Multi-omics Analysis Revealing the Impact of Excessive Maternal Folate Supplementation on Offspring Brain Development [spatial transcriptome]

Folate is crucial for diverse biological processes including neurogenesis. While folate supplementation during pregnancy is standard for preventing neural tube defects (NTDs), concerns are growing over the potential risks of excessive maternal intake. In this study, we employed spatial transcriptomics and single-nucleus multi-omics techniques to investigate the impact of increased maternal folate intake on offspring brain development. Elevated folate intake broadly affected gene pathways linked to neurogenesis and neuronal axon myelination across multiple brain regions. Furthermore, specific gene expression alterations related to learning and memory processes emerged in thalamic and ventricular regions. Single-nucleus multi-omics analysis revealed that maturing excitatory neurons in dentate gyrus are particularly vulnerable to suboptimal maternal folate intake. Aberrant gene expression and chromatin accessibility changes were primarily centered on pathways governing ribosomal biogenesis, which is critical for synaptic formation. Altogether, our findings provide novel insights into how excessive maternal folate supplementation affects offspring brain development, notably by influencing gene expression and chromatin accessibility. Adult female mice (8-12 weeks old) were randomly assigned to either a control folic acid (FA) diet (2mg/kg) or an excessive FA diet (20mg/kg) for two weeks before mating with males. These diets were maintained throughout pregnancy and lactation. Pups were harvested on postnatal day 21 (P21). To profile spatial transcriptome, the mouse brains were freshly isolated to prepare FFPE tissue blocks, then coronally sectioned and placed onto the Visium slides for Visium Spatial Gene Expression library preparation. To simultaneously profile the transcriptome and chromatin accessibility at single cell resolution, hippocampus tissues were micro-dissected from the freshly isolated mouse brain tissues for single nuclei suspension preparation and subsequently loaded onto the Chromium iX using the single nucleus multiome kit from 10x genomics. Single nucleus RNA-seq (snRNA-seq) and single nucleus ATAC-seq (snATAC-seq) libraries were simultaneously constructed from the same nucleus following the manufactures' instructions.