Zeiser, Eva A. et al. (2009) International Worm Meeting "MosSCI and Gateway compatible toolkit for germline expression of transgenes."

Frokjaer-Jensen, Christian, Ahringer, Julie, Jorgensen, Erik, Zeiser, Eva A.

[International Worm Meeting, 2009]

Transgene silencing in the germline has hampered research in the germline and early embryo. The creation of "complex" extrachromosomal arrays using genomic DNA and then the development of technology for low copy number insertions using microparticle bombardment finally allowed germline expression of transgenes (Mello et al., 1991; Praitis et al., 2001). However, bombardment is very labor intensive and complex extrachromosomal arrays often are silenced. Recently, the Mos1 mediated Single Copy Insertion (MosSCI) method was developed to insert single copies of transgenes into defined sites in the genome of Caenorhabditis elegans (Frokjaer-Jensen et al., 2008). The technique is based on the MosTIC technique (Robert & Bessereau, 2007). Directed integration of a single copy avoids problems like overdosage, passage related loss of transgenes and locus mediated phenotypes. It also avoids extrachromosomal array related gene silencing in the germline which makes it especially attractive for studies on the gonad, and early embryos. We are using the MultiSite Gateway system to generate transgenes for germline gene expression. This method allows easy fusion of three different sequences in a defined order (5'', middle, and 3''). We have generated a set of 5'' constructs containing the germline active promoter pmex-5 (Merritt et al., 2008) on its own or fused to the sequences of GFP, EGFP, Citrine and Tag RFP-T. Similarly, we have generated a set of 3'' constructs by fusing GFP, EGFP or Citrine to the tbb-2 3''UTR (Merritt et al., 2008). Using an appropriate combination of 5'' and 3'' constructs with a middle ORF of one''s choice allows easy generation of N- or C-terminal fluorescently tagged transgenes for expression in the germline or early embryo. We also show that the hsp-16.2 and hsp-16.41 heat shock promoters can be used for inducible germline and early embryo gene expression as single copy MosSCI generated insertions.

D'Souza SA et al. (2016) PLoS Genet "The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans."

D'Souza SA, Bagg R, Roy PJ, Barbier L, Rajendran L, Moshiri H, van Pel DM

[PLoS Genet, 2016]

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

Chan KK et al. (2014) PLoS Genet "EVA-1 functions as an UNC-40 Co-receptor to enhance attraction to the MADD-4 guidance cue in Caenorhabditis elegans."

Bagg R, Chan KK, Seetharaman A, Zhang Y, Kim J, Selman G, Roy PJ

[PLoS Genet, 2014]

We recently discovered a secreted and diffusible midline cue called MADD-4 (an ADAMTSL) that guides migrations along the dorsoventral axis of the nematode Caenorhabditis elegans. We showed that the transmembrane receptor, UNC-40 (DCC), whose canonical ligand is the UNC-6 (netrin) guidance cue, is required for extension towards MADD-4. Here, we demonstrate that MADD-4 interacts with an EVA-1/UNC-40 co-receptor complex to attract cell extensions. EVA-1 is a conserved transmembrane protein with predicted galactose-binding lectin domains. EVA-1 functions in the same pathway as MADD-4, physically interacts with both MADD-4 and UNC-40, and enhances UNC-40's sensitivity to the MADD-4 cue. This enhancement is especially important in the presence of UNC-6. In EVA-1's absence, UNC-6 interferes with UNC-40's responsiveness to MADD-4; in UNC-6's absence, UNC-40's responsiveness to MADD-4 is less dependent on EVA-1. By enabling UNC-40 to respond to MADD-4 in the presence of UNC-6, EVA-1 may increase the precision by which UNC-40-directed processes can reach their MADD-4-expressing targets within a field of the UNC-6 guidance cue.

Fujisawa K et al. (2007) Science "The slit receptor EVA-1 coactivates a SAX-3/Robo mediated guidance signal in C. elegans."

Wrana JL, Fujisawa K, Culotti JG

[Science, 2007]

The SAX-3/roundabout (Robo) receptor has Shiga-like toxin 1 (SLT-1)/Slit-dependent and -independent functions in guiding cell and axon migrations. We identified enhancer of ventral-axon guidance defects of unc-40 mutants (EVA-1) as a Caenorhabditis elegans transmembrane receptor for SLT-1. EVA-1 has two predicted galactose-binding ectodomains, acts cell-autonomously for SLT-1/Slit-dependent axon migration functions of SAX-3/Robo, binds to SLT-1 and SAX-3, colocalizes with SAX-3 on cells, and provides cell specificity to the activation of SAX-3 signaling by SLT-1. Double mutants of eva-1 or slt-1 with sax-3 mutations suggest that SAX-3 can (when slt-1 or eva-1 function is reduced) inhibit a parallel-acting guidance mechanism, which involves UNC-40/deleted in colorectal cancer.

Kazuko Fujisawa et al. (2002) East Coast Worm Meeting "Identification of Genes Specifically Required for Ventralward Axon Guidance in C. elegans"

Kazuko Fujisawa, Joe Culotti

[East Coast Worm Meeting, 2002]

Several conserved classes of guidance molecules direct axon guidance in C. elegans and other organisms. The UNC-6/netrin diffusible guidance cue is required for axon guidance along the dorsal-ventral axis in C. elegans, Drosophila and vertebrates. The UNC-5 guidance receptor in combination with UNC-40/DCC mediates repulsive response to the UNC-6 which is expressed in ventral nerve cord and ventral epidermis, to orient growing axons in a dorsal direction. Attractive responses to UNC-6 require UNC-40 but not UNC-5. We and the laboratory of C. Bargmann have reported that SAX-3/Robo mediates repulsive response to SLT-1/Slit which is expressed in dorsal body wall muscles, to orient axons in a ventral direction. eva-1, enhancer of ventralward axon guidance-1, has AVM ventralward axon guidance defects. We have rescued eva-1 mutations with overlapping cosmids. We have found that the eva-1 mutation enhances the AVM and PVM guidance defects of unc-6 null or unc-40 null alleles, suggesting that EVA-1 functions in an axon guidance pathway that acts in parallel with UNC-6 and UNC-40. We have also found that eva-1 mutation doesn't enhance the AVM nor PVM guidance defects of slt-1 null allele or sax-3 strongest allele (ky123), suggesting that EVA-1 functions in the same pathway as SLT-1 and SAX-3.

Tegha-Dunghu J et al. (2014) Methods Mol Biol "Measuring microtubule growth and gliding in Caenorhabditis elegans embryos."

Srayko M, Tegha-Dunghu J, Gusnowski EM

[Methods Mol Biol, 2014]

Microtubule plus-tip tracking is a powerful method to measure microtubule growth dynamics in vivo. Here we outline an approach that exploits live confocal microscopy of a GFP-tagged EB1-like protein to measure microtubule growth behavior and minus-end-directed microtubule motor activity at the cortex of Caenorhabditis elegans embryos. The EB1 velocity assay (EVA) provides a method to reproducibly monitor motor- and non-motor-assisted microtubule movements.

Marcus, Nancy et al. (2011) International Worm Meeting "The Rac-GEF UNC-73/TRIO Mediates Multiple and Genetically Distinct Pathways to regulate excretory cell migration."

Stringham, Eve, Marcus, Nancy

[International Worm Meeting, 2011]

The actin cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell (Stringham, et al., 2002; Schmidt et al., 2009) while the kinesin like motor VAB-8 is essential for most posteriorly directed migrations in C. elegans (Wightman et al., 1996). Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell specific rescue experiments, suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double mutant combination with either UNC-53 or VAB-8. Collectively, our results suggest a bipartite model for UNC-73/TRIO function, where the UNC-73E isoform, together with UNC-53 and UNC-71 functions cell autonomously within the excretory cell to promote outgrowth, while the UNC-73B isoform through partnering with VAB-8 and the receptors SAX-3 and EVA-1 has a cell non-autonomous function in excretory cell outgrowth.

Bulow HE et al. (2008) Curr Biol "Extracellular sugar modifications provide instructive and cell-specific information for axon-guidance choices."

Tjoe N, Bulow HE, van Kuppevelt TH, Townley RA, Didiano D, Hobert O

[Curr Biol, 2008]

Heparan sulfates (HSs) are extraordinarily complex extracellular sugar molecules that are critical components of multiple signaling systems controlling neuronal development [1-3]. The molecular complexity of HSs arises through a series of specific modifications, including sulfations of sugar residues and epimerizations of their glucuronic acid moieties. The modifications are introduced nonuniformly along protein-attached HS polysaccharide chains by specific enzymes [4]. Genetic analysis has demonstrated the importance of specific HS-modification patterns for correct neuronal development [2, 3]. However, it remains unclear whether HS modifications provide a merely permissive substrate or whether they provide instructive patterning information during development. We show here with single-cell resolution that highly stereotyped motor axon projections in C. elegans depend on specific HS-modification patterns. By manipulating extracellular HS-modification patterns, we can cell specifically reroute axons, indicating that HS modifications are instructive. This axonal rerouting is dependent on the HS core protein lon-2/glypican and both the axon guidance cue slt-1/Slit and its receptor eva-1. These observations suggest that a changed sugar environment instructs slt-1/Slit-dependent signaling via eva-1 to redirect axons. Our experiments provide genetic in vivo evidence for the "HS code" hypothesis which posits that specific combinations of HS modifications provide specific and instructive information to mediate the specificity of ligand/receptor interactions [3, 5, 6].

Marcus-Gueret N et al. (2012) Genetics "Distinct cell guidance pathways controlled by the Rac and Rho GEF domains of UNC-73/TRIO in Caenorhabditis elegans."

Marcus-Gueret N, Stringham EG, Schmidt KL

[Genetics, 2012]

The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell while the kinesin-like motor VAB-8 is essential for most posteriorly directed migrations in Caenorhabditis elegans. Null mutations in either unc-53 or vab-8 result in reduced posterior excretory canal outgrowth, while double null mutants display an enhanced canal extension defect, suggesting the genes act in separate pathways to control this posteriorly directed outgrowth. Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell-specific rescue experiments suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit, and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double-mutant combination with either unc-53 or vab-8. On the basis of these results, we propose a bipartite model for UNC-73/TRIO activity in excretory canal extension: a cell autonomous function that is mediated by the Rho-specific GEF domain of the UNC-73E isoform in conjunction with UNC-53 and UNC-71 and a cell nonautonomous function that is mediated by the Rac-specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1.

Rella L et al. (2021) Proc Natl Acad Sci U S A "A switch from noncanonical to canonical Wnt signaling stops neuroblast migration through a Slt-Robo and RGA-9b/ARHGAP-dependent mechanism."

Schoppink L, Mars J, Fernandes Povoa EE, Rella L, Betist MC, Ebbing ALP, Korswagen HC

[Proc Natl Acad Sci U S A, 2021]

Members of the Wnt family of secreted glycoproteins regulate cell migration through distinct canonical and noncanonical signaling pathways. Studies of vertebrate development and disease have shown that these pathways can have opposing effects on cell migration, but the mechanism of this functional interplay is not known. In the nematode <i>Caenorhabditis elegans</i>, a switch from noncanonical to canonical Wnt signaling terminates the long-range migration of the QR neuroblast descendants, providing a tractable system to study this mechanism in vivo. Here, we show that noncanonical Wnt signaling acts through PIX-1/RhoGEF, while canonical signaling directly activates the Slt-Robo pathway component EVA-1/EVA1C and the Rho GTPase-activating protein RGA-9b/ARHGAP, which are required for migration inhibition. Our results support a model in which cross-talk between noncanonical and canonical Wnt signaling occurs through antagonistic regulation of the Rho GTPases that drive cell migration.