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Figure 6 | Neural Development

Figure 6

From: Sp8 exhibits reciprocal induction with Fgf8 but has an opposing effect on anterior-posterior cortical area patterning

Figure 6

Sp8 directly binds the Fgf8 promoter and Emx2 represses Sp8 induction of Fgf8. (a) Sequence of a 585 bp fragment, containing the 555 bp immediate upstream region of the mouse Fgf8 transcription start site and the 30 bp 5' UTR of Fgf8 having six putative Sp8 binding sites. Putative Sp8 binding sites predicted from Sp1-binding motifs (GGGGCGG or CCCCGCC) are underlined. (b) Gel retardation assay of Sp8 and Fgf8 promoter fragments. P32-labeled Fgf8 promoter fragments show slow mobility after incubation with the Sp8-expressed lysate (black arrow) compared to the unbound DNA (gray arrow). These bands are not detected in the control lysate sample and are diminished in the presence of an equal (×1) or five-fold (×5) amount of unlabeled DNA compared to labeled probes. (c) Sp8 binding to the oligonucleotide of the Fgf8 promoter region. HA-Sp8 was co-precipitated with biotinylated non-mutated oligonucleotide corresponding to a putative Sp8 binding site, but not with oligonucleotides with a mutated core recognition sequence. (d) Luciferase reporter assay for induction of Fgf8. Control or test vectors (Sp8, Sp9, Emx2) were transfected independently or in combinations into C3H10T1/2 cells also transfected with an Fgf8 reporter construct consisting of a promoter element for Fgf8 and a firefly luciferase reporter vector. The relative effectiveness of Fgf8 induction was assessed by measuring luciferase activity. Sp8 robustly induces expression of the Fgf8 reporter construct; this Sp8 induction is suppressed by co-expression of Emx2. Sp9 modestly induces expression of the Fgf8 reporter construct; again, this is suppressed by Emx2. Emx2 alone has no effect on Fgf8 induction, similar to a control empty vector.

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