Fig. 4

Identification of Otp as a direct target gene of Dlx1/2. a Location of Dlx1 ChIPseq peaks in the genome. b, c De novo motif analyses revealed that binding sites for two transcription factors, NHLH1 and Dlx1/2, are enriched in our Dlx1 ChIPseq peaks (b), which are mostly located in the summit area of Dlx1 ChIPseq peaks (c). d, e Identification of Dlx1 ChIPseq peak P29 associated with Otp (d) and validation of Dlx1 binding to this peak in ChIP using anti-Dlx1 antibody and E16.5 hypothalami (e). Bars represent mean, error bars indicate the SD (e). f Luciferase assays in HEK293 cells, in which Dlx1 activates the activity of Dlx5/6-P27-Luc reporter and represses the activity of Otp-P29-Luc reporter in a dose dependent manner (5 and 10 ng of Dlx1 expression vector). Bars represent mean, error bars indicate the SD. The reporter assays were repeated four times, which produced similar results. A representative set of results is as shown. g In ovo expression of GFP by a reporter directed by P29 is strongly suppressed by coexpressed Dlx1. h Schematic model for Dlx1/2 to suppress Otp expression via direct binding of Otp-DlxRE by Dlx1/2. i IHC analyses of Otp/Dlx1 expression in E13.5 and E14.5 embryos using our home-made antibodies against Otp and Dlx1, in which Dlx1 and Otp were expressed in a mutually exclusive pattern. j In IHC analyses, the number of Otp⁺ cells was significantly increased in Dlx1/2^cKO mice (n = 4 for E16.5, n = 3 for P28) relative to their littermate controls (n = 4 for E16.5, n = 3 for P28). In both stages, p-values in Student’s t-test were <0.01. Quantification was performed with multiple embryos as indicated and at least three sections from each embryo, and representative images for only one side of the ARC are as shown. Scale bars, 100 μm. Bars represent mean, error bars represent the SEM