Ralf Schnabel et al. (2002) European Worm Meeting "Improvement of the biolistic transformation of C. elegans"

Cathrin Struck, Ralf Schnabel

[European Worm Meeting, 2002]

Transformation of the worm by microinjection is now a rate-limiting step in many labs during the analysis of genes. We find microinjection still quite unreliable and cumbersome. At least in our hands the transformation rate is in average quite low, we get only one stable array per 30 injected hermaphrodites. In 1999 we demonstrated that the worm can be reliably transformed using ballistic bombardment with gold particles using a helium gun. However, the efficiency by which stable arrays were formed was still rather low, approximately one stable line per ten shots was isolated. Meanwhile we improved the method by a factor of 100; typically we now isolate 10 stable arrays per shot. One or two of these lines show a stable transmission of the transformed DNA, indicating that integration into the genome occurred. The in and outs of biolistic transformation will be discussed.

Ralf Schnabel et al. (2001) International C. elegans Meeting "A High Resolution Digital 4-D-Microscopy"

Anja-Kristina Schulz, Ralf Schnabel

[International C. elegans Meeting, 2001]

A High Resolution Digital 4-D-Microscope Anja-Kristina Schulz and Ralf Schnabel Institut fur Genetik, Technische Universität Braunschweig, Spielmannstraße 7, D-38106 Braunschweig, Germany The small size and transparency of the C. elegans embryo in combination with the known lineage offers the unique chance to analyse the consequences of manipulations, mutations or RNAi experiments and expression patterns of genes on the single cell level. 4D-microscopy is an ideal tool to follow and analyse the fate of many cells. The disadvantage of 4D-microscopes so far was that the high resolution required to "completely" analyse embryos was coupled to very expensive and special equipment as well as high running costs for the storage media. We recently designed a new system which can be built from commercially available parts and which stores high quality pictures. A 4D-microscope requires an excellent Nomarski optics, a matching analog camera (e.g. Hamamatsu Newvicon, PCO VX44), a frame grabber (Inspecta-2), a very precise control of the stage for the z-scans and a shutter for the light beam. The microscope is controlled by a PC (Windows NT) and the pictures are stored on a large hard disc as a series of bitmap files (BMP format). To save space on the hard disk an algorithm compresses the pictures by a factor of eight to ten without any visible quality loss (Wavelet compression, Luratech, Berlin); this works much better than for example JPEG compression. A full recording of 20.000 pictures only has 800 MB with this compression algorithm. When the Zeiss Axioplan Imaging with its new stage control is used the precision is high enough to acquire very precise z-series. Former models can be equipped with a piezo mover and an external shutter for the light beam. The new program allows to control the motorized functions of the Zeiss microscope. A special setup menu helps to find the optimal parameters for the recordings. Parameters like time, increment and position of the upper level can be corrected any time during the recording. The video window contains functions for an online enhancement of the pictures. The program also offers the necessary functions to document fluorescent pictures. We also designed a dual channel microscope which permits the alternate recording of Nomarski and fluorescence pictures which is suitable for the precise analysis of GFP expression patterns. The lineage analysis program SIMIBiocell was adapted to read the digital pictures.

Rothhamel, Sophie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Three cib-genes, three functions, one phenotype Embryonic germline development"

Schmidt, Henning, Rothhamel, Sophie, Wiekenberg, Anne, Schnabel, Ralf

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In C. elegans, the embryonic germ line is defined during the first four rounds of division. In contrast to the soma, the germ line cells (P-cells) divide asymmetrically in a stem-cell-like manner. To understand the mechanisms underlying this asymmetry, we analysed maternal lethal mutants in three genes with major defects in the embryonic germ line. In cib-1 (changed identity of blastomeres, Schnabel and Schnabel, 1990), cib-2 and cib-3 the germ line cells P1 to P3 divide symmetrically instead of asymmetrically after a delay of up to one cell cycle. In cib-2, after a prolonged cytokinesis during the first mitosis, no nuclei are formed in the 2-cell-stage. This first defect results from failed chromosome condensation and causes DNA fragmentation during cell division. As a consequence, the cell cycle control atl-1 chk-1 (Brauchle et al., 2003) is activated. We confirmed, that the activation of the cell cycle control depends on the damaged DNA by laser irradiation of one pronucleus in the wild-type embryo, which phenocopies the cib-2 mutant. Since cib-1 codes for the only homologe of a thymidylate synthase (Winska et al., 2005) in worms, we assume the P-cell phenotype caused by mutations in cib-1 and cib-2 are due to a reduced DNA integrity. To our surprise the phenotype is germ line specific, which suggests the existence of a germ line specific DNA integrity checkpoint. CIB-3, in contrast, is essential for the organisation of the asymmetric cytoplasmic distribution of germ line specific factors. Nevertheless, CIB-2 and CIB-3 have identical interaction proteins in a Yeast-Two-Hybrid screen. This indicates a linkage between DNA integrity and the organisation of cytoplasmic polarity. Thus, the system may serve to survey integrity of the worm population, by eliminating embryos with defect germ line DNA.

von Elsner, Sophie et al. (2009) International Worm Meeting "Three cib-genes, three functions, one phenotype News in germ line development."

von Elsner, Sophie, Schmidt, Henning, Schnabel, Ralf, Wiekenberg, Anne

[International Worm Meeting, 2009]

In C. elegans, the embryonic germ line is defined by the first four rounds of division. In contrast to the soma, the germ line cells (P-cells) divide asymmetrically in a stem-cell-like manner. To understand the mechanisms underlying this asymmetry, we analysed mutants in three genes with major defects in the embryonic germ line. In cib-1 (Schnabel and Schnabel, 1990), cib-2 and cib-3 (changed identity of blastomeres) the germ line cell P1 to P3 divide, after a delay up to one cell cycle, symmetrically. The initial defect in cib-2 is that the mitotic chromosomes do not condense. As a consequence, in cib-2, DNA is shredded during mitosis. Therefore the delay occurs (as a secondary defect) in the activation of the cell cycle checkpoint. We confirmed, that the activation of the cell cycle control depends on the damaged DNA by laser irradiation of one pronucleus in the wildtype embryo. This phenocopies the cib-2 mutant. Since cib-1 codes for the only thymidylate synthase (Winska et al., 2005) in the worm, the P-cell problem caused by mutations in both genes are probably due to DNA integrity. The germ line phenotype suggests maybe the existence of a germ line specific DNA integrity checkpoint. CIB-3 is essential for the organisation of the cytoplasmic distribution of germ line specific factors. Nevertheless, CIB-2 and CIB-3 have identical interaction partners. This indicates a linkage between DNA integrity and the organisation of the cytoplasmic polarity. Thus, the system may serve to survey integrity of the worm population, by eliminating embryos with defect germ line DNA.

Memar, Nadin et al. (2009) International Worm Meeting "A screen for temperature-sensitive embryonic lethal mutations affecting cell focussing in Caenorhabditis elegans."

Wiekenberg, Anne, Schnabel, Ralf, Martin, Katharina, Memar, Nadin

[International Worm Meeting, 2009]

Schnabel et al. (2006) proposed cell focussing as mechanism for global cell sorting by cell identity in the C. elegans embryo. It should be helpful to find mutants to define the mechanism of this process. Interestingly a very large collection of nonconditional embryonic lethal mutants did not contain a mutant affecting cell focussing. We supposed that genes affecting this process might be highly pleiotropic, since it should also keep larvae and adults in shape and/or maternal rescue may occur in the embryo. Therefore, ts mutants may be the tool of choice to identify genes. We designed a new screen for ts mutants using the worm sorter, since a very large number of mutants may be required but the yield is less than 1%. Identification of specific mutants is very cumbersome since candidates have to be subjected to a careful 4D analysis. Only mutants in which fates are wild-type - but the positioning of subsets of cells is wrong - qualify as potential cell focussing mutants. A first screen yielded two candidates in approx. 1000 ts mutants. In the next round we isolated again 1000 mutants, which now await analysis. We expect that up 10000 ts mutants may be required to define a cell focussing pathway. However, the mutant collection may be also a valuable resource for the worm community.

Domeier ME et al. (1997) International C. elegans Meeting "EXPRESSION OF pha-1 DURING EMBRYOGENESIS"

Domeier ME, Mango SE

[International C. elegans Meeting, 1997]

At about the 200 cell stage of embryogenesis, 27 precursors are born that will ultimately produce the 80 cells of the pharynx. Initial studies using a pha-1::lacZ construct determined that pha-1 was expressed at the 200 cell stage, in pharynx and body wall muscle precursors1. To visualize the pharyngeal precursors in living embryos, we created two pha-1::GFP constructs. The first construct was a translational fusion that contained 3.7 kb of upstream sequences plus the entire pha-1 coding domain fused to GFP. The second was a transcriptional fusion carrying pha-1 5! sequences and a nuclear-localized GFP::lacZ cassette. GFP expression was assayed from stable lines in N2 embryos. Surprisingly, both GFP constructs were expressed in multiple cell types in addition to pharynx and body wall muscle precursors. For example, hypodermis, neurons and intestinal cells all expressed pha-1::GFP. Thus, pha-1 may be expressed more globally than first reported. Furthermore, the pha-1::GFP protein fusion was found predominantly in the cytoplasm. This construct was able to rescue pha-1 mutants, suggesting that endogenous PHA-1 may be a cytoplasmic protein as well. To determine the expression pattern of endogenous PHA-1, we are making anti-PHA-1 antibodies. 1.Granato M., Schnabel H. and Schnabel R. (1994) Development 120, 3005-3017. We thank Heinke and Ralf Schnabel for generously sending us alleles of pha-1 as well as pha-1 expression constructs.

Ofenbauer, Andreas et al. (2015) International Worm Meeting "A high-throughput EMS screen identifies a potential muscle linage mutant."

Schnabel, Ralf, Ofenbauer, Andreas, Tursun, Baris

[International Worm Meeting, 2015]

Having multiple cell types with distinct functions has been a prerequisite for the evolutionary success of most multicellular organisms. Accordingly, understanding the mechanisms that form the basis of this strategy has always been of big interest and has historically been the topic of developmental biology. Nowadays several other disciplines deal with the question of how one cell type differentiates into another, such as stem cell biology, epigenetics, systems biology, RNA biology, or even biomaterial research.Transdifferentiation is the process by which an already specialized cell is directly converted/reprogrammed into another specialized cell. This naturally not very abundant process is the major focus of our lab. We try to identify and characterize genetic factors that play a role in induced transdifferentiation by mis-expressing transcription factors (TFs). Currently there are several selector genes known that can activate specific cell fates such as hlh-1, the worm homolog of the myogenic bHLH TF MyoD. When mis-expressed, HLH-1 induces muscle fate in early embryonic cells but terminally differentiated cells in older animals are resistant to hlh-1-induced direct reprogramming. In order to identify mechanisms that antagonize selector genes, we apply both forward and reverse genetics. We use transgenic lines allowing ectopic expression of a specific selector gene in combination with the appropriate cell fate reporter.To complement manual microscopic screening, we use a semi-automated high-throughput forward genetics screen combining EMS mutagenesis with the Biosorter system (Union Biometrica). Using this approach we identified a mutant showing additional cells at the posterior end of the pharynx that express a myo-3 reporter. To our surprise, these cells show muscle reporter expression independently of ectopic induction of hlh-1. Although our screen aims to find mutants that allow induced direct reprogramming, our finding was quite striking as the phenotype shows 100% penetrance. To elucidate the underlying mechanism, which could be a missing apoptosis event, an additional cell division or a lineage conversion event, we are currently tracing lineages and trying to identify the mutated locus using whole genome sequencing. .

Bezler, Alexandra et al. (2009) International Worm Meeting "Characterization of a Novel Mutation Activating the Spindle Assembly Checkpoint in C. elegans ."

Schnabel, Ralf, Bezler, Alexandra, Gonczy, Pierre

[International Worm Meeting, 2009]

Correct bipolar attachment of sister chromatids is crucial for faithful segregation of the genetic material in mitosis. This process is regulated by the spindle assembly checkpoint (SAC), which inhibits the anaphase promoting complex/cyclosome (APC/C) until proper attachment is achieved. We are characterizing a mutant strain, tentatively named emb-86(t1565), in which embryos exhibit variable spindle defects (multipolar or anastral monopolar) and altered M phase duration. We found that emb-86(t1565) mutant sperm contains aberrant centriole numbers and DNA content, likely explaining the observed spindle defects in the resulting embryos. Moreover, we observed that anteroposterior axis specification is defective in emb-86(t1565) mutant embryos fertilized by acentrosomal sperm, as anticipated from previous work. Importantly in addition, we found that M phase duration is approximately 5-fold longer in emb-86(t1565) mutant embryos with anastral monopolar mitosis than in the wild-type. By contrast, incorrect chromosome attachment in the wild-type causes only a 2-fold delay in M phase. We established that the 5-fold delay in emb-86(t1565) is of maternal origin and caused by SAC engagement, since inactivation of MDF-1 or MDF-2 by RNAi rescues proper M phase duration in emb-86(t1565) mutant embryos. We mapped the emb-86(t1565) locus to a region on the right arm of LGIII comprising 21 annotated genes, including such-1, an APC5 related subunit. Previous work has shown that such-1(h1960) mutant embryos show a slight M phase delay. Here, we established that monopolar anastral mitosis in such-1(h1960) embryos leads to a 5-fold increase in M phase duration, as in emb-86(t1565) mutants. Moreover, emb-86(t1565) fails to complement such-1(h1960) under conditions where the SAC is activated, suggesting that emb-86(t1565) is a novel allele of such-1. Taken together, compromising the APC/C may cause a SAC dependent M phase delay, in addition to the previously described SAC independent delay. Our unexpected findings raise the possibility that the APC/C may act as negative regulator of the SAC.

Martin, Katharina et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A screen for temperature-sensitive embryonic lethal mutants affecting cell migration in Caenorhabditis elegans."

Wiekenberg, Anne, Martin, Katharina, Memar, Nadin, Schnabel, Ralf, Hennig, Christian, Schiemann, Sabrina

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Schnabel et al. (2006) proposed cell focussing as mechanism for global cell sorting by cell identity in the C. elegans embryo. A collection of embryonic lethal mutants could give us a deeper understanding of this barely described process. As genes affecting cell focussing may be highly pleiotropic, temperature-sensitive mutants are the tool of choice. Our group designed a large screen for ts mutants. Unfortunately a very strict and consistent inspection leads to a yield of less than 1 %. Nevertheless, we have almost 2000 mutant strains by now with a great variety of interesting phenotypes. So far 538 strains were analysed via 4D microscopy and 82 of them seem to have defects in morphogenesis and cell adhesion, which are the most valuable phenotypes for our project. After a short analysis of a defined subset of cells with SIMI Biocell, our bioinformatical tools show if cells or whole regions migrate not correctly. Continuative methods give us a deeper insight in cell migration, cell fates, time of development and many more aspects of the embryogenesis. This way, we hope to find mutants with defects in cell migration and sorting, identify corresponding genes and thus get a molecular view about cell focussing. Moreover the mutant collection will be hopefully a valuable source for the worm community.

Memar, Nadin et al. (2009) International Worm Meeting "A view on the embryogenesis of the Caenorhabditis Family."

Martin, Katharina, Schnabel, Ralf, Memar, Nadin

[International Worm Meeting, 2009]

The molecular differences of the four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri are currently of great interest, however little is known about development. Zhao et al. (2008) reported an automatic lineage of C. briggsae and came - based mostly on the cleavage pattern and cell positions - to the conclusion that the embryogenesis of the two species is very similar. We now present detailed 4D analyses of the species including the terminal differentiation patterns. All analyses including bioinformatical quantifications of cell behaviour show a huge similarity between those species. Immunochemical analyses of the tissue distributions only reveal a difference in the intestinal differentiation of C. brenneri. Interestingly hybrid embryos always appear to fail in different ways in embryogenesis.