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

0114476Sater拟议的工作解决了非洲爪哇神经规范背后的细胞机制。BMP-4抑制神经命运，细胞外BMP-4拮抗剂如noggin和chordin启动前神经外胚层的诱导。成纤维细胞生长因子参与了神经发育的启动；目前的研究结果表明，成纤维细胞生长因子效应蛋白激酶是神经指定所必需的。BMP-4的抑制作用是通过转录抑制介导的：关键的神经特异基因的表达在原肠形成之前启动，然后在非神经外胚层中被BMP-4抑制。最近发现MAP能在哺乳动物细胞中抑制Smad1，提示MAP可能在体内神经分化过程中拮抗BMP-4/Smad1信号。这项拟议的工作检验了这一假设，并检验了这些相互作用的功能意义。AIM I的研究将确定内源性Smad1的转录调控是否受到MAPK活性变化的影响。在AIM II上的研究将使用在MAPK位点上携带突变的Smad1蛋白的过度表达，以防止抑制磷酸化。这些研究将确定Smad1中MAPK位点的抑制性磷酸化是否参与神经规范和模式形成。他们还将提出这样一种假设，即抑制Smad1是后脑神经规范所必需的。AIM III的研究将确定成纤维细胞生长因子/MAPK和BMP-4通路之间的拮抗性相互作用是否会影响原肠胚外胚层中基因表达的边界。这些研究将考察暴露于局部来源的成纤维细胞生长因子和/或骨形态发生蛋白-4的外胚层中，这些通路的激活以及Smad1依赖的基因表达的空间模式。这项研究将比较高表达WT-Smad1的外胚层和表达突变的Smad1不受MAPK影响的外胚层中Smad1依赖的转录模式。这种比较将显示出对抗相互作用是否有助于空间边界的建立。初步研究结果表明，在非洲爪哇外胚层中，MAPK和BMP-4信号是相互拮抗的：Smad1中阻止MAP激酶抑制的突变共同起到功能获得突变的作用，而BMP-4则通过TAK1依赖的途径抑制MAP激酶的活性。由于BMP和FGFs在许多发育过程中具有相反的作用，分析这些途径之间的相互作用可能会阐明发育调控的一个关键细胞机制。

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.