Fig. 6

FGF signaling is required during the late blastula period to regulate BMP transcription. Embryos were injected with iFGFR-1 and activated with AP20187 at the 512-cell stage. Embryos were collected at early and mid-gastrula stages for real-time PCR. Embryos at early gastrulation (shield stage) (A) were tested for their fold change (y-axis) of bmp2, bmp4, and bmp7 (A), and for their expression of otx2, hoxb1b, and gata2 (B) at mid-gastrulation (70% epiboly). Activating FGF signaling at the 512 cell stage results in an early decrease in bmp2, bmp4, and bmp7 expression at shield stage. At mid-gastrulation, these embryos have increases in otx2 and hoxb1b transcript levels and a decrease in gata2 levels. (C) FGF signaling was inhibited with the pharmacological drug SU5402 at 8-cell, 128-cell, 512-cell, early blastula, and late blastula, and collected at shield stage (early gastrula) for real-time PCR analysis. Graphed is the fold change (y-axis) for bmp2, bmp4, and bmp7 levels relative to control-treated embryos. These results show that the effectiveness of the SU5402 treatment in blocking repression of BMP transcript levels diminishes between the 512-cell and early blastula (sphere stages). (D) FGF signaling was either inhibited or activated with SU5402 or AP20187 at the 512-cell stage. Embryos were collected at the shield stage (early gastrula) for their fold change (y-axis) of vox and eve1. Activating FGF signaling results in a 25% and 50% reduction in transcript levels for vox and eve1, while inhibiting FGF results in a 30% and 50% increase in vox and eve1 transcript levels. These results show that inhibiting or activating FGF at the 512-cell stage results in loss of BMP target expression.