The Role of MAP Kinase in Neural Specification in Xenopus

对非洲爪蟾神经诱导分子机制的新研究已经确定了神经命运的积极和消极调节因子。其中包括神经抑制因子骨形态发生蛋白-4 （BMP-4）及其拮抗剂，神经诱导剂noggin和chordin，它们被认为通过结合和隔离BMP-4起作用。此外，一些研究表明碱性成纤维细胞生长因子（bFGF）可诱导后神经外胚。由于FGF信号主要通过丝裂原活化蛋白激酶（MAP激酶）途径转导，这些结果暗示MAP激酶参与神经诱导反应。尽管取得了这些进展，但神经命运和前后神经模式建立的分子机制仍然知之甚少。本实验室的初步结果提供了具有挑衅性的证据，表明MAP激酶途径调节原胚外胚层神经命运的承诺。先前的研究表明，外胚层细胞在内源性诱导信号的作用下，细胞内pH值（pHi）增加。最近的研究结果表明，碱化对于原肠胚外胚层中前神经特异性基因表达的启动是必要和充分的（Uzman等人，已提交）。此外，神经诱导和人工碱化未诱导的外胚层都导致MAP激酶磷酸化和活性比未诱导的外胚层在同一阶段增加。初步数据进一步表明，在过表达MAP激酶磷酸酶-1的诱导外胚层中，神经特异性基因的表达大大减少或缺失，该基因在体内使MAP激酶去磷酸化和失活。这些发现，结合早期对bFGF诱导作用的研究，提出了以下两种假设：1)MAP激酶在原肠形成过程中的神经规范中起关键作用；2)新诱导的神经外胚层中MAP激酶活性水平的升高导致后侧位置认同的增加。本研究旨在探讨MAP激酶在爪蟾神经命运和前后模式建立中的作用。第一组实验将通过量化未诱导外胚层的MAP激酶活性，确定神经命运和MAPK活性之间是否存在相关性，这些外胚层接受了已知的神经命运调节因子。第二组将通过检查外胚层的神经规范来测试神经诱导对MAP激酶活性的要求，其中MAP激酶信号被MKP-1的过度表达破坏。第三项研究将探讨MAP激酶在原胚外胚层的激活是否足以导致神经规范，以及在新诱导的神经外胚层中，MAP激酶活性的升高是否会导致位置身份的后移。最后，第四项研究将确定MAP激酶通路是否在细胞内碱化反应中激活前神经特异性基因表达。这项工作将揭示细胞内调节机制的基础上建立的神经命运。

Sater 97-23183 The renewed investigation of molecular mechanisms underlying neural induction in Xenopus has identified both positive and negative regulators of neural fate. These include the neural repressor Bone Morphogenetic Protein-4 (BMP-4) and its antagonists, the neural inducers noggin and chordin, which are thought to act by binding and sequestering BMP-4. Moreover, several studies have shown that basic Fibroblast Growth Factor (bFGF) can induce posterior neural ectodemm. Since FGF signals are transduced primarily by the Mitogen-Activated Protein Kinase (MAP kinase) pathway, these results implicate MAP kinase in the response to neural induction. Despite these advances, the molecular mechanisms underlying the establishment of neural fate and anteroposterior neural pattern remain poorly understood. Preliminary results from this laboratory provide provocative evidence that the MAP kinase pathway regulates commitment to neural fate in gastrula ectoderm. Previous work has shown that ectoderm cells undergo an increase in intracellular pH (pHi) in response to endogenous inductive signals. More recent results indicate that this alkalinization is necessary and sufficient for the initiation of anterior neural-specific gene expression in gastrula ectoderm (Uzman et al., submitted). Moreover, both neural induction and artificial alkalinization of uninduced ectoderm lead to an increase in MAP kinase phosphorylation and activity over that observed in uninduced ectoderm at the same stage. Preliminary data further show that neural-specific gene expression is greatly reduced or absent in induced ectoderm overexpressing MAP Kinase Phosphatase-1, which dephosphorylates and inactivates MAP kinase in vivo. These findinga, together with the earlier studies on the inductive effects of bFGF, suggest the following two hypotheses: 1) MAP kinase plays a key role in neural specification during gastrulation; and 2) increasing levels of MAP Kinase activity in newly induced neural ectoderm lead to an increasingl y posterior positional identity. The proposed work addresses the role of MAP kinase in the establishment of neural fate and anteroposterior pattern in Xenopus. The first set of experiments will determine whether there is a correlation between neural fate and MAPK activity by quantifying MAP kinase activity in uninduced ectoderm treated with known regulators of neural fate. The second set will test for a requirement for MAP kinase activity in neural induction by examining neural specification in ectoderm in which the MAP kinase signals are disrupted by overexpression of MKP-1. The third study will investigate whether activation of MAP kinase in gastrula ectoderm is sufficient to bring about neural specification, and whether elevating MAP kinase activity in newly induced neural ectoderm leads to a posterior shift in positional identity. Finally, the fourth study will determine whether the MAP kinase pathway underlies the activation of anterior neural-specific gene expression in response to intracellular alkalinization. This work will reveal intracellular regulatory mechanisms that underlie the establishment of neural fate.