Poster #45 - Shikhar Gupta

Prenatal Hypoxia Reprograms Alternative Splicing Landscapes in the Developing Cortex

Gupta, Shikhar. (Undergraduate Researcher), University of Pennsylvania, Philadelphia, PA, USA. Cassidy, Maggie. (BA), Cell & Molecular Biology Graduate Group, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA. Cristancho, Ana. (MD/PhD), Department of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Prenatal hypoxia is a leading cause of neonatal morbidity and detrimental fetal development, yet its molecular consequences are not fully elucidated. Alternative splicing, a critical mechanism for generating transcriptomic and proteomic diversity, is tightly regulated during brain development and frequently disrupted in neurological disease, but its role in hypoxic brain injury has not been established. To investigate whether oxygen deprivation alters splicing programs in the fetal cortex, we applied computational analyses to single-cell RNA sequencing data derived from a murine prenatal hypoxia model. Using rMATS on pseudo-bulked single-cell transcriptomes, we identified over 1,200 significant differential splicing events across all five major categories, including exon skipping, intron retention, and alternative splice site usage. These changes were bidirectional, with increases and decreases in exon inclusion. Gene ontology analysis revealed enrichment patterns: transcripts with higher exon inclusion under normoxia were enriched for chromatin remodeling and transcriptional regulation, while hypoxia-associated isoforms were enriched for RNA processing and metabolic pathways. Notably, chromatin regulators such as KDM5A, SMARCA2, and CHD8 exhibited condition-specific splicing, suggesting that hypoxia modifies not only gene expression but also the isoform composition of epigenetic regulators themselves. To investigate cell type-specific dynamics, we are using a single-cell splicing framework using the MARVEL R pipeline in conjunction with SingCellaR preprocessing and UMAP-based cortical cell type annotation. Ongoing work will test whether splicing alterations are uniform across excitatory neurons, inhibitory neurons, and glial populations, or whether they represent distinct adaptive strategies to hypoxic stress.