Fig. 5. FGF8 delays RP maturation. Posterior (A-E) and lateral (F-G) views of HH14-15 (A-C), HH16-17 (D-F') and HH18 (G) chick embryos treated by in situ hybridization for the detection of RP markers. The expression of noggin (A), Id3 (B) and Gdf7 (C) is transiently downregulated in the caudal midbrain at stage HH14-15 (arrowheads). A mature expression pattern of Gdf7 is observed in the caudal midbrain, beginning at stage HH16 (D). Insertion of a FGF8-soaked bead (E, red dot) in the caudal midbrain prolongs Gdf7 downregulation beyond HH17. (F,G) The same delay in Gdf7 expression is observed on the ectopic RP induced by FGF8 beads (red dots in F,G). Embryos fixed 24 (F,F') or 30 (G) hours after FGF8 bead insertion were treated for the detection of Gdf7 in purple and Wnt1 in red. (F,F') Same embryo shown in bright and dark field. At 24 hours, an ectopic preRP is already labeled for Wnt1 (F,F'), but Gdf7 expression lags behind (F). At 30 hours Gdf7 and Wnt1 are co-expressed in the induced RP (G). (H-J) The hypothetical consequences of an imbalance between the positive and negative influences of FGF8 on RP differentiation. The competence factors Lmx1b and Wnt1 (purple) are targets of FGF8 signaling; their expression is sufficient to induce slowly maturing RP markers (see Fig. 4). Conversely, high levels of FGF8 signaling (red) inhibit the expression of RP maturation markers. (H) A balance between these two influences of FGF8 maintains a progress zone of RP maturation (orange). (I) An imbalance in favor of competence factors should result in RP widening, whereas (J) increasing the inhibitory activity of FGF8 at the expense of competence results in the formation of gaps in the developing RP. We propose that the lack of a RP structure in the caudal part of rotated midbrain vesicles (Alexandre and Wassef, 2003; Marin and Puelles, 1994) is the consequence of this last configuration.