Hippo/YAP1 signaling regulates the Oligodendrocyte–Astrocyte fate switch and ependymal gene expression in adult spinal cord stem cells

The adult mammalian spinal cord harbors ependymal cells with latent neural stem-cell properties, including the capacity to generate oligodendrocytes. Yet following injury, these cells overwhelmingly commit to an astrocytic fate, and the molecular cues governing this lineage choice remain poorly understood. To address this gap, we investigated the emergence of PDGFRA⁺ oligodendrocyte progenitor cells (OPCs) in neurosphere cultures derived from adult spinal cord ependymal cells. We found that proliferating neurosphere cells adopt a hybrid transcriptional identity, co-expressing astrocytic (NFIA, SOX9) and oligodendrocytic (OLIG1/2, SOX4, NKX2.2, TCF4) transcription factors. Upon differentiation, oligodendrocytic factors were selectively retained in newly specified OPCs while declining in the remaining cell population. Leveraging Pdgfra H2B−GFP knock-in mice, we isolated nascent OPCs from neurospheres and subjected them to multi-omic profiling. OPC formation was accompanied by upregulation of chromatin remodeling factors and downregulation of stem-cell-associated markers, including EGFR, HES1, and TNC. Strikingly, it also coincided with reduced expression of YAP1 and its partner TEAD1, core effectors of the Hippo signaling pathway. Functional studies revealed that YAP1 loss promoted oligodendrocytic differentiation while suppressing astrocytic and ependymal/cilia-associated gene expression. Conversely, constitutive YAP1 activation triggered abortive cell-cycle activation accompanied by cell death, blocked differentiation along both glial lineages, and drove an ependymal-like transcriptional program including upregulation of the ependymal marker CD24a and cilia-associated proteins such as CROCC (Rootletin). Collectively, these findings uncover unrecognized roles for YAP1 in adult spinal cord stem-cell fate decisions and provide a molecular framework for leveraging these cells in regenerative strategies targeting spinal cord repair.