Organizing signal and the tectal and cerebellar differentiation

地峡就像一个组织者，Fgf8模仿了这种组织活动。峡组织者分别在中脑顶盖和后脑小脑的发育中起作用。非常有趣的是，在一个共同的信号下，顶盖和小脑的结构完全不同。我们注意到Fgf8的8个亚型中，Fgf8a和Fgf8b在峡部表达，并通过卵内电穿孔对Fgf8a和Fgf8b进行错表达。Fgf8a对间脑顶盖的诱导作用与预期一致。令人惊讶的是，Fgf8b改变了中脑鼻翼板到小脑的命运。Fgf8b抑制Otx2在中脑的表达，但诱导通常在中脑表达的Gbx2和Irx2的表达。定量研究发现，Fgf8a和Fgf8b的差异可归因于信号强度的差异。提示Fgf8的强烈信号使基因表达级联向小脑分化方向发展。然后我们对Ras-ERK通路作为Fgf8信号下游的信号转导通路感兴趣。在正常胚胎中，ERK在Fgf8 mRNA定位的位置被激活。Fgf8b激活ERK，而Fgf8a或低剂量Fgf8b在mes/metencephalon中没有激活ERK。Ras的显性阴性形式（Ras^<S17N>）对Ras- erk信号通路的破坏改变了后颅翼板从小脑到顶盖的命运。Ras^<S17N>消除了Fgf8b的作用，而Fgf8a和Ras^<S17N>共转染产生了加性作用。结果表明，假定的后脑接收到强烈的Fgf8信号，激活Ras-ERK通路并向小脑分化。

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.