Christina Tuskey et al. (2003) International Worm Meeting "Wnt signaling orients the mitotic spindle in the early C. elegans embryo"

Jeff Hardin, Timothy Walston, Christina Tuskey, Bruce Bowerman, Greg Ellis

[International Worm Meeting, 2003]

Wnt signaling in the early C. elegans embryo plays an essential role in specifying cell fate and positioning the mitotic spindle. At the 4-cell stage, the posterior-most blastomere, P2, induces its neighbor, EMS, to specify the endoderm of the entire animal. The induction of EMS is dependent on a Wnt signaling pathway that requires both SRC-1-mediated phosphotyrosine signaling as well as MAP kinase signaling within EMS. P2/EMS signaling also directs the mitotic spindle of EMS to rotate from its initial L/R orientation to ultimately align and divide along the A/P axis. The orientation of the EMS mitotic spindle has been shown to involve a noncanonical Wnt signaling pathway that is independent of gene transcription.Wnt signaling has also been shown to be vital for the orientation of the mitotic spindle of the ABar blastomere. In the 8-cell embryo, the mitotic spindle of ABar is oriented perpendicular to the other three granddaughters of AB. Several mom mutants have been shown to disrupt the orientation of the ABar mitotic spindle such that it orients and divides parallel to the other AB granddaughters. We show that the orientation of ABar is dependent on a noncanonical Wnt signaling pathway that branches at GSK-3. We also show that SRC-1 is involved in orienting the ABar spindle. In contrast to EMS spindle orientation, the removal of Wnt signaling components without removing SRC-1 is sufficient to cause ABar to divide improperly. Interestingly, depletion of WRM-1/-catenin causes an additional phenotype affecting the orientation of the ABar spindle. We show that depletion of WRM-1 or LIT-1, a MAP kinase pathway member, delays the rotation of ABar. Unlike the orientation of EMS, the ABar rotation is dependent on gene transcription through the POP-1 transcription factor. Thus, both a noncanonical and canonical Wnt signaling pathway is required for proper orientation and rotation of the ABar mitotic spindle.

Allison M Lynch et al. (2007) International Worm Meeting "A Feeding RNAi Screen Reveals Putative Interactors of the C. elegans Cadherin-Catenin Complex."

Allison M Lynch, Jeff Hardin, Christina Tuskey

[International Worm Meeting, 2007]

Morphogenesis is a fundamental process to the development of an organism that requires cells to be able to dynamically alter cell shapes and positions. The cadherin-catenin complex in worms, consisting of HMR-1/cadherin, HMP-1/alpha-catenin, and HMP-2/beta-catenin, was discovered through its role in regulating the morphogenesis of the hypodermis during ventral enclosure. A previously isolated hypomorphic hmp-1 allele, fe4, exhibits ~70% lethality and body shape defects due to a perturbed actin cytoskeleton. Worms homozygous for the fe4 mutation provided a sensitized genetic background that allowed for the isolation and characterization of molecules that contribute to the function of the cadherin-catenin complex (Pettitt, et al., 2003, J. Cell Biol. 162, 15-22). In continuing efforts to identify other regulators, we have employed the same rationale and conducted a genome-wide RNAi screen to identify genes whose loss of function enhances the fe4 phenotype. We have recently completed a screen of chromosome IV, and identified several genes that when knocked down by RNAi exhibit no phenotype in wild-type worms, but enhance the penetrance and severity of the fe4 mutation. We have focused our attention on MAGI-1, a MAGUK protein that is highly conserved across species. MAGI-1 localizes to adherens junctions and can interact with HMP-2 (Stetak, et al. European Worm Meeting Abstract. 2006) and actin-binding proteins (Patrie, et al., 2002, J. Biol Chem. 277, 30183-90). magi-1(RNAi) also enhances the severity of a hmp-2 hypomorph (qm39) that has not been well characterized to date. All magi-1(RNAi);hmp-1(fe4) and magi-1(RNAi);hmp-2(qm39) embryos arrest with a characteristic humpback phenotype. This suggests MAGI-1 may function with the cadherin-catenin complex and our current goal is to explore the role of MAGI-1 during epidermal morphogenesis of C. elegans.

Tim Walston et al. (2004) Mid-west Worm Meeting "The Dishevelleds and Casein Kinase I Regulate Spindle Orientation Through a Wnt Pathway Independent of Transcription"

Gregory Ellis, Bruce Bowerman, Tim Walston, Jeff Hardin, Nancy Hawkins, Christina Tuskey

[Mid-west Worm Meeting, 2004]

Cell division in the early Caenorhabditis elegans embryo is a highly coordinated event involving several signaling pathways. Components of Wnt signaling play a role in positioning the mitotic spindle in the EMS and ABar blastomeres independent of b -catenin. Wnt signaling also regulates endoderm induction through b -catenin and gene transcription. Because Dishevelleds act as integrators of multiple Wnt pathways, determining their roles in cell division and differentiation is key to understanding early development. We identify novel components of a Wnt signaling pathway that controls spindle orientation in EMS and demonstrate that the same pathway orients the spindle in ABar. We show that all three C. elegans Dishevelleds act redundantly to position the spindles of both blastomeres. We identify KIN-19/CKI as a member of the Wnt spindle alignment pathway, which does not require WRM-1/ b -catenin or gene transcription. We also show that Src signaling affects spindle orientation in ABar. Finally, we show that a separate Wnt/ b -catenin signaling pathway controls the timing of spindle rotation in ABar. We conclude that Wnt signaling, through the Dishevelleds, KIN-19/CKI and GSK-3 regulate spindle orientation independent of b -catenin and transcription. Wnt signaling also regulates the timing of spindle rotation through a separate pathway requiring b -catenin and gene transcription.

Christina Dittrich et al. (2006) European Worm Meeting "Identifying Novel Genes Involved in DNA Damage Response Pathways in C. elegans"

Michael O. Hengartner, Christina Dittrich

[European Worm Meeting, 2006]

Christina Dittrich and Michael O. Hengartner1. Upon DNA damage, a cell responds by arresting its cell cycle, changing its transcription patterns, recruiting the repair machinery or, most severely, by executing its own death by apoptosis. Malfunction of any of these pathways allows cells to replicate in spite of DNA lesions, and thus their genomes accumulate mutations and becomes unstable, a hallmark of tumor cells. Therefore, it is not only of paramount biological, but also of high medical interest, to understand the molecular pathways underlying DNA damage responses. Genotoxic stress induces in the germ line of Caenorhabditis elegans a spectrum of DNA damage responses similar to that observed in humans. Because important genes are often conserved throughout evolution, studies in C. elegans might improve our understanding of the molecular programmes safeguarding the genome of human cells. Wild-type C. elegans animals carry out proper DNA damage responses following exposure to ionizing radiation (IR), thus most of their progeny survive. In contrast, radiation-sensitive (Rad) mutants, which are defective in these pathways, show high levels of embryonic lethality upon IR. Taking advantage of this phenotype, we performed a forward genetic screen and identified 17 mutants whose embryos show reduced viability following IR treatment. Two of those mutants, op442 and op444, display a strong Rad phenotype. We are currently mapping the affected genes taking advantage of molecular polymorphisms between the Bristol and the Hawaii isolate of C. elegans. Additionally, we are characterising those two mutants further in order to learn more about the molecular nature underlying the observed defects.

Kavita Babu et al. (2005) International Worm Meeting "Function And Regulation Of Cell Adhesion Molecules At The C. elegans Synapse"

Joshua Kaplan, Kavita Babu

[International Worm Meeting, 2005]

Adhesion molecules play important roles at the synapse, which are the sites of communication between neurons and their targets. They are required for maintaining the integrity of the junction and promoting the stability of the synapse by linking the pre-synaptic and post-synaptic membranes. Target recognition, differentiation of pre-synaptic and post-synaptic specialisations and regulation of synaptic structure and function are perceived as important functions of synaptic adhesion molecules. My experiments aim to study the function of Cell Adhesion Molecules (CAMs) on synaptic growth/homeostasis and activity dependent plasticity. In addition my project has two long-term goals: to examine how CAMs are targeted to synapses and how the abundance of CAMs is regulated at the synapses, as relatively little is known about factors that mediate targeting and turnover (recycling/degradation) of CAMs at the synapse. To identify CAMs that regulate the body wall neuromuscular junction (NMJ) I have started an RNAi screen for changes in aldicarb sensitivity in the known set of 46 cell adhesion molecules which are encoded by the C. elegans genome (Cox et al., 2004). Aldicarb is an acetylcholine esterase inhibitor that causes hypercontraction of muscles in wild type animals. Mutants with defects in synaptic transmission have altered responses to aldicarb. Cell adhesion molecules that show either enhanced sensitivity or resistance to aldicarb will be further characterised using pre-synaptic and post-synaptic markers as well as soluble GFP markers. Further, protein fusions will be made of CAMs with GFP and CAMs that are expressed at neuron-neuron synapses or the NMJs will then be used for large scale RNAi screens looking for molecules that change the subcellular localisation of the CAM (seen as changes in CAM-GFP expression). This screen will help identify molecules involved in CAM localisation at the NMJ and further give insights on the regulation of CAMs at the synapse. Cox, E. A., Tuskey, C., and Hardin, J. (2004) J Cell Sci 117, 1867-1870.