Activation of Ras-ERK pathway by Fgf8 and its downregulation by Sprouty2 for the isthmus organizing activity.

In the previous studies, we showed that strong Fgf8 signaling activates the Ras-ERK pathway to induce cerebellum. Here, we show importance of negative regulation following activation of this pathway for proper regionalization of mesencephalon and metencephalon in chick embryos. 'Prolonged' activation of ERK by misexpression of Fgf8b and dominant-negative Sprouty2 (dnSprouty2) did not change the fate of the mesencephalic alar plate. Downregulation of ERK activity using an MEK inhibitor, U0126, or by tetracycline-dependent Tet-off system after co-expression of Fgf8b and dnSprouty2 forced the mesencephalic alar plate to differentiate into cerebellum. We then paid attention to Mkp3. After misexpression of dnMkp3 and Fgf8b, slight downregulation of ERK activity occurred, which may be due to Sprouty2, and the mesencephalon transformed to the isthmus-like structure. The results indicate that ERK must be once upregulated and then be downregulated for cerebellar differentiation and that differential ERK activity level established by negative regulators receiving Fgf8 signal may determine regional specificity of mesencephalon and metencephalon. Copyright 2009 Elsevier Inc. All rights reserved.