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Analysis of signalling networks during the generation of unique cell fates in animal development

Final Activity Report Summary - SIGNALING NETWORKS (Analysis of signalling networks during the generation of unique cell fates in animal development)

The principal aim of this project was to unravel the functional signalling networks established within the cell during the specification of cellular identities. Nowadays, it has become apparent that signal transduction pathways do not act individually, as linear cascades. Conversely, complex signalling networks with multiple nodes of cross-talk between signalling pathways are established. Such networks must be highly regulated to allow specific and accurate cell responses. However, the mechanisms by which signal transduction pathways are integrated and modulated, within these networks, are poorly understood. Thus, to comprehend the acquisition of unique cell fates during development, as well as the failures that underlie many diseases, it is crucial to further investigate the functional signalling networks that are built and regulated in vivo. The nodes of crosstalk between signalling cascades within these networks constitute key points of regulation.

PDZ (PSD-95, Dlg, ZO-1) domains are involved in protein-protein interactions. Multiprotein complex formation around PDZ-based scaffolds at specific sub-membrane locations is known to be decisive for signal transduction rate and fidelity. Hence, PDZ proteins are excellent candidates as modulators of signalling networks. In this project, we aimed to investigate the function of the Drosophila PDZ domain-containing protein Canoe/AF-6 and other PDZ proteins as key nodes of regulation of signalling networks. Specifically, we carried out this analysis during cell identity generation throughout development, in processes such as progenitor cell specification and asymmetric cell division.

We have shown that, during the process of muscle/heart progenitor specification, the PDZ protein Canoe/AF-6, a PDZ protein normally associated with cellular junctions, is a key modulator of Wingless/Wnt, Ras-MAPK and Notch signalling pathways cross-communication. Our data show a repressive effect of Canoe/AF-6 on these three highly conserved signalling pathways through physical interactions with activated Ras, Notch and the cytoplasmic protein Dishevelled, another PDZ protein and key Wingless/Wnt effector. We propose a model in which Canoe/AF-6, through those interactions, actively coordinates at the membrane level Ras-MAPK, Notch and Wingless/Wnt signalling pathways during progenitor specification. This work was published in PLoS ONE (PLoS ONE, 2006, 1(1): e66. doi:10.1371/journal.pone.0000066).

Our results also strongly support a new function for the PDZ protein Canoe/AF-6 during the asymmetric division of neuroblasts, the neural stem cells of the Drosophila central nervous system. This work shows how Canoe/AF-6 is functionally networking with different proteins previously identified as key elements during neuroblasts asymmetric division. For example, we found that Canoe/AF-6 acts as a key effector of the proteins Inscuteable, Partner of Inscuteable and the subunit Ga of the heterotrimeric G proteins. As all these proteins, Canoe/AF-6 is required to asymmetrically distribute cell-fate determinants, to properly orientate the mitotic spindle and to generate unequal-sized daughter cells, the most characteristic features of an asymmetric cell division. This work is in preparation for its publication. Finally, we also have the results of a yeast two-hybrid screen in which we used Canoe as bait to isolate new partners of Canoe. We have to reassess the positive clones to confirm direct interactions with Canoe. Then, we will further characterise the selected clones and analyze them in the context of the processes we are analysing to determine its functional relevance. This opens a lot of new work for the future.