Wildwater, Marjolein et al. (2011) International Worm Meeting "Cell Shape and Wnt Signaling redundantly Control the Division Axis of C. elegans Epithelial Stem Cells."

de Vreede, Geert, Wildwater, Marjolein, Sander, Nicholas, Galli, Matilde, Van den Heuvel, Sander

[International Worm Meeting, 2011]

Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight regulation of the orientation and plane of cell division is critical in this process. Here, we study the reproducible pattern of anterior-posterior oriented stem cell-like divisions in the Caenorhabditis elegans seam epithelium. In a genetic screen, we identified an alg-1 Argonaute mutant with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of the miRNA-Induced Silencing Complex (miRISC) and was previously shown to regulate the timing of postembryonic development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively interferes with cell adherence, orientation and timing of seam cell division, cell shape and Wnt signaling. We found that Wnt inactivation, through mig-14 Wntless mutation, disrupts tissue polarity but not anterior-posterior division. However, combined Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. Thus, we identified additional alg-1 regulated processes and uncovered a previously unknown function of Wnt ligand signaling in seam tissue polarity. Furthermore, we showed that Wnt signaling and geometric cues redundantly control the seam cell-division axis. In addition to the division axis, we observed that seam cells displace their mitotic spindle off center during asymmetric division, giving rise to a smaller anterior daughter cell (with a hyp7 fate) and a larger posterior daughter cell (with a seam cell fate). Interestingly, this spindle displacement is not observed during symmetric seam cell division. Initial observations revealed that the conserved coiled-coil protein LIN-5 (NuMA in mammals) regulates the orientation of the spindle within the seam cells. In addition, the non-muscle myosin motor NMY-2 localized posteriorly just prior to anterior movement of the spindle, suggesting that myosin II motors could be involved in the inhibition of spindle displacing forces. Currently we are investigating the mechanisms that control spindle displacement. Taken together, our findings establish a novel system to study the division plane in the context of an epithelial tissue.

Wildwater, Marjolein et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "ALG-1/ Argonaut ensures proper development by coordinated regulation of cell polarity, cell-cell contacts, spindle positioning, and developmental timing"

Van den Heuvel, Sander, Sander, Nicholas, Wildwater, Marjolein

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

Proper development of an organism requires both cell division and cell differentiation. Asymmetric cell division leads to the formation of two daughters with unequal fates. C. elegans epidermal seam cells are an excellent model system to study asymmetric division as seam cells divide in an asymmetrical manner once during every larval stage. To understand the regulatory mechanisms of asymmetric seam cell divisions we performed a mutagenesis screen to identify new components involved in asymmetric seam cell division control. We identified a mutation in alg-1 /Argonaut, an essential component of the RISC complex and important in regulation of miRNA function. With a newly developed in vivo time-lapse system for larvae we inve stigated the importance of alg-1 /miRNAs in asymmetric seam cell division control. We observed a sequential range of developmental aberrations, emphasizing the importance of miRNA function during C. elegans development. Reduction of alg-1 first causes mislocalisation of Wnt signaling pathway components and cell fate defects. Then, cell-cell contacts were lost, cell membranes showed hyper motility, cell shape of seam cells became increasingly round and cells started to divide in random directions. As a late effect, we observed developmental timing defects. We discovered that miRNAs responsible for regulation of developmental timing do not regulate division polarity or membrane integrity. We furthermore found epidermal seam cell shape to be critical in division orientation control by miRNAs. Our results highlight the important role of miRNAs during several steps of developmental cell division events and shows that like in mammalian systems miRNAs are crucial factors to re gulate development at different branch points.

Marjolein Wildwater et al. (2008) Development & Evolution Meeting "Regulation Of Asymmetric Cell Division During Development Of The Post-Embryonic Seam Cell Lineages"

Wytse Bruinsma, Matilde Galli, Sander Van Den Heuvel, Marjolein Wildwater

[Development & Evolution Meeting, 2008]

During growth and development an animal has to generate a proper number of cells with the right cell fates. One way to simultaneously generate new and different cells is through asymmetric cell division, in which a cell generates two daughters with different fates. Tight regulation of asymmetric cell division frequency is critical for normal development. Defects in this process have major consequences in tissue regeneration and repair and likely contribute to tumour formation. C. elegans hypodermal seam cells are an excellent model system to study the regulatory mechanisms of asymmetric cell division since most seam cells divide in an asymmetric manner once during each larval stage. Interestingly, the majority of seam cells also include a single round of symmetrical division during the second larval stage. To study seam cell divisions in detail we first aimed to set up a method for in vivo time-lapse recording. Although in vivo time-lapse recordings have been extensively used in studies of C. elegans embryonic divisions, a proper method to record larval divisions was never reported. To facilitate filming we generated several strains expressing GFP::H2B (to mark DNA), GFP::PH (to mark membranes), GFP::a-tubulin and GFP::g-tubulin (to mark the spindle) specifically in the seam lineages. We use these tools to study spindle positioning during seam cell division. We could show that spindle poles migrate in a flexible but limited number of directions in both asymmetric and symmetrically dividing seam cells before they end up at the anterior and posterior side of the cell. In the L2 stage, only asymmetric cell division results in two differently sized daughter cells, indicating active spindle positioning. In order to study how division frequency and polarity is controlled in seam cells we have initiated two already successful approaches, a candidate gene approach and a mutagenesis screen. Mutants derived from this latter screen can be subdivided in three different classes: mutants of the first class are affected in division frequency, mutants of the second class are affected in division polarity and mutants of the third class are affected in both. To understand how our candidate genes of interest and our novel mutants control asymmetric seam cell division we are currently introducing reporter constructs into the mutant background to be able to perform in vivo time-lapse studies.

Kerkhof, Engelien et al. (2015) International Worm Meeting "C. elegans as robust, high throughput in vivo system for hazard assessment."

Lokman, Christien, Wildwater, Marjolein, Pieters, Raymond, Kerkhof, Engelien

[International Worm Meeting, 2015]

Innovative toxicity test systems that enable identification of adverse outcome pathways (AOP) relevant for humans, are highly interesting to allow improved hazard identification and characterization. The nematode Caenorhabditis elegans (C. elegans) has been thoroughly established as a developmental biological test system and shows a high level of conserved molecular pathways and cellular mechanisms compared to man. The behavior of the 959 somatic cells of which the hermaphrodite nematode exists have been mapped in detail. The nematode transparency, short turnover time, small size, conserved organs, high throughput screening ability and the available technological resources allow rapid, reproducible testing of compounds and easy identification of AOPs and molecular initiating events (MIE). Furthermore, in the view of developmental and reproductive toxicology (DART) which is amongst the most difficult toxicological outcomes to assess, and which requires the highest numbers of vertebrates for testing, C. elegans can be a suitable alternative test system as it has a full life cycle. We have tested 22 well documented and well characterized DART compounds from the ECHA, Staatscourant and EPA list and show that the C. elegans test model has a predictive value of ~80% for DART related compounds, indicating the suitability of the C. elegans test system for DART assessment. To understand AOPs underlying DART toxicity in C. elegans, a RNA-sequencing approach was followed. RNA sequencing showed alterations in gene expression profiles of genes orthologous to humans. Data analysis consisted of community based profiling and a posttranscriptional gene silencing approach (RNAi). Two individual genome wide RNAi libraries are available in C. elegans that allow down regulation of gene expression of most genes in the C. elegans genome. If particular up regulated genes (identified by RNA-sequencing) are critically responsible for DART effects, posttranscriptional gene silencing by RNAi should reduce the DART effects, thus enabling the identification of critical AOP genes. Alternatively, community based alignment of DART responses enables the identification of global gene expression patterns responsive to DART effects.

Monique van der Voet et al. (2006) European Worm Meeting "The Function of LIN-5 in Chromosome Segregation and Asymmetric Division"

Matilde Galli, Christian Berends, Monique van der Voet, Justin Peters, Sander Van Den Heuvel, Mike Boxem, Marjolein Wildwater, Adri Thomas, Inge The

[European Worm Meeting, 2006]

Monique van der Voet1, Marjolein Wildwater1, Mike Boxem, Justin Peters1, Christian Berends1, Matilde Galli1, Inge The1, Adri Thomas1 and Sander van den Heuvel. The position of the spindle apparatus determines the plane of cell division. During asymmetric division, proper positioning of the spindle is needed in the generation of daughter cells that differ in size, cytoplasmic determinants and developmental fate. We have focused our attention on the C. elegans gene lin-5 to identify novel regulatory mechanisms and molecules that control spindle-mediated functions.. Our previous studies demonstrated that lin-5 is generally required for chromosome segregation and spindle positioning during meiotic and mitotic M phase. lin-5 encodes a coiled-coil protein that localizes to the cell cortex as well as spindle asters, and recruits the G protein regulators GPR-1/GPR-2 to the same locations. Results from our lab as well as others support that LIN-5 and GPR act in concert with the GOA-1/GPA-16 G?i/o subunits of heterotrimeric G proteins in an evolutionarily conserved pathway for spindle positioning. Many aspects of this process remain poorly understood. In particular, it is unclear how polarity determinants at the cortex affect LIN-5/GPR/G?i/o function to create asymmetry in the pulling forces that act on the spindle asters. In addition, the downstream targets of the LIN-5/GPR/G?i/o pathway remain unknown. To better understand the molecular mechanisms of these processes, we continue to use a combined genetic and biochemical approach. We have identified novel candidate partners of LIN-5 through immunoprecipitation from embryonic lysates followed by mass spectrometry. In addition, we identified multiple residues of LIN-5 and GPR that are phosphorylated in vivo. We are currently testing the in vivo relevance of these interactions and modifications and hope to present our results at the meeting.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .