Figure 2: Ctip2 directly represses Unc5C expression.
From: Unc5C and DCC act downstream of Ctip2 and Satb2 and contribute to corpus callosum formation
(a) Immunohistochemical staining against Unc5C and L1. Unc5C expression is absent in Satb2−/− brains when compared with wild-type and Ctip2−/− brains. The expression of Unc5C is restored in Satb2−/−;Ctip2−/− double-mutant brains. (b) Unc5C expression in E17.5 wild-type embryos in cells electroporated at E12.5 with Ctip2/EGFP or EGFP control within the electroporated as well as contralateral hemisphere. Subsequent section stained for GFP, Ctip2, Draq5 (a nuclear marker) IHC. Unc5C mRNA expression is lost in the region electroporated with Ctip2 when compared with either the control electroporation or the corresponding region in the contralateral hemisphere. (c) Scheme showing the two regions in the Unc5C promoter where Ctip2 was predicted to bind. (d) ChIP from P0 cortex of wild-type and Satb2−/− mutants show that Ctip2 binds to the Unc5C promoter region. 2.78±0.2722-fold enrichment for E2 compared with 1.23±0.73 for E1 in Satb2−/−, n=3, P-value=0.027 (Student’s t-test) and 1.67±0.42-fold enrichment for E2 compared with 1.18±0.14 for E1 in wild type n=4, NS. (e) Luciferase assay to demonstrate that Ctip2 binds to Unc5C genomic region and represses its expression. In the presence of full-length Ctip2, 1.9- and 1.7-fold decrease in luminescence ratio was observed in case of E1 and E2, respectively. E1 and E2 being two putative Ctip2-binding sites. All *P-values were ≤0.05, Student’s t-test n=3. Scale bar, 100 μm.
