analyses with weighted gene co-expression network analysis and single-cell regulatory network interference and clustering based on single-nucleus transcriptomes identify myocyte enhancer factor 2C (MEF2C) as a shared transcriptional regulator consistently downregulated in excitatory and inhibitory neurons across mid-term gestation, which is validated in NSCLP fetal brain tissues. MEF2C expression is negatively correlated with synaptophysin immunofluorescence intensity. In MEF2C-deficient primary cortical neurons, impaired synaptic formation, reduced postsynaptic density protein-95 expression, and weakened excitatory postsynaptic transmission without altering intrinsic excitability are found. Upstream regulators of MEF2C are enriched for pathways controlling neuronal differentiation, synaptic plasticity, and epigenetic regulation, suggesting broad disruption of neurodevelopmental programs. Together, this study provides molecular evidence of disrupted brain development in NSCLP and implicates MEF2C as a potential mediator of neurodevelopmental impairments.