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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 dngsk3β 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.
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