The laminar structure, a unique feature of the mammalian cerebrum, is formed by a number of genes in a highly complex process. The pyramidal neurons that make up each layer of the cerebrum are functionally characterized by specific gene expressions. In particular, Cux1 and Cux2, which are specifically expressed in layer II-IV neurons, are known to regulate dendritic branching, spine morphology, and synapse formation. However, it is still unknown how their expression is regulated transcriptionally. Here we constructed Cux2-mCherry transgenic mice that reproduce the cortical layer II-IV-specific expression of Cux2, a member of the Cut/Cux/CDP family, using BAC transgenesis and a variety of coordinated cortical layer markers that are known to date. Our immunohistochemistry analysis shows that mCherry was expressed in cortical layer II-IV and the corpus callosum in the same way as endogenous Cux2 without ectopic expression. We also identified a region of 220 bp that is highly conserved in mammals and controls specific cerebral expression of Cux2, using comparative genome analysis and in vivo reporter assays. Furthermore, we confirm that Lhx2, whose expression in cortical layer II-IV is similar to that of the Cux2 enhancer, can act as a transcriptional activator. These results suggest that cortical layer II-IV expression of Cux2 can be regulated by the interaction of Cux2-E1 and Lhx2, and that their failure to co-regulate is associated with neurodevelopmental disorders such as autism and schizophrenia. (C) 2019 Elsevier Inc. All rights reserved.