Fig. 4. The combination of vhnf1 expression and Fgf signal is sufficient to activate posterior hindbrain gene expression. (A) The experimental method consisted of injection of the indicated RNA into one cell at the two-cell stage, then implantation of a protein-coated bead after the shield stage. Embryos were aged to about the three-somite stage and fixed for analysis. (B,C) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead resulted in significant induction of krox20 expression both within the neural plate (B) and in lateral ectoderm (C). Arrowhead indicates the location of endogenous krox20 expression. (D) Injection of vhnf1 RNA and implantation of a BSA-coated bead does not induce krox20 expression. (E) The Fgf8-coated bead alone is not sufficient to induce ectopic krox20 expression. (F) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead induces valentino (val) expression. Arrowhead indicates the location of endogenous val expression. (G) Injection of vhnf1 RNA into a val mutant embryo (no endogenous r5 krox20 expression) and implantation of an Fgf8-coated bead does not induce krox20 expression. This dorsal view of the neural plate shows that r3 krox20 is repressed by vhnf1. Arrowhead indicates endogenous r3 krox20 on the uninjected side. (H) Injection of val RNA and implantation of a Fgf8-coated bead is not sufficient for induction of krox20 expression. (I) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead results in localized repression of hoxb1a expression. (J) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead does not induce expression of the axial mesoderm marker no tail (ntl). (K) Summary of data. The combination of Fgf8+vhnf1 is sufficient to induce val and krox20 expression, and val function is required along with other Fgf+vhnf1-inducible factor(s) (X) for krox20 induction.