ABSTRACT: Epigenetic regulation is essential for cell fate determination, yet its role in spinal motor neuron (MN) development remains poorly understood. SETD5, an epigenetic regulator, is mutated in SETD5 syndrome, which is characterized by intellectual disability and motor dysfunction. However, the mechanisms underlying these locomotor deficits remain elusive. Here, we integrated in ovo knockdown in chick embryos, conditional knockout strategies in mice and in vitro MN differentiation system to investigate Setd5 function during MN development. Setd5 ablation in MN progenitors (pMNs) with Olig2-Cre markedly reduced the Olig2+ progenitor pool by attenuating proliferation, as evidenced by diminished Ki67 and BrdU incorporation, while concurrently promoting premature differentiation, as shown by elevated Neurod1 expression and precocious generation of Mnx1+ newborn MNs.This imbalance disrupted MN subtype diversification, dysregulating key identity markers (Mnx1, Isl1, Lhx3, and Foxp1). Notably, late-stage Setd5 deletion with Isl1-Cre revealed a cell-context-specific requirement for Setd5 in maintaining Foxp1 expression selectively within thoracic preganglionic motor column neurons. These developmental defects resulted in aberrant axonogenesis, impaired neuromuscular junction formation, and severe locomotor phenotypes, including reduced spontaneous movement, poor motor coordination, and abnormal gait. Transcriptomic profiling of Setd5-deficient pMNs identified downregulation of cell-cycle genes and upregulation of neural differentiation programs, mechanistically linking Setd5 loss to the observed cellular phenotypes. Heterozygous animals displayed intermediate defects, confirming a dose-sensitive requirement. Collectively, our findings establish Setd5 as a critical epigenetic coordinator of MN proliferation and differentiation, and provide a mechanistic framework for understanding the motor abnormalities associated with SETD5 syndrome.
