The Role of MAP Kinase in Neural Specification in Xenopus

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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.