A transient sex-biased transcriptional program shapes early postnatal L2/3 neuron development

Abstract Most neurodevelopmental disorders (NDDs), including intellectual disability, epilepsy, and autism spectrum disorder (ASD), exhibit marked sex differences in risk, incidence, prognosis, and clinical presentation. Increasing evidence suggests that these disorders often originate from alterations in circuit formation that occur during critical developmental windows preceding the onset of symptoms. However, the extent to which sexual dimorphism emerges during neuronal differentiation remains largely underexplored in the context of NDD research. In the cerebral cortex, layer (L) 2/3 excitatory pyramidal neurons, which integrate information across cortical areas and hemispheres, have been broadly implicated in NDDs. In this study, we investigate the transcriptional expression dynamics of L2/3 neurons across three developmental stages—embryonic day (E) 19, postnatal day (P) 4, and P7—and identify a window of transient, sex-biased gene expression at P4, possibly related to the process of brain masculinization. Furthermore, gene expression analysis following knockout (KO) mutations of Cux1 and Cux2 in L2/3 neurons reveals sex-biased dysregulation, with male neurons exhibiting heightened susceptibility and amplified transcriptional disruption, resulting in a molecular fingerprint that is distinct from their WT counterparts. These findings underscore the need to incorporate studies of sex-dependent differentiation trajectories as a biological variable in NDD research and may inform the development of more targeted, sex-specific therapeutic strategies.