Sustained interactive Wnt and FGF signaling is required to maintain isthmic identity

Fibroblast growth factor 8 (FGF8) is expressed at the mid-hindbrain boundary and is an important signal emanating from the isthmic organizer. Wnt1 is expressed in the caudal midbrain juxtaposed to Fgf8 expression and has been implicated in its regulation. In this study, we examine the requirement for continuous Wnt signaling in the maintenance of Fgf8 expression at the isthmus. We demonstrate that prior to HH10, ongoing Wnt signaling is required to maintain the normal pattern of isthmic Fgf8 expression in ovo. Similarly, in explant assays, sustained Wnt signaling is essential to maintain Fgf8 expression in rhombomere 1. The mechanism by which Wnt signaling regulates isthmic Fgf8 expression is likely to be a maintenance response rather than an inductive effect. Finally, we show that Wnt maintenance of Fgf8 expression is dependent upon positive feedback by FGF signaling itself, and that rhombomere 1 does not receive instructive cues from the posterior hindbrain. In summary, these findings establish that a sustained reciprocal interaction between Wnt and FGF signaling is essential to maintain isthmic identity.