Homeobox transcription factors are widely conserved in all kingdoms of life including human beings and regulate complex developmental programs. In the C. elegans genome 100 homeobox genes have been found versus approx. 250 in humans. We apply multi-channel 4D (time lapse) microscopy to analyze gene expression in vivo in the developing embryo at single cell level. The method has been developed and optimized in our laboratory. Gene activity is visualized by promotor GFP fusion constructs. Additionally, we are able to study subcellular localization of the target protein with translational GFP fusion constructs. We use gamma irradiation to integrate the transgenic lines which is crucial to obtain consistent expression. Additional integrated reporter strains were kindly provided by several other worm labs. Cell lineage data is obtained from DIC imaging during early embryogenesis. We suppress motion artifacts during late embryogenesis by an anesthesia protocol. We have so far analyzed the temporal expression patterns for several homeobox genes. Early expression before comma stage is present for ceh-5, ceh-13, ceh-20, ceh-26, ceh-33, ceh-34, ceh-40, ceh-45, mls-2 and ZC376.4. Patterns that start during morphogenesis are found for ceh-6, ceh-8, npax, ceh-30, ceh-41 and ceh-44. Other genes only showed larval expression: ceh-19, dsc-1 and T13C5.4. We also identified the expressing cells for some of these genes. ceh-5, ceh-6, ceh-8 show a spatially distinct pattern, involving small numbers of cells. ceh-20, ceh-40 and ZC376.4 show expression in large portions of the embryo and exclude only few tissue types. Promotor activity in ceh-5, ceh-8, ceh-19 appears to be linked to neuronal development. All analyzed expression patterns have a highly dymamic expression level over time. Our aim is to collect a comprehensive set of expression patterns with focus on embryogenesis and larval development in a virtual C. elegans embryo model. Virtual-Worm Base is developed by our research group. We are trying to correlate the expression and co-expression of particular genes to the known differentiation and function of the respective target cell in C. elegans. Our screening method returns more dynamic data than any currently available system can do. It will provide new insights into the complex transcriptional network around the homeobox transcription factors.

Authors: Jurgen Hench, Johan Henriksson, Krai Meemon, Konstantin Cesnulevicius, Akram Abou-Zied, Johan Dethlefsen, Martin Luppert, David Baillie, Thomas Burglin