GROWTH AND PATTERN · Coordinated Control of Patterning and Growth: Mechanisms of Transcriptional Regulation and Dynamics of Dpp Signaling in Flies

Coordinated control of patterning and growth: mechanisms of transcriptional regulation and dynamics of Dpp signaling in flies

My proposal was formed of two parts. Below I summarise what I have proposed and report the current progress for both parts. Proposed aim I: Test the hypothesis that the T-box genes dorsocross 1, 2 and 3 encode cofactors of Mad/Medea-mediated transcription Dorsocross 1, 2, and 3 (Docs) are direct targets of Dpp signalling with expression patterns similar to that of Dpp in multiple tissues and their loss-of-function phenotypes also resemble that of Dpp. Therefore, we will test the hypothesis that Docs might be the long sought after co-factors for Mad and Medea, the effectors of Dpp mediated transcription in Drosophila. First, we will screen known Dpp targets and determine whether they are co-regulated by Docs. Then, we will identify target genes that are directly regulated by Docs as well as Mad using a genome-wide approach. And, finally, we will test if Docs can facilitate Mad-mediated transcription off of candidate promoters. When the proposed experiments are completed, we will have clarified the connection between Docs and Dpp, and will have acquired a better understanding of transcriptional regulation by the T-box family. Results: So far, I have completed the first part of this aim. I have recombined a deficiency that removes all three Doc genes onto an FRT chromosome and using this tool performed clonal analysis in wing imaginal discs. I have found that the known target genes of Dpp are not co-regulated by Dorsocross genes. This finding does not support our initial hypothesis, but does not rule it out either. Additionally, I observed that the clones of cells lacking Doc function don't have a growth disadvantage as they grow similarly to their twin spots. It is possible that although expressed there Docs may not have any critical function in wing imaginal discs. Proposed aim II: Analysis of the dynamics of Dpp signalling in vivo Dpp regulates its target genes via various means (activation versus repression; different threshold levels) and therefore every enhancer region found on the various Dpp targets provides us with a unique tool. We will fuse Dpp responsive elements to a cDNA construct that encodes a highly unstable version of GFP allowing us to detect small and transient fluctuations in Dpp response. These experiments aim to achieve a detailed spatio-temporal analysis of the dynamics of Dpp signalling in vivo. Since Dpp signalling serves as a paradigm to study growth and patterning of epithelial tissues, such in vivo studies are absolutely crucial, they will allow putting forward or excluding certain models of how Dpp exerts its multiple functions. Results: I have taken this part of my proposal a lot further. First of all, this aim requires establishing live imaging of wing imaginal discs. Specifically, establishing techniques for imaging through larval cuticle is more advantageous as the same animal can be imaged over time. Now, I am able to obtain images from live larvae placed under a confocal microscope. As the most problematic part is keeping larvae immobile for extended periods of time without killing them, I have tried different anaesthetic drugs. I have recently obtained transgenic flies that carry unstable fluorescent protein constructs driven by a dad enhancer which was characterized in detail in the lab. Simultaneously, I have generated different versions of the fluorescent protein that are not as unstable as the original proposal to make sure that we can detect expression. Currently, I am in the process of establishing lines for future analysis. In the mean time, I have worked with available lines and made new lines and practiced dissection of early discs. Finally, I will be collaborating with computational biologists for the analysis of my data. To this end, we established a collaboration with Dr Sven Bergmann of Universit¿ de Lausanne. During the course of this fellowship, apart from working on my proposed aims, I also brought some preliminary results obtained during my PhD to publication in the Journal of Cell Science. I am happy to report that an image I have acquired was featured on the cover. Additionally, I have contributed as first and corresponding author to an invited mini-review in Science Signalling. Both publications acknowledge the FP7 Marie Curie Fellowship support.

Data: CORDIS, © European Union