Figure 6: DCC is negatively regulated by Satb2 and is upregulated in the Satb2−/− cortex.
From: Unc5C and DCC act downstream of Ctip2 and Satb2 and contribute to corpus callosum formation
(a) Immunohistochemical staining for DCC in rostral and caudal sections shows an upregulation of DCC expression in Satb2−/− mutant brains as compared with wild-type littermates. Arrows point to the region of altered DCC expression between wild-type and Satb2−/− sections. ISH reflects an upregulation of DCC in E18.5 Satb2−/− cortex when compared with Satb2 heterozygous brains. (b) Series of images show DCC expression in E14.5 wild-type embryos in cells electroporated with either Satb2/Ski/EGFP at E11.5 or with EGFP alone at E12.5. GFP IHC delineates the region electroporated. DCC ISH demonstrates a downregulation of DCC mRNA within cells electroporated with Satb2/Ski compared with DCC expression in the contralateral hemisphere, or in the control electroporation. (c) Schematic diagram showing two putative binding MAR sites for Satb2 upstream of the DCC transcription-initiation site. ChIP from P0 cortices showed a 5.27±2.75-fold enrichment in n=6 wild-type compared with 1.72±0.015 in n=2 Satb2−/− mutants for DCC MAR1, *P-value=0.025 (Student’s t-test) and for DCC MAR2, n=7 wild-type showed a range of 0.91–10.83-fold enrichment with an average of 4.69±4.38, as compared with 1.47±0.39 in n=2 Satb2−/− mutants, P-value=0.10 (Student’s t-test). (d) Luciferase assay to demonstrate that Satb2 binds to the DCC genomic region and represses its expression. In the presence of full-length Satb2, DCC MAR1 showed a 1.5-fold decrease in luminescence ratio while DCC MAR2 did not shown any change (Student’s t-test, **P-value=0.0035). Scale bar, 100 μm, unless specified differently in the figure.
