Differential and dose-dependent regulation of gene expression at the mid-hindbrain boundary by Ras-MAP kinase signaling.

Recent experiments suggest that activation of the Ras-MAP kinase pathway at the mid-hindbrain boundary (MHB) induces cerebellar development, whereas tectal development occurs in the absence of Ras-MAP kinase activity. To test this model we have stimulated or inhibited Ras-MAP kinase signaling in chick embryos through targeted misexpression of a constitutive active (Ras(V12)) or dominant negative (Ras(N17)) form of Ras. The consequence of these manipulations on the expression of several genes that are expressed in distinct patterns at or around the MHB organizer, including En1, Pax2, Pax3, Pax5, Wnt1, Meis2, and ephrin-A2, -A5, and -B1, was assessed. Extending previous findings we show that inhibition of Ras-MAP kinase signaling differently affects Pax3 expression in different regions of the mid-hindbrain territory, inhibiting its expression in the midbrain but inducing it in the MHB region. Expression of the midbrain specific marker gene Meis2 was not affected by Ras(N17) at first but later upregulated concomitantly with the morphological transformation of hindbrain to midbrain. In addition, we show that different dosages of Ras-MAP kinase activity are required for transcriptional activation of Wnt1 and En1 at the MHB. Collectively, these results validate and extend previous findings on the molecular changes associated with Fgf8 loss-of-function or gain-of-function phenotypes at the MHB, demonstrate that gene expression at the MHB is regulated by Ras-MAP kinase signaling in a spatially and temporally distinct manner and provide evidence for a dosage dependent function of Fgf8 signaling at the MHB.