Abstract
<title>Abstract</title> <p>Human cortical development depends on tightly regulated transcriptional and signaling networks, while their disruption can lead to neurodevelopmental disorders. Meanwhile, the roles of key transcription factors in human-specific cortical patterning remain incompletely understood. Aberrant function of regulatory factor X4 (RFX4) has been implicated in diverse neurodevelopmental and neuropsychiatric disorders, although the mechanistic contribution remains unclear. This study aimed to investigate RFX4 using gain- and loss-of-function approaches in human embryonic stem cell (hESC)-derived cortical organoids (COs). RFX4 overexpression induced neural differentiation of hESCs in the absence of exogenous patterning cues and activated transcriptional programs associated with cortical development. Conversely, RFX4 deficiency caused profound defects in early CO development, resulting in an excitatory–inhibitory (E/I) imbalance. Single-cell transcriptomic and molecular analyses revealed that RFX4 knockout suppresses expression of multiple WNT ligands and disrupts WNT signaling, a pathway essential for cortical patterning. Pharmacological activation of WNT signaling rescued ventralization and growth defects in RFX4-deficient COs. Furthermore, RFX4 directly binds to regulatory regions of WNT genes, supporting its role as a transcriptional regulator. Collectively, these findings identify RFX4 as a critical regulator of human cortical patterning by modulating WNT signaling and provide mechanistic insight into its contribution to neurodevelopmental disorders.</p>