Impaired phrenic nerve axon development and diaphragm neuromuscular junction formation in embryonic Cyfip2-null mice

Abstract Pathogenic variants in CYFIP2 cause developmental and epileptic encephalopathy 65 (DEE65) and have been predominantly investigated in the context of central nervous system dysfunction. However, emerging clinical evidence suggests that peripheral nervous system (PNS) involvement may also contribute to disease manifestations. To explore this possibility, we examined the role of CYFIP2 in the development of the phrenic neuromuscular system, which is essential for neonatal respiration. Because conventional Cyfip2-null (Cyfip2 −/− ) mice exhibit perinatal lethality, we analyzed phrenic nerve axon development and diaphragm neuromuscular junction (NMJ) formation in embryonic mice. At embryonic day 16.5, Cyfip2 −/− embryos displayed significantly reduced phrenic nerve axon length and branching compared to wild-type controls. Postsynaptic acetylcholine receptor (AChR) clustering in Cyfip2 −/− diaphragms showed spatial heterogeneity: sparse regions exhibited a significant increase in endplate bandwidth, whereas dense regions showed a decreasing trend. Further analysis using synaptophysin and α-bungarotoxin labeling revealed reduced pre- and post-synaptic puncta density and decreased colocalization, despite preserved puncta intensity and volume, indicating impaired synaptic organization. Together, these findings demonstrate that CYFIP2 is required for proper phrenic nerve innervation and NMJ organization during embryonic development. This study extends the functional scope of CYFIP2 to the PNS and establishes the diaphragm as a tractable model for investigating peripheral mechanisms underlying CYFIP2-associated neurodevelopmental disorders.