Regulation of BMP-4/Smad1 Signals by MAP Kinase During Xenopus Neural Specification

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0114476SaterThe proposed work addresses the cellular mechanisms underlying neural specification in Xenopus. Neural fate is inhibited by BMP-4, and induction of anterior neural ectoderm is initiated by extracellular BMP-4 antagonists such as noggin and chordin. FGF has been implicated in the initiation of neural development; current findings indicate that the FGF effector MAP kinase is necessary for neural specification. The inhibitory effects of BMP-4 are mediated by transcriptional repression: expression of key neural-specific genes is initiated prior to gastrulation and later inhibited in non-neural ectoderm by BMP-4. The recent finding that MAP kinase can inhibit Smad1 in mammalian cells suggests that MAP kinase may antagonize BMP-4/Smad1 signals during neural specification in vivo. The proposed work tests this hypothesis, and examines the functional significance of these interactions. The studies in Aim I will determine whether transcriptional regulation via endogenous Smad1 is affected by alterations in MAPK activity. The studies in Aim II will use overexpression of Smad1 proteins carrying mutations at MAPK sites that prevent inhibitory phosphorylation. These studies will determine whether inhibitory phosphorylation of MAPK sites in Smad1 participates in neural specification and patterning. They will also address the hypothesis that inhibition of Smad1 is necessary for posterior neural specification. The studies in Aim III will determine whether antagonistic interactions between the FGF/MAPK and BMP-4 pathways can affect boundaries of gene expression within gastrula ectoderm. These studies will examine the spatial patterns of activation of these pathways, and of Smad1-dependent gene expression, in ectoderm exposed to localized sources of FGF and/or BMP-4. This investigation will compare the pattern of Smad1-dependent transcription in ectoderm overexpressing WT-Smad1 with that of ectoderm expressing a mutant Smad1 impervious to MAPK. The comparison will show whether antagonistic interactions contribute to the establishment of spatial boundaries. Preliminary findings indicate that MAPK and BMP-4 signals are mutually antagonistic in Xenopus ectoderm: mutations in Smad1 that prevent inhibtion by MAP kinase act collectively as a gain-of-function mutation, while BMP-4 inhibits MAP kinase activity via a TAK1-dependent pathway. Since BMPs and FGFs have opposing effects in many developmental processes, analysis of the interactions between these pathways may elucidate a key cellular mechanism of developmental regulation.