Meningeal Cxcl12 expression is reduced in Foxc1hith/hith mutants and directly regulated by Foxc1. (A, B) Cxcl12 expression analysis on coronal sections of wild-type (WT) and Foxc1hith/hith embryos at E13.5 shows transcripts in the meninges, cortical SVZ/IZ, and periocular mesenchyme (green asterisks). Meningeal Cxcl12 expression is reduced or absent (red asterisk in (B)) throughout the forebrain meninges of the mutant embryo while expression in the cortical SVZ/IZ and facial mesenchyme is not affected (green asterisks in (B)). (C) A scheme of the Cxcl12 gene with exons shown as boxes and the coding region darkened. The sequence of the fragment amplified in the ChIP analysis is shown below with the reverse complement of the Foxc1 binding consensus sequence GTAAATAAA highlighted in red. (D) The results of the ChIP analysis where in lanes 1 and 2 the fragment containing the Foxc1 binding site was amplified from DNA templates pulled down with two different Foxc1 antibodies. Genomic DNA was used in the positive control (lane 3), DNA pulled down with an IgG antibody in the negative control (lane 4), and no template DNA in lane 5. (E, F) Transfection of meningeal fibroblasts in primary culture with an expression construct for Foxc1 (F) and control plasmid (E). (G) Illustration of the parameters used to quantify the average ratio of Cxcl12 immunofluorescence signal intensity [3xFLAG-Foxc1 (Average) = A/(B1 + B2 + B3)/3] or maximum ratio of signal intensity [3xFLAG-Foxc1 (Max) = A/Max (B1 + B2+ B3)]. A, Foxc1-transfected cell; B1 to B3, neighboring not transfected cells. (H) The results of the analysis and significant upregulation of Cxcl12 in 3xFLAG-Foxc1-transfected cells. Error bars indicate the average deviation. Scale bars: 200 μm (A, B); 20 μm (D, E). Cx, cerebral cortex; LGE, lateral ganglionic eminence; Me, meninges.