It has long been recognized that cells in a developmental field respond to changes in position by organizing growth and changes in developmental fate according to positional information which is derived and interpreted via interactions with their neighbors"""""""". Understanding the molecular basis of those interactions is central to understanding the impressive ability of tissues to respond to a myriad of insults by repairing and regenerating complex structures. The elegant and orderly process of regeneration provides clues to the mechanisms of pattern formation but also offers the hope that the process might one day be manipulated to replace damaged body parts. To manipulate the process, it will be necessary to understand the genetic basis of the process. In the case of the insect leg, we are coming close to such a level of understanding and many of the lessons learned are relevant to vertebrate systems. A dynamic web of gene regulatory networks appears to create a robust self organizing system that is at once extremely intricate but also perhaps simple in its reliance on a few key signaling pathways and a few simple processes e.g. autoactivation and cross inhibition. The focus of this application is to understand the mechanisms and the logic of these processes at the molecular as well as tissue levels. Studies during the last period of this project reveal that two key morphogens, wg and dpp, form a self organizing system by suppressing each other's expression and perhaps by autoactivating their own expression. We have found that the repression of dpp expression by Wg is direct and mediated by dTcf and have identified sites in the wg regulatory region that mediate its autoactivation and repression by dpp. Here we propose to continue our studies to determine whether the mechanisms of autoactivation and cross inhibition are direct or indirect. We propose to determine the biochemical specificity and function of different dTcf isoforms to better understand the role of this protein in these regulatory events. We will seek to identify a putative co-repressor. Preliminary experiments suggest a new level of complexity in the response to positional confrontations alluded to by Wolpert. Namely, cells that are unable to respond to Wg are still able to adopt what are traditionally thought of as Wg dependent cell fates although this is revealed only if production of ectopic morphogens is blocked. This raises possibilities with respect to alternative mechanisms of cell fate determination and regeneration responses that will be tested. Finally, we have identified a collection of mutants affecting disc growth patterns that serve as an enriched collection of new candidate genes to participate in these events.
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