Bruce Bowerman et al. (2006) Development & Evolution Meeting "Anterior-Posterior Asymmetry in Beta-Catenin Localization During Embryonic Cell Divisions in the Spiral-Cleaving Polychaete Platynereis dumerilii"

Stephan Schneider, Bruce Bowerman

[Development & Evolution Meeting, 2006]

Beta-catenin functions in the canonical Wnt-signaling pathway by activating target gene expression in the nucleus. Early roles for beta-catenin signaling are surprisingly different among animal phyla. To gain insight into the ancestral role of beta-catenin, we are using the polychaete Platynereis dumerilii. This annelid's slowly evolving gene sequences, and its phylogenetic position as a lophotrochozoan, make it an attractive model, both for inferring ancestral bilaterian functions and for linking invertebrate and vertebrate model systems. Platynereis embryos deploy a highly stereotypic cell division program called spiral cleavage that generates blastomeres of defined size and cell fate, in a series of asynchronous anterior-posterior (a-p) oriented cell divisions. We observed a-p asymmetries in the levels of detectable beta-catenin during the first 70 embryonic cell divisions, from fertilization, through the spiral cleavage stages, and up to the emergence of bilateral symmetry. In every a-p cell division, beta-catenin accumulated to higher levels in the nuclei of posterior daughters. This asymmetry occurs independent of cell size, cell cycle, cell fate, and cell lineage. Interestingly, the asymmetry is lost only in transverse/bilateral cell divisions, and is reversed only in the germline lineage. These results are strikingly reminiscent of the a-p oriented divisions and Wnt pathway asymmetries that occur throughout embryonic and post-embryonic development in C.elegans. We also find similarities to beta-catenin signaling in early deuterostome and insect embryos. We propose that this a-p asymmetry of beta-catenin in daughter cells constitutes an ancestral and versatile developmental module that has been deployed and modified throughout evolution in metazoan embryos.

Robin Schneider et al. (2006) European Worm Meeting "The Loss of the Single APC Homolog apr-1 Causes Hyperplasia in the C. elegans Intestine"

Olaf Bossinger, Gisela Helbig, Robin Schneider

[European Worm Meeting, 2006]

Robin Schneider, Gisela Helbig and Olaf Bossinger. In humans APC is a powerful tumour suppressor that is mutated in most cases of sporadic or congenital colorectal cancer. In these forms of cancer, cells lose their ability to control the cell cycle and cell migration along the crypt-villus axis is disturbed. The C. elegans intestine is a simple epithelial tube that consists of only 20 cells (E-cells). Depletion of the C. elegans APC homolog APR-1 (Hoier et al., 2000) by bacterial RNAi causes hyperplasia of E-cells. In contrast, apr-1(RNAi) embryos contain up to 40 E-cells, which in most cases achieve to arrange into a tube-like structure. The use of gut-specific RNAi against APR-1 and the gain-of-function phenotype of the cycline dependent kinase CDC-25.1 (Clucas et al., 2002) suggests that intestinal cell proliferation is under organ-specific control. Preliminary data indicate that C. elegans homologs of the vertebrate WNT pathway are also involved in the regulation of endodermal cell proliferation. A detailed analysis of gut-specific hyperplasia phenotypes will be presented and discussed with regard to the involved pathways.

Jacques Pecreaux et al. (2006) European Worm Meeting "Mechanical Oscillations during Asymmetric Spindle Positioning Require a Threshold Number of Active Cortical Force Generators"

Jonathon Howard, Frank Julicher, Stephan W. Grill, Anthony A. Hyman, Karsten Kruse, Jens-Christian Roper, Jacques Pecreaux

[European Worm Meeting, 2006]

Jacques Pecreaux1, Jens-Christian Rper2, Karsten Kruse3, Frank Julicher3, Anthony A. Hyman1, Stephan W. Grill, Jonathon Howard1 Background. Asymmetric division of the C. elegans zygote is due to the posterior-directed movement of the mitotic spindle during metaphase and anaphase. During this movement along the anterior-posterior axis, the spindle oscillates transversely. A theoretical analysis indicates that oscillations might occur as a result of the concerted action of many cortical force generators that pull on astral microtubules in a tug-of-war situation. This model predicts a threshold of motor activity below which no oscillations occur. Results: We have tested the existence of a threshold by using RNA interference to gradually reduce the levels of GPR-1 and GPR-2 that are involved in the G-protein-mediated regulation of the force generators. We found an abrupt cessation of oscillations as expected if the activity drops below a threshold. Furthermore, we could account for the complex choreography of the mitotic spindle - the precise temporal coordination of the build-up and die-down of the transverse oscillations with the posterior displacement - by a gradual increase in the processivity of the force generators during metaphase and anaphase. Conclusions: The agreement between our results and modeling suggests that the same motor machinery underlies two different spindle motions in the embryo: the equal and opposite motors on each side of the AP axis drive oscillations whereas the imbalanced motors in the two halves of the embryo drive posterior displacement.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Mihail Sarov et al. (2008) European Worm Meeting "A first version of the C. elegans Transgeneome: genome scale set of tagged fosmid transgenes"

Vlarie Reinke, Stewart Kim, Anthony Hyman, Francis Stewart, John Murray, Robert Waterston, Michael Snyder, Mihail Sarov, Mei Zhong

[European Worm Meeting, 2008]

Protein tagging with fluorescent and/or affinity epitopes provides a. generic way to address gene function. Recently we have developed a 96 well. format recombineering pipeline that allows us to rapidly convert large. genomic DNA clones such as BACs or fosmids into tagged transgenes [1]. Due. to their large size, these transgenes usually contain most cis regulatory. elements and correctly recapitulate the endogenous patterns of gene. expression. Ballistic transformation of fosmid transgenes allows us to. routinely generate low copy integrated worm lines, with stable and. reproducible expression of the tagged protein in variety of tissues and. developmental stages including germline and early embryos.. The high efficiency and fidelity of this approach allows us to engineer. genome scale sets of fosmid transgenes with unprecedented throughput.. Generation of a comprehensive "Transgeneome" resource with a tagged. transgene for most C. elegans genes is now within reach. As a first step. towards this goal we started with several targeted projects that aim to. determine the physical interactions and the localization of more than 1000. proteins involved in transcription regulation, chromatin structure and. function and cell division. As a part of the ModENCODE consortium we have. already generated hundreds of transgenic constructs and tens of integrated. transgenic worm lines. That allowed us to test more than 20 different tag. combinations and we now have validated tags that perform very well for. protein localization, purification and chromatin immunoprecipitation. At. the meeting we will present results illustrating the power of the approach. as well as some of the lessons we learned from the large-scale application. of the method. We will discus how the community can access these resources. and we hope to initiate collaborations that will translate this research to. other functional gene sets.. 1. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang,. A.A Hyman, and A.F Stewart. 2006. A recombineering pipeline for. functional genomics applied to Caenorhabditis elegans. Nature Methods 3:. 839-844.

Mihail Sarov et al. (2007) International Worm Meeting "Recombineering based high throughput protein tagging in C. elegans."

Mihail Sarov, Francis Stewart, Anthony Hyman

[International Worm Meeting, 2007]

The methods for protein tagging in C. elegans so far have been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. The recent generation of a genomic fosmid library[1] opened the possibility for direct modification of any gene of interest in its native genomic environment by recombineering in E. coli. We recently published a method for precise in vivo engineering of large genomic clones into GFP tagged transgenes for ballistic transformation[2]. Using such transgenes, we reproduce known and document new expression patterns. We further demonstrate that the tagged protein can complement the function of its endogenous counterpart. Applying the latest developments of the recombineering field and introducing several innovations of our own we have now streamlined the protocol to allow rapid 96 well format liquid culture processing in a continuous recombineering pipeline that takes just four days. This unprecedented throughput and the achieved economy of scale allow us to rapidly produce thousands of tagged transgenes. Our method has become the basis for a large-scale project aimed at comprehensive description of the expression pattern (through fluorescent reporters) and the DNA binding sites (through affinity tag based chromatin immunoprecipitation) of more than 400 C. elegans transcription factor genes (as part of the NIH funded ModENCODE program). At the meeting we will discus the advantages of a distributed, community based collaboration model that will bring this technology to any worm lab and will make the genome wide protein tagging a feasible goal. 1. Perkins, J., K. Wong, R. Warren, J.E. Schein, J. Stott, R. Holt, S. Jones, M.A. Marra, and D. Moerman. 2005. A Caenorhabditis elegans fosmid library. In 15th International C. elegans Conference, University of California, Los Angeles. 2. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A.A. Hyman, and A.F. Stewart. 2006. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods 3: 839-844.

Li, Rachel C.K. et al. (2011) International Worm Meeting "Genetic and molecular investigation of PKD-2 and LOV-1 involved in male C. elegans perception of sex pheromone."

Li, Rachel C.K., Chow, King L.

[International Worm Meeting, 2011]

Transient receptor potential polycystin (TRPP) complex PKD1/PKD2 in human are important in kidney's function, and their mutations are associated with polycystic kidney disease. The C. elegans counterparts, PKD-2/LOV-1 localized to the ciliated endings of the male specific neuron CEMs, however, are indispensable for the male response to sex pheromone. In this study, we aim to identify additional PKD-2/LOV-1 polycystin signaling molecules required for sex pheromone perception. atp-2 and cwp genes are potential pkd-2 and lov-1 interacting partners based on their roles in male sensory behavior and their co-expression in CEMs. In our pheromone assays, all cwp mutant males respond normally while males with atp-2 knocked-down are defective, which suggest the distinct requirement of atp-2 in this sex-specific behavior. The interdependence between pkd-2, lov-1 and atp-2 will be ascertained by testing atp-2 RNAi males or its cell specific over-expression in pkd-2 and/or lov-1 mutants by pheromone assay. In addition, a pkd-2 promoter deletion analysis has been performed to identify upstream regulatory components of PKD-2/LOV-1 TRPP channel synthesis. As genes co-expressed in a specific cell type may share a common cis-regulatory motif, identifying the signature motif for CEM-specific expression may help uncover potential pkd-2 interacting partners by a genome-wide search for genes with a CEM-signature sequence. For the cellular function of PKD-2/LOV-1 TRPP complex in CEMs, we hypothesize that this complex is directly activated by sex pheromone and the signal is relayed intracellularly. This hypothesis will be evaluated by an in vitro system using Drosophila Schneider cells to investigate if physiological response can be triggered via PKD-2/LOV-1 TRPP complex by sex pheromone stimulation. Results from this experiment would be informative in defining the functional activity of these TRPP channels in the sex pheromone perception, shedding light on the biology and signaling of polycystin complex. (This study is supported by Research Grants Council, Hong Kong.).

Rodriguez, Josana et al. (2011) International Worm Meeting "Novel players of cell polarity and asymmetric cell division in C. elegans identified through analysis of a polarity genetic network."

Fievet, Bruno, Ahringer, Julie, Rodriguez, Josana

[International Worm Meeting, 2011]

Cell polarity and asymmetric cell divisions are essential for the generation of cellular diversity. The importance of these processes during development has been emphasized by recent findings, which show that defects lead to the formation of tumors. Towards identifying most of the factors involved in cell polarity and asymmetric cell division, we carried out 17 large scale RNAi suppressor screens of temperature sensitive mutants of genes involved in actomyosin contraction, par polarity and microtubule pulling forces governing spindle positioning. This allowed us to generate a polarity genetic interaction network of 186 genes with 229 genetic interactions (see Fievet et al., abstract). Of these genes, 23 have been previously shown to have a role in the asymmetric first cell division, validating our approach. For the rest of the candidates (87% of our network), no early polarity phenotype has been reported for knock-down in a wt background. To identify functions of novel candidates, we have been studying effects of their knockdown in sensitized backgrounds. This strategy has successfully identified specific functions for genes in the network. For example, out of the 24 identified suppressors of nmy-2(ts) (non-muscle myosin II), we found that eight enhance embryonic lethality when knocked-down in a gain of function mutant of act-2 (actin) and seven out of these eight enhancers show defects in the actomyosin cytoskeleton during the first cell division. In collaboration with Stephan Grill we are using biophysical methods to characterize actomyosin dynamics upon loss of function of these candidates. We are using a similar approach to characterize the suppressors of par-2(ts) and pkc-3(ts). Functional antagonism between PAR-2 and PKC-3 is critical for C. elegans asymmetric first cell division and robustly found in our screens. Therefore, we are studying the enhancement phenotype of par-2 suppressors in pkc-3(ts) and vice versa. In another strategy, we have been using patterns of genetic interactions to predict functions for novel genes in the network. This analysis identified several potential new components and/or regulators of the anaphase-promoting complex (APC). In addition, the network functionally implicates different signalling pathways in polarity. For example, we have identified a role for PKA signalling in the regulation of the asymmetric first cell division, which we are currently studying. Due to the conservation of cell polarity mechanisms, we expect that the C. elegans polarity network we have built will be relevant for cell polarity events in other model organisms.