Stefan Zappe 

Assistant Professor of Biomedical Engineering

Integrated Technologies for High-Throughput Drosophila Embryo Functional Genomics Screens

Prof. Zappe's group is developing automated technologies for Drosophila embryo injection, imaging, and image analysis in order to determine the functions of genes inferred from the Drosophila genome sequences. Current screen technologies are optimized for RNA interference (RNAi), a powerful method for silencing of specific genes. RNAi is based on insertion of designed, double-stranded RNA (dsRNA) into an organism. Inside cells, dsRNA is triggering molecular events that lead to deactivation of a specific gene. Analysis of a phenotype due to gene silencing indicates when the silenced gene is active, in which tissue it is active, and what its function might be. 

Drosophila genome projects. The genome projects have brought unprecedented opportunities to understand molecular mechanisms of development and disease. The genome sequence of the fruit fly Drosophila melanogaster has been particularly valuable because the past 100 years of studies of this organism can now be combined with the new molecular data to determine gene functions [1]. Drosophila serves as a model organism for developmental and cellular processes common to higher eukaryotes, including humans. Comparative genomics studies have revealed that D. melanogaster has orthologs to 177 out of 289 examined human disease genes [2], including genes implicated in various cancers, Parkinson’s and Alzheimer’s. In total, D. melanogaster has approx. 13,600 genes. In addition to the genome sequence of D. melanogaster, the genome sequences of 11 other Drosophila species have been made publicly available over the past two years [3, 4]. Automated functional genomics screens yield a better understanding of molecular mechanisms of diseases and new strategies for therapies.

Automated screen technologies. In support of high-throughput, Drosophila embryo-based screens, Prof. Zappe’s group has developed systems for automated Drosophila embryo injection based on MEMS microinjectors (Fig.1, left) [5].  A fully automated LEICA TCS SP5 confocal fluorescence microscope is used for time-lapse imaging of injected embryos that express fluorescent markers in tissue of interest (Fig.1, center).  In collaboration with Prof. Minden (Drosophila researcher, CMU) and Prof. Kovacevic (Center for Bioimage Informatics), initial algorithms were developed for automated recognition of phenotypes due to gene silencing, e.g. delayed or absent formation of the ventral furrow during early embryonic development (Fig1, right) [6].

Figure 1:  Automated technologies in support of high-throughput Drosophila embryo functional genomics screens encompass  a) embryo injection technologies based on MEMS microinjectors (left),  b) time-lapse confocal fluorescence microscopy of embryos that express fluorescent markers in tissue of interest (center), and   c) image analysis software that automatically predicts phenotypes (normal and abnormal ventral furrow formation) due to gene silencing (right).  

1. Adams, M.D., et al., The Genome Sequence of Drosophila melanogaster. Science, 2000. 287(5461): p. 2185-2195.
2. Rubin, G.M., et al., Comparative genomics of the eukaryotes. Science, 2000. 287: p. 2204-2215.
3. Richards, S., et al., Comparative genome sequencing of Drosophila pseudoobscura: Chromosomal, gene, and cis-element evolution. Genome Research, 2005. 15(1): p. 1-18.
4. Crosby, M.A., et al., FlyBase: genomes by the dozen. Nucleic Acids Research, 2007. 35(suppl_1): p. D486-491.
5. Zappe, S., et al., Automated MEMS-based Drosophila embryo injection system for high-throughput RNAi screens. Lab Chip, 2006. 6: p. 1012–1018.
6. Kellogg, R.A., et al., Towards an image analysis toolbox for high-throughput Drosophila embryo RNAi screens, in International Symposium on Biomedical Imaging (ISBI). 2007, IEEE: Metro Washington, DC.

Lab Webpage: http://zappe.bme.cmu.edu/