Kerstin Reichert et al. (2002) European Worm Meeting "Development of a bio-monitoring screening system based on the xenobiotically induced gene expression of Caenorhabditis elegans - The Celegans Toxchip"

Ralph Menzel, Kerstin Reichert, Rudolf Achazi

[European Worm Meeting, 2002]

From an ecotoxicological point of view it is essential at which concentration a contaminant becomes a pollutant, implying a toxic effect. At high environmental concentrations reproduction and mortality are used to define toxicity. But what about lower concentrated contaminants? In that case no significant effect could be found using classical ecotoxicological test systems. This presentation shows the development of a bio-monitoring screening system based on the xenobiotically induced gene expression of Caenorhabditis elegans - The Celegans Toxchip. Such a system should be more sensitive than classical ecotoxicological tests.

Nadine Saul et al. (2003) International Worm Meeting "Comparison of reproduction and xenobiotically induced gene expression as ecotoxicological test parameters in the nematode Caenorhabditis elegans."

Matthias Rodel, Nadine Saul, Kerstin Reichert, Ralph Menzel

[International Worm Meeting, 2003]

The nematode Caenorhabditis elegans satisfies all typical criteria to function as a test organism for ecotoxicological studies. In the current postgenomic era C. elegans gains its significance in particular through the fact that its whole genome is decoded. Here we present two approaches for testing toxic effects on the nematode comparing the endpoint reproduction in liquid medium with the specific levels of xenobiotically induced gene expression. Differently expressed genes were measured by semi-quantitative RT-PCR as well as a low density DNA array.For the reproduction test, age-synchronized L1 larvae were cultivated in liquid medium and exposed to different xenobiotics. Every substance was tested in six different concentrations versus controls. After an exposure of 96 h the number of offspring was determined from the number of second-generation juveniles of all stages. The test showed in all cases a strong concentration dependent relationship. It revealed tributyltin, endosulfan and fluoranthene as most toxic substances in the test. In a parallel set of experiments the specific messenger RNA expression of four different cytochrome P450 genes (CYP) was analyzed by semi-quantitative RT-PCR. Cytochromes P450 have been particularly implicated in the biotransformation of different xenobiotics. The threshold of the semi-quantitative RT-PCR experiments showed a significant higher sensitivity than the reproduction test. Atrazin, PCB52, fluoranthene, -naphthoflavone and lansoprazol were found to induce CYP35A1, A2, A5 and C1 gene expression at concentrations far below the EC50 for reproduction. The parallel determination of a variety of stress inducible genes by using a DNA microarray will considerably enlarge the implementation of this in vitro approach. Using a whole genomic approach 64 xenobiotically induced genes were selected so far. For the so called "Celegans Toxchip" corresponding cDNA fragments of these genes were spotted on poly-L-lysine coated glass slides. The typical hybridization experiment was performed with Cy3/Cy5 labeled cDNA samples. First experiments using the Celegans Toxchip confirmed the RT-PCR based results and revealed this C. elegans gene expression test as a suitable tool for bio-monitoring. The main advantages of the gene expression test compared to the reproduction test are the increased sensitivity and its potential for a substance class specific effect determination depending on the selected marker genes.

Kirchner, Marcel et al. (2015) International Worm Meeting "Correlation of centrosome size and spindle architecture in early C. elegans embryos."

Muller-Reichert, Thomas, Kirchner, Marcel, Hyman, Anthony

[International Worm Meeting, 2015]

Centrosomes are the main microtubule organizing centers (MTOC) in most animal cells and are involved in setting up the mitotic bipolar spindle. They consist of a pair of centrioles surrounded by an electron dense matrix called pericentriolar material (PCM). Although mitotic spindles show little variation in size within a single cell type, spindle length can vary at least 10-fold between different species. Recent studies have shown that centrosome size sets mitotic spindle length in the early C. elegans embryo and that the centrosome size correlates with cell size due to limiting amounts of cytoplasmic components incorporated into the PCM [1, 2].Therefore we aim to understand how centrosome architecture correlates with the number of nucleated microtubules and find out how the density and length distribution of spindle microtubules is altered due to changes in centrosome size.Using the early C. elegans embryo as a model system, we deplete centrosome-associated proteins by RNAi to generate centrosomes of different sizes within a single embryo. Our work focuses on SAS-4, a centriole component required for centriole duplication in the two-cell embryo. Partial depletion of SAS-4 results in structurally defective centrioles, which recruit proportionally less PCM and therefore show centrosomes of various sizes in the second mitotic division [3].Although light microscopic data suggest a correlation between centrosome size and the number of microtubules nucleated from the centrosome, there is currently no ultrastructural data to proof this hypothesis. We use a correlative light and electron microscopy (CLEM) approach by combining live-cell imaging and 3D-reconstruction by electron tomography. Therefore, we image a fluorescently labeled SPD-2::GFP strain to analyze centrosome size and immediately apply high pressure freezing after imaging to preserve the ultrastructural organization of mitotic spindles [4]. After subsequent freeze-substitution and semi-thick sectioning (300 nm), tomographic reconstruction is applied to visualize the organization of spindle microtubules.Using this approach, we will develop a functional model explaining how centrosome size is linked to spindle architecture.[1] Decker et al. 2001. Current Biology. 21(15):1259-1267[2] Greenan et al. 2010. Current Biology. 20(4):353-358[3] Kirkham et al. 2003. Cell. 112:575-587[4] Muller-Reichert et al. 2003. Journal of Microscopy. 212:71-80.