Mouse anti-Human EN2 monoclonal antibody, clone 2F2

The mid-hindbrain boundary (MHB) organizer is among the best studied local organizers that patterns the midbrain and cerebellum in vertebrates and is established by a regulatory network of several transcription factors and signals, including Wnt signaling. In the present study, we found that Xenopus Nkx6.1 and Nkx6.2, two transcription factors of the Nkx homeobox family, are required for MHB formation. In Xenopus embryos, nkx6.1 and nkx6.2 are expressed in the MHB, and knockdown of either nkx6.1 or nkx6.2 by specific morpholinos disrupts the MHB expression of en2 as well as wnt1, a key regulator for vertebrate MHB formation. In the nkx6.1/nkx6.2 morphants, co-injection of wnt1 or dngsk3 beta mRNA, which activates Wnt signaling, rescues the expression of en2. Nkx6.1 and Nkx6.2 activate canonical Wnt signaling in reporter assays in both cultured mammalian cells and Xenopus embryos. An Nkx6.2 deletion construct, which inhibits the ability of wild type Nkx6.2 to activate Wnt signaling, also reduces the MHB expression of en2 in Xenopus embryos. These results suggest that Nkx6.1 and Nkx6.2 are involved in MHB formation in Xenopus embryos, likely by modulating wnt1 expression.

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. Cotransfection 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.