Sex-Biased Transcription in Early Postnatal Layer 2/3 Cortical Neurons: Interplay Between Sex and Cux1/2 Mutations

Despite many neurodevelopmental disorders (NDDs) exhibiting pronounced sex differences, the role of sex in shaping the genetic regulation of the brain remains barely explored. Brain development relies on a precise control of gene expression across time and disruptions in this process, especially during critical periods, can result in NDDs. In the neocortex, layer (L) 2/3 excitatory pyramidal neurons stand out for integrating information across cortical areas and hemispheres. Here, we show a transient window of sex-biased gene expression of L2/3 neurons at postnatal day 4 (P4), mainly characterized by autosomal genes. Loss-of-function experiments targeting Cux1 and Cux2, key transcription factors in L2/3 development, lead to sex-dependent transcriptional deregulation in these neurons, with a significant male-biased sensitivity. Moreover, differentially expressed genes shift substantially between developmental stages, reflecting the dynamic regulation of Cux-mediated gene expression. Overall, our study identifies P4 as a key determinant of early sex differences and highly vulnerable stage for the emergence of NDDs, and highlights the importance of sex-specific analyses for understanding brain development and disease.