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. Author manuscript; available in PMC: 2014 Jan 9.
Published in final edited form as: Neuron. 2013 Jan 9;77(1):83–98. doi: 10.1016/j.neuron.2012.11.035

Figure 5.

Figure 5

Dlx1&2 are required for Zfhx1b expression in the SVZ of the subpallium. (A-B’) Coronal hemisections of the telencephalon comparing Zfhx1b expression in Dlx1/+/- and Dlx1/2-/- at E12.5 (A-A’) and E15.5 (B,B’). Note the greatly reduced Zfhx1b expression in the SVZ of the LGE and MGE. X notes reduction in Zfhx1b expression in the SVZ. (C-K) Regulatory elements near Zfhx1b that drive subpallial expression are bound by DLX2 in vivo, and are positively regulated by DLX2. (C) Relative genomic position of two ultraconserved DNA elements near the Human Zfhx1b (Zeb2) locus, #675 (D) and #649 (E) (data from http://genome.ucsc.edu/). (D and E) Genomic alignment of enhancers #675 and #649; each contain a number of conserved consensus homeobox sites (asterisks). (F and G) Base-resolution view of regions with homeobox sites within #649 and #675, which are heavily conserved across vertebrate species and are similar to known DLX2 binding sites (Potter et al., 2008). (H and I) Whole mount E11.5 enhancer-lacZ transgenic mouse embryos that demonstrated lacZ expression (X-Gal staining) in the ganglionic eminences (subpallium) (H’ and I’). (J) Luciferase assay demonstrating DLX2-dependent transcriptional activation (pCAGGS vector) mediated by enhancers #649 and #675 upstream of luciferase (pGL4.23 vector). (K) Blue bars: DLX2 ChIP qPCR assay (N=3) demonstrates anti-DLX2 binding to chromatin from E13.5 ganglionic eminences to subdomains of enhancers #675 and #649 and the positive control (Dlx5/6 enhancer), and not to the negative control region (a non-conserved domain upstream of Dlx2). Red bars: Addition of a DLX2 peptide blocks the anti-DLX2 binding. Abbreviations: Cx: cortex; LGE: lateral ganglionic eminence; Luc. Luciferase; MGE: medial ganglionic eminence; MZ: mantle zone; SP: subpallium; SVZ: subventricular zone, VZ: ventricular zone. Scale bar equals 500μm (A and B). ***: p<.001; n.s. : not significant.