Cortical RORβ is required for layer 4 transcriptional identity and barrel integrity

Retinoic Acid-Related Orphan Receptor Beta (RORβ) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORβ is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age. Loss of RORβ delays excitatory input and disrupts gene expression and chromatin accessibility, with downregulation of L4 and upregulation of L5 genes, suggesting a shift in cellular identity. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORβ, Thsd7a , is downregulated without RORβ, and Thsd7a knockout alone disrupts TCA organization in adult barrels.