Non-cell-autonomous regulation of Bhlhb5 expression in cortical projection neurons by GABAergic interneuron development and position.

The functional architecture of the cerebral cortex relies on the coordination between excitatory projection neurons and inhibitory GABAergic interneurons. While intrinsic programs governing their specification are well-characterized, it remains unclear whether the proper development of interneurons is required to maintain the molecular identity of projection neurons. We generated double conditional knockout (DCKO) mice lacking the transcription factors Sp8 and Sp9 specifically in the GABAergic lineage using the Dlx5/6-Cre driver. Cortical cytoarchitecture and neuronal identity were assessed via immunohistochemistry at postnatal days 4 (P4) and 11 (P11). Sp8&Sp9-DCKO mice exhibited severe defects in the development and laminar positioning of cortical interneurons, resulting in their abnormal distribution and reduced abundance in the cortex. Despite these inhibitory defects, the general laminar organization, checked by the expression of TBR1, CTIP2, FOXP2, FOXP1, CUX1, and NeuN, remained intact. Strikingly, the expression of BHLHB5, a key regulator of sensory area identity, was specifically downregulated in projection neurons at P11. Crucially, BHLHB5 expression was indistinguishable from controls at P4, indicating that the proper development of cortical interneurons is essential for stabilizing the molecular identity of projection neurons. These findings reveal a novel non-cell-autonomous mechanism where proper GABAergic interneuron positioning and development are essential for the maintenance, but not the acquisition, of specific projection neuron molecular identities.