Novel Tools and Technologies

FlyORF/ORFeome/phiC31

This project focuses on the generation of fly strains that allow the controlled overexpression of Drosophila ORFs with the Gal4/UAS system. The incentive to offer such a “gain-of-function” collection comes from the finding that the majority of genes have no readily detectable loss-of-function phenotypes. For many genes, this may be due to functional redundancy between related genes; for others, the reason may be the lack of suitably sensitive assays. Given that in Drosophila - and other organisms as well - many genes still have no function assigned, alternative approaches are needed to reveal gene functions. The collection of fly strains expressing individual ORFs is such an alternative for ”gain-of-function“ analyses. The overexpression of a gene often results in an observable effect, even when the loss of that gene has no detectable consequence. Gene activation is thus a powerful complementary tool to elucidate gene functions.

Initially we created a very efficient and easy-to-use ΦC31 integrase system for germ-line transformation in Drosophila. This ΦC31 integrase system has put us into the position to generate a large ORFeome library that we generate in collaboration with Dr. Bjorklund’s group in Dundee. In particular, it is now possible to inject large collections of constructs, as this method guarantees efficient integration. Targeted insertion eliminates the need for mapping, and selection of a suitable landing site minimizes unpredictable position effects. Consequently, the effects of transgenes can be reliably compared. Currently we focus on generating a 3xHA-tagged library. To increase the versatility of this library, we have further introduced ‘swapping’ elements (i.e. modified FRT sites flanking the ORFs) to facilitate in vivo exchange of epitope tags and regulatory regions. Thus, any sequence-encoded N- or C-terminal modification can be precisely swapped onto a target protein. All the generated and confirmed fly strains are available at FlyORF.

We think that overexpression analyses will have a profound impact on Drosophila research in the future and lead to the identification of previously unknown components in various signaling pathways. Gain-of-function screens are still underexplored compared to loss-of-function, thus representing a largely unexplored potential.

Selected References

Bischof, J., Maeda, R.K., Hediger, M., Karch, F., and Basler, K. (2007). An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proceedings of the National Academy of Sciences of the United States of America 104, 3312-3317.

Bischof, J., Björklund, M., Furger, E., Schertel, C., Taipale, J., and Basler, K. (2013). A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila. Development (Cambridge, England) 140, 2434-2442.

Bischof, J., Sheils, E.M., Björklund, M., and Basler, K. (2014). Generation of a transgenic ORFeome library in Drosophila. Nature Protocols 9, 1607-1620.

Schertel, C., Huang, D., Björklund, M., Bischof, J., Yin, D., Li, R., Wu, Y., Zeng, R., Wu, J., and Taipale, J., et al. (2013). Systematic screening of a Drosophila ORF library in vivo uncovers Wnt/Wg pathway components. Developmental Cell 25, 207-219.

 

EpiTools: An Open-Source Image Analysis Toolkit for Quantifying Epithelial Growth Dynamics

Epithelia grow and undergo extensive rearrangements to achieve their final size and shape. To the capture of the dynamic nature of a growing epithelium and to help reveal novel properties of developing epithelial organs live imaging and automated image processing software are needed. To this end we have designed a new open-source image analysis toolkit called EpiTools. It allows automated image analysis tools to extract quantitative such that one can image the dynamics of tissue growth and morphogenesis and take advantages of the advances in time-lapse microscopy. EpiTools provides user-friendly graphical user interfaces for accurately segmenting and tracking the contours of cell membrane signals obtained from 4D confocal imaging. It is designed for a broad audience, especially biologists with no computer-science background. Quantitative data extraction is integrated into a larger bioimaging platform, Icy, to increase the visibility and usability of our tools. We demonstrated the usefulness of EpiTools by analyzing Drosophila wing imaginal disc growth, revealing previously overlooked properties of this dynamic tissue, such as the patterns of cellular rearrangements.

The Epitools Pipeline

Selected References

Heller, D., Hoppe, A., Restrepo, S., Gatti, L., Tournier, A.L., Tapon, N., Basler, K., and Mao, Y. (2016). EpiTools: An Ope{Willecke 2011 #100}n-Source Image Analysis Toolkit for Quantifying Epithelial Growth Dynamics. Developmental cell 36, 103-116.

Zartman, J., Restrepo, S., and Basler, K. (2013). A high-throughput template for optimizing Drosophila organ culture with response-surface methods. Development (Cambridge, England) 140, 667-674.