Inductive signal and tissue responsiveness defining the tectum and the cerebellum.

The mes/metencephalic boundary (isthmus) has an organizing activity for mesencephalon and metencephalon. The candidate signaling molecule is Fgf8 whose mRNA is localized in the region where the cerebellum differentiates. Responding to this signal, the cerebellum differentiates in the metencephalon and the tectum differentiates in the mesencephalon. Based on the assumption that strong Fgf8 signal induces the cerebellum and that the Fgf8b signal is stronger than that of Fgf8a, we carried out experiments to misexpress Fgf8b and Fgf8a in chick embryos. Fgf8a did not affect the expression pattern of Otx2, Gbx2 or Irx2. En2 expression was upregulated in the mesencephalon and in the diencephalon by Fgf8a. Consequently, Fgf8a misexpression resulted in the transformation of the presumptive diencephalon to the fate of the mesencephalon. In contrast, Fgf8b repressed Otx2 expression, but upregulated Gbx2 and Irx2 expression in the mesencephalon. As a result, Fgf8b completely changed the fate of the mesencephalic alar plate to cerebellum. Quantitative analysis showed that Fgf8b signal is 100 times stronger than Fgf8a signal. Co-transfection of Fgf8b with Otx2 indicates that Otx2 is a key molecule in mesencephalic generation. We have shown by RT-PCR that both Fgf8a and Fgf8b are expressed, Fgf8b expression prevailing in the isthmic region. The results all support our working hypothesis that the strong Fgf8 signal induces the neural tissue around the isthmus to differentiate into the cerebellum.