Visualizing the molecular basis for stem cell niche function and Hh signal transduction
Final Report Abstract
During the course of the project I addressed two independent topics, namely the link between Hh signalling and stem cell niche function in the Drosophila testis, and the visualization of the molecular basis of Hh pathway activation. Both subprojects involved technology development or the import of cutting edge technologies into the lab. While this was successful for the first subproject, where we established genome editing by Crispr/Cas9 as well as the in vivo mapping of transcription factor binding by DamID, we were only partially successful in the second subproject. There, we were able to establish automated, FACS based screening for Smo activation as orignally envisaged, but both my lab and our collaborators failed to adapt our cell lines for genome wide RNAi. However, in terms of results both subprojects were successful. In the first subproject, we focussed on the targets of the transcriptional repressor zfh-1, which is the key transcription factor regulated by the Hedgehog and upd/Jak-Stat niche signals. Using damID both in S2 cells and in vivo we identified the hippo pathway as the major regulator of stem cell proliferation. More importantly, in combination with previous observations and results from other labs our results demonstrate that different niche signals directly impinge on different aspects of stem cell biology, and that these aspects can be genetically separated at the level of niche signalling input. In our upcoming manuscript we are therefore going to challenge the canonical view of stem cell niche function, whereby the niche provides a complex signalling microenvironment that is integrated by the stem cells into a binary decision between stemness and differentiation. Instead we will propose a new model of niche function, whereby individual niche signals "micromanage" separate aspects of stem cell behaviour, and it is these cellular responses that we as observes sum up as stemness. Importantly, our experiments point us at an extremely interesting link between niche signalling and stem cell metabolism that will form the basis of upcoming grant applications. In the second subproject the RNAi resistance of our reporter cell lines prevented the original strategy of combining genome wide RNAi with automated fluorescence microscopy at the Sheffield facility. Instead, we used pharmacological inhibition using a library of compounds with known molecular targets We could thus also switch to FACS based analysis that provided a much better quantitation of reporter fluorescence. Using this backup approach we identified three kinase cascades not previously implicated in Smo activation. Due to time constraints we have decided to split the already completed in vitro work from the ongoing validation of our results in the living organism, and have again started preparing a corresponding manuscript.
Publications
- 2013. Phosphorylation of the Smo tail is controlled by membrane localisation and is dispensable for clustering. Journal of Cell Science. 126:4684-4697
Kupinski, A.P., I. Raabe, M. Michel, D. Ail, L. Brusch, T. Weidemann, and C. Bökel
(See online at https://doi.org/10.1242/jcs.128926)
