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The discovery of "Legless" and "Pygopus" in the group of Professor Basler
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| Normal facet eye of fruit fly |
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| Fly overexpressing the Wingless protein |
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| Effect can be dampened by reducing the amount of Wingless signal transducing components (e.g. legless). |
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Genes that play a role in developmental signaling pathways can be identified through so-called "mutagenesis screens". The principle of these screens is to randomly induce mutations in flies and check which of these disturb the biological process to be studied. The mutated gene can then be identified and isolated. The final proof that the correct gene was isolated is obtained by reconstituting mutant flies with an artificial "transgene" that can substitute the previously lost genetic information.
Erich Brunner, a former Ph.D. student in Basler's laboratory, used female flies carrying a "reporter transgene" that led to expression of the Wingless protein in the fly's compound eye, a site where it is normally not present. The resulting pathway activation disrupts the normally regular arrangement of the eye, leading to a rough eye. He then crossed these females to males with randomly mutated sperm cells. If by chance a mutation in the sperm cell results in a disabled signal transducing protein, the wingless signal transduction is weakened. This results in a smoother eye compared to the female mutant's eyes. In a next step Erich looked at about 200,000 offspring from such a cross, and looked for those few individuals that had smoother eyes than their siblings. He found five such flies among the 200,000 and could show that they all carried mutations in the same gene. He isolated the responsible gene and bred flies that express only the mutated version of this gene. As all of these flies displayed a defect in leg development, Erich called the gene "legless".
A second graduate student, Oliver Peter, identified and isolated the affected gene, and determined the complete gene sequence. Computer-analysis of the protein structure revealed some similarity with a human gene called B cell lymphoma 9 or BCL9, which had formerly been shown to be over-expressed in certain forms of immune system cancer. Unfortunately, nothing was known about putative molecular functions of the BCL9 protein to reveal more about Legless, which could provide a clue as to the function of Legless.
Following on from Oliver, Thomas Kramps joined Konrad Basler's group, and started to characterize the function of the Legless protein. Thomas looked for available pathway components that might associate with Legless, and found that it bound to Armadillo, the central Wingless signal transducer. Normally, Armadillo is rapidly degraded in a cell through APC and other proteins. Upon wingless signaling, however, it is stable and can modify gene activity. For completing this task, Armadillo apparently needs the help of Legless. Genetic evidence suggested that there was yet another Legless-binding protein besides Armadillo. Thomas identified this second partner and named it Pygopus (due to an Australian legless lizard that exemplifies some of the phenotypic defects in mutant flies). Further studies in flies showed that the only important function of Legless is to effectively tether Pygopus to Armadillo, and that this function is absolutely required for activating genes in response to Wingless signals. Most likely, the same situation occurs in humans, where homologues of these proteins exist. In fact, Thomas succeeded in showing that the corresponding human genes are completely equivalent in function to the fly's genes.
The requirement of Legless' and Pygopus' associating with Armadillo or its human counterpart b-Catenin opens new ways for developing drugs that might interfere with Wingless signaling in disease. Once an over-activating event such as the loss of the APC gene has occurred in a human tissue, for example in the colon, b-Catenin can only be stopped by inactivating the accessory protein machinery that it depends on. As all three proteins, b-Catenin, Legless and Pygopus form part of a ternary complex, ending their association through administering drugs is a simple and promising concept for therapy.
Thomas Kramps
Reference:
Kramps, T., Peter, O., Brunner, E., Nellen, D., Froesch, B., Chatterjee, S., Murone, M., Züllig, S., and Basler, K.
"Wnt/Wingless Signaling Requires BCL9/Legless-Mediated Recruitment of Pygopus to the Nuclear b-Catenin-TCF Complex"
Cell (2002), in press -> PDF |
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