Towards defining the mechanisms that regulate Zfhx1b expression in the developing subpallium, we identified two regulatory elements near the Zfhx1b locus that drive expression in the developing subpallium. These enhancers, here named #649 and #675, were identified by virtue of their extremely strong evolutionary conservation (Figures 5C-G) and their reproducible enhancer activity in the forebrain of mouse embryos in transgenic experiments (Figures 5H-I) (Visel et al., 2007; Visel et al., 2008). The other genes in this region do not have known expression in the developing subpallium. Analysis of enhancer activity at E11.5 in transgenic whole mounts and sections showed that both enhancers drive LacZ expression in the subpallium, including the SVZ of the MGE (Figures 5H,I). The spatial overlap of enhancer activities and Zfhx1b mRNA expression suggests that these two elements are distant-acting transcriptional activators of Zfhx1b in the developing subpallium. Computational analysis identified multiple candidate homeobox binding sites (asterisks in Figures 5D,E and highlighted regions in 5F,G).
