Single-cell transcriptomics reveal oxidative phosphorylation and oxidative stress in the superior temporal plane of non-syndromic cleft lip and palate fetuses.

AIMNon-syndromic cleft lip and palate (NSCLP) is a common congenital disability that causes morphological and functional abnormalities in the craniofacial region. Many studies have focused on clinical treatments and surgical aspects, but there has been limited investigation into the neural mechanisms in individuals with NSCLP. Therefore, it remains unclear whether developmental abnormalities in the brains of NSCLP fetuses exist.

MATERIALS AND METHODSTo investigate the presence of developmental abnormalities in the brains of NSCLP fetuses, we performed single-nucleus RNA sequencing (snRNA-seq) on the superior temporal plane (STP) from three NSCLP fetuses and three normal fetuses. Samples were collected at 17-23 gestational weeks. Real-time quantitative PCR (RT-qPCR) and immunofluorescence were performed to validate further phenotypes identified in the sequencing data.

KEY FINDINGSFollowing dimensionality reduction and clustering, we identified seven distinct cell types. Cell abundance changes revealed that excitatory neurons, inhibitory neurons, and astrocytes varied dramatically. We further identified the abnormally increased levels of oxidative phosphorylation (OXPHOS) and oxidative stress in the STP of NSCLP fetuses. Further investigation determined that a specific subpopulation of inhibitory neurons, InN6, is closely associated with altered OXPHOS and oxidative stress, potentially contributing to abnormal brain development in NSCLP. Finally, immunofluorescence and RT-qPCR results confirmed the abnormal increase in OXPHOS and oxidative stress in the NSCLP fetuses' brain.

SIGNIFICANCEThis study provides a single-cell atlas of STP in the NSCLP fetuses, revealing increased OXPHOS and oxidative stress in the NSCLP fetal brain. These findings provide new insights into the pathological mechanisms of the brain in NSCLP.