Organizing signal and the tectal and cerebellar differentiation

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The isthmus works as an organizer, and Fgf8 mimics the organizing activity. The isthmic organizer plays a role for the development of the tectum and the cerebellum in the mesencephalon and in the metencephalon, respectively. It is very interesting that completely different architecture of the tectum and the cerebellum differentiates under a common signal. We paid attention to that among 8 isoforms of Fgf8, Fgf8a and Fgf8b are expressed in the isthmus, and carried out misexpression of Fgf8a and Fgf8b by in ovo electroporation. Fgf8a induced the tectum in the diencephalon as expected. It was surprising that Fgf8b changed the fate of the mesencephalic alar plate to the cerebellum. Fgf8b repressed Otx2 expression in the mesencephalon, but induced expression of Gbx2 and Irx2, which are normally expressed in the metencephalon. The quantitative study revealed that the difference between Fgf8a and Fgf8b could be attributed to the difference in the intensity of the signal. It was suggested that strong signal of Fgf8 turns the gene expression cascade toward the cerebellar differentiation.We were then interested in Ras-ERK pathway as the signal transduction pathway downstream of the Fgf8 signal. In normal embryos ERK is activated at the site where Fgf8 mRNA is localized. Fgf8b activated ERK while Fgf8a or lower dose of Fgf8b did not activate ERK in the mes/metencephalon. Disruption of the Ras-ERK signaling pathway by dominant negative form of Ras (Ras^<S17N>) changed the fate or the metencephalic alar plate from cerebellum to tectum. Ras^<S17N> canceled the effects of Fgf8b, while co-transfection of Fgf8a and Ras^<S17N> exerted additive effects. The results indicate that the presumptive metencephalon receives strong Fgf8 signal that activates the Ras-ERK pathway and differentiates into the cerebellum.