3385_Ch20 - Chapter 20 Wnt Signaling in Xenopus Embryos...

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20 © 1999 by CRC Press LLC Chapter Wnt Signaling in Xenopus Embryos François Fagotto Contents I. Introduction A. Generalities B. A Summary of Early Patterning During Development C. Molecular Nature of the Inducers D. The Wnt Pathway II. Experimental Manipulations of Axis Formation III. Testing Dorsalizing and Ventralizing Activities by Injection of mRNA IV. Interfering With Endogenous Components: An Overview V. Preparation of Embryo Extracts VI. Subcellular Distribution: Microscopic and Biochemical Methods VII. Detecting Molecular Interactions VIII. Detecting GSK Activity IX. Analyzing β -Catenin Stability X. Detection of Gene Expression XI. Concluding Remarks mRNA Concentrations, Expression Levels, Activity Levels This is Not a “Clean” System! Further Considerations on Expression/Overexpression Experiment Taking Advantage of a Complex System: “Playing” on Different Backgrounds Acknowledgments References
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© 1999 by CRC Press LLC I. Introduction A. Generalities The very same conserved signal transduction pathways active in adult differentiated cells also regulate embryonic development. Inhibition of a pathway, or its ectopic activation, will often perturb embryonic patterning and cause morphological abnor- malities. Because such phenotypes can be readily observed and are highly repro- ducible, they constitute an exquisite read-out for the activity of the underlying signaling pathways. One spectacular phenotype in Xenopus embryos is obtained by modulating the maternal Wnt pathway, involved in early specification of the dorsoventral axis: inhibition of the pathway leads to loss of body axis, while its ectopic activation causes formation of a complete second body axis ( Figure1 ). Several laboratories have exploited this system extensively to dissect the molecular mechanism of the Wnt pathway. embryos are particularly well fitted for this purpose: straight- forward mRNA injections allow rapid screens of mutants and coexpression of various molecules in various doses and combinations. Only a few of these large embryos are sufficient for analysis by standard biochemical/cell biological tech- niques, and molecular data can be directly related to functional analysis (phenotype read-out). Because the stage and localization of signaling activities is known, it is possible to test signaling molecules in various contexts (e.g., upstream signal “on” or “off”), and determine possible changes in molecular interactions, cellular distri- bution, posttranslational modifications, and activity. Finally, despite the lack of genetics, and thus of real “knock-outs” in Xenopus, several methods are now avail- able to deplete or inhibit specific molecules. This chapter contains a short update on early induction in Xenopus and on the Wnt pathway, provides guidelines and strategies to characterize and analyze components of FIGURE1 The Wnt pathway regulates axis formation in embryos. A. Ectopic activation of the pathway (here by expression of an Axin dominant negative construct) in the ventral side of early cleaving embryos results in induction of a secondary axis. B. Inhibition of the endogenous pathway (here by overexpression
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3385_Ch20 - Chapter 20 Wnt Signaling in Xenopus Embryos...

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