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.

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.

Rella, L. et al. (2019) International Worm Meeting "A switch from non-canonical to canonical Wnt signaling stops QR descendant migration through a Slt/Robo and RGA-9/RhoGAP dependent mechanism."

Fernandes Povoa, E., Ebbing, A., Betist, M.C., Korswagen, H.C., Rella, L.

[International Worm Meeting, 2019]

Members of the Wnt family of secreted signaling proteins are key regulators of cell migration, but the underlying signaling mechanisms that determine whether the cell moves, changes direction or stops are still poorly understood. The C. elegans QR lineage provides a powerful model system to study Wnt-dependent cell migration at single cell resolution. We have previously shown that the migration of the QR descendants (QR.d) relies on the non-canonical CAM-1/Ror and MOM-5/Frizzled pathways that separately control polarity and migration. Moreover, we found that once the QR.d reach their final position, they stop their migration by switching to canonical, beta-catenin dependent Wnt signaling. Here, we show that canonical Wnt signaling counteracts the MOM-5/Fz-dependent migration pathway. To gain insight into this cross-talk mechanism, we performed mRNA sequencing on isolated QR.d and found that canonical Wnt signaling induces a specific transcriptional program. Among the target genes that are upregulated upon activation of canonical Wnt signaling are two genes that are both necessary and sufficient for migration termination: eva-1, which encodes a component of the Slt/Robo pathway, and rga-9, which encodes a conserved Rho GTPase activating protein (GAP). Consistent with the role of EVA-1 in Slt/Robo signaling, slt-1/Slt, sax-3/Robo and the putative downstream effector srgp-1/srGAP are also required. Rho GTPases are important regulators of cell polarity and migration, and several family members function partially redundantly in QR.d migration. Our results indicate that these Rho GTPases are at the center of the interaction between the MOM-5/Fz and canonical Wnt pathways. Thus, epistasis analysis suggests that MOM-5/Fz acts through PIX-1, a guanine nucleotide exchange factor (GEF) that promotes Rho activity. Canonical Wnt signaling, on the other hand, induces RGA-9 expression and may activate SRGP-1 through Slt/Robo signaling, both of which are predicted to inhibit Rho activity. Based on these results, we propose that cross-talk between canonical and non-canonical Wnt signaling at the level of Rho activators and inhibitors controls the highly stereotypic migration of the QR descendants.

Eva Brozova et al. (2006) European Worm Meeting "NHR-40, a Caenorhabditis elegans Supplementary Nuclear Receptor Regulates the Development of Embryos and L1 Larvae"

Katerina Simeckova, Zdenek Kostrouch, Eva Brozova, Marta Kostrouchova

[European Worm Meeting, 2006]

Eva Brozova1, Katerina Simeckova1, Zdenek Kostrouch2, Marta Kostrouchova1 In this study, we functionally characterized NHR-40, a member of an extended group of supplementary nuclear receptors (Robisnon-Rechavi et al, J. Mol. Evol. 2005, 60: 577-586). We show that nhr-40 is expressed in 3 isoforms that are regulated by two promoters organized in tandem. The function of NHR-40 was studied by RNA interference, by overexpression of nhr-40 and using a mutant strain, RB840, which carries a deletion in the first intron. We show that NHR-40 regulates embryonic and early larval development. This phenotype is connected with defective movement and muscle cell development. This adds NHR-40 to the growing list of nematode NHRs involved in the regulation of development.. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi, M.W. Krause for support and advice, The C.elegans Gene Knockout Consortium for preparation of the deletion strain RB840. The work was supported by grants 303/03/1115 and 301/ 05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Petr Liby et al. (2006) European Worm Meeting "Inhibition of bir-1, the Homologue of Human Survivin, Induces Changes of Expression of Developmentally Active Collagen Genes in L1 Larval Stage"

Marta Kostrouchova, Eva Brozova, Marketa Kostrouchova, Michal Pohludka, Jaroslav Vohanka, Petr Liby, Zdenek Kostrouch

[European Worm Meeting, 2006]

Petr Liby1, Marketa Kostrouchova1, Michal Pohludka1, Jaroslav Vohanka2, Eva Brozova2, Marta Kostrouchova2 and Zdenek Kostrouch1. BIR-1 is a member of the Inhibitors of Apoptosis protein family that is involved in the regulation of microtubule organization, condensation of chromosomes and cell division in C. elegans as well as in vertebrates. In C. elegans, bir-1 is organized in an operon together with SKIP, a transcription and splicing cofactor. bir-1 inhibition induces developmental defects including dpy and egl phenotypes. Here we studied the effect of bir-1 inhibition by RNAi in L1 larval stage using whole genome microarrays (Affymetrix). Microarrays detected changes in expression of several developmentally active collagen genes in bir-1 inhibited larvae. We further characterized the effect of bir-1 inhibition on selected collagen gene expression and induction of dpy phenotype. Acknowledgement: We thank Drs. A. Fire for GFP construct, vectors and host used in RNAi and M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Katerina Simeckova et al. (2006) European Worm Meeting "NHR-60, a Caenorhabditis elegans Supplementary Nuclear Receptor residing at the Nuclear Periphery, Regulates Embryonic Development in Connection with Acyl-Coenzyme a Binding Protein"

Zdenek Kostrouch, Eva Brozova, Marta Kostrouchova, Jaroslav Vohanka, Katerina Simeckova, Michal Pohludka

[European Worm Meeting, 2006]

Katerina Simeckova1, Eva Brozova1, Jaroslav Vohanka1, Michal Pohludka2, Zdenek Kostrouch2, Marta Kostrouchova1 . NHR-60 is a Caenorhabditis elegans supplementary nuclear receptor that belongs to a subset of 18 of the 284 nuclear hormone receptors characterized by the P-box sequence CNGCKT. We show that NHR-60 is expressed ubiquitously and is predominantly localized at the nuclear periphery. The expression of NHR-60 is accented in seam cells, gland cells and in the germline. In seam cells, the expression of NHR-60 is dependent on CHR3 (NHR-23). Our research also focused on the possible interaction of Acyl-CoA-binding protein, which is known to interact with several nuclear hormone receptors, and NHR-60. The interaction in vivo between these two proteins was confirmed by co-immunoprecipitation and co-localization using confocal microscopy. acbp-1 inhibition by RNAi induced developmental defects similar to nhr-60 RNAi. These results indicate that regulation of development by nuclear hormone receptor NHR-60 is functionally linked to Acyl-CoA-binding protein and may constitute a connection with fat metabolism. Acknowledgement: We thank Drs. A. Fire for vectors and host used in RNAi and M.W. Krause for support and advice. The work was supported by grants 303/03/1115 and 301/05/0859 from the Czech Science Foundation and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.

Sanchez, Sophia et al. (2021) International Worm Meeting "Systematic Behavioral Screen of 21st Chromosome Gene Overexpression in C. elegans"

Bigham, Caymee, Syed, Briana, Smith, Sofia, Nordquist, Sarah, Pierce, Jon, Perks, Katherine, Sanchez, Sophia

[International Worm Meeting, 2021]

Down syndrome (DS) is caused by overexpression of 21st chromosome (Hsa21) genes due to the presence of an extra copy of Hsa21. A major goal in DS research is to understand the individual contribution of each Hsa21 gene to the numerous phenotypes that accompany DS. Progress is being made using mouse models to understand the role of certain Hsa21 genes. However, most Hsa21 genes have not been studied in any detail. To circumvent the time and cost of mouse models for single-gene studies, we are leveraging the speed and affordability of the nematode C. elegans. Our lab found that C. elegans has 51 highly-conserved orthologs of Hsa21 genes. By studying loss-of-function mutants and performing RNAi for these orthologs, we found that 14 are essential and 10 are required for neural or muscular function in C. elegans -- 3 of which had not previously been studied. Through epistasis analysis, we found that the novel molecule, MTQ-2, appears to modify G-protein signaling to regulate rates of cholinergic synaptic transmission. Additionally, to probe how overexpression (OE) of individual Hsa21 genes contributes to neuronal, muscular, and developmental phenotypes related to DS, we are generating a complementary set of 51 C. elegans transgenic strains that each overexpress a different Hsa21 ortholog. We are performing quantitative behavioral analyses with the OE strains to deduce which genes cause neural or muscular dysfunction when overexpressed. Thus far, we discovered a specific behavioral defect for six OE strains including EVA-1, a novel slit receptor involved in axon guidance recently discovered in C. elegans. By identifying Hsa21 genes that cause phenotypes when overexpressed in C. elegans, this study will highlight individual Hsa21 genes and pathways to prioritize for further study in other models and to consider as potential therapeutic targets for improving health in those with DS.

Sanchez, S. et al. (2019) International Worm Meeting "Systematic functional analysis of 21st chromosome genes using C. elegans."

Pierce, J., Sanchez, S., Nordquist, S., Smith, S.

[International Worm Meeting, 2019]

Down syndrome (DS), caused by trisomy of the 21st chromosome, leads to lifelong cognitive impairment. Efforts to understand this disorder first require an understanding of the genes encoded on the 21st chromosome (HSA21). While progress has been made using traditional mouse models, C. elegans represents a complementary model to uncover the in vivo function of genes. We analyzed all 213 predicted protein-coding genes on HSA21 and found that, excluding the keratin genes, approximately half have orthologs represented in worm. Using RNAi and loss-of-function mutants, we systematically investigated the role of all orthologs in viability and neuronal function. We identified ten HSA21 orthologs that are required for neuromuscular behaviors: cle-1 (COL18A1), cysl-2 (CBS), dnsn-1 (DONSON), eva-1 (EVA1C), mtq-2 (N6ATM1), ncam-1 (NCAM2), pad-2 (POFUT2), pdxk-1 (PDXK), rnt-1 (RUNX1), and unc-26 (SYNJ1). We also found that three of these genes are required for normal release of the neurotransmitter acetylcholine. This includes a known synaptic gene unc-26 (SYNJ1), as well as uncharacterized genes pdxk-1 (PDXK) and mtq-2 (N6ATM1). Furthermore, we found that the glutamine methyltransferase MTQ-2 localizes to cholinergic synapses where it appears to regulate neurotransmission via methylation of a G?/o signaling protein. Going forward, we are overexpressing each of the HSA21 orthologs in worm. In some cases, overexpression of orthologs causes highly penetrant behavioral phenotypes. Overexpression of other orthologs causes no phenotype for the majority, but a severe phenotype for a minority of worms. At least one gene showed no obvious phenotype when overexpressed. We are therefore investigating which of the orthologs are subject to either direct or indirect forms of compensation when overexpressed. Differences in compensation could underlie this phenotypic disparity in worms, and potentially may account for the puzzling variable penetrance and expressivity in individuals with DS. As the first systematic functional analysis of HSA21 orthologs, our study may serve as a platform to understand genes that underlie phenotypes associated with DS.

Kosalka, J. et al. (2015) International Worm Meeting "Mechanism and regulation of piRNA biogenesis in the C. elegans germline."

Weick, EM., Miska, E., Kosalka, J.

[International Worm Meeting, 2015]

Mechanism and regulation of piRNA biogenesis in the C. elegans germlineJoanna Kosalka, Eva-Maria Weick, Eric A MiskaWellcome Trust/CRUK Gurdon Institute/Department of Genetics, University of Cambridge, Cambridge, United KingdomPiwi-interacting RNAs (piRNAs) are the most recently identified and by far the largest class of endogenous small regulatory RNAs (sRNAs). In C. elegans, piRNAs are synthesised as single strand precursors from specific genomic loci known as piRNA clusters, processed into mature 21 nucleotides long, 5'U sRNAs that are recognised by the PIWI clade of Argonaute proteins. piRNAs are germline-expressed and are essential for fertility and genome stability. piRNAs can align to transposons and repetitive sequences in an antisense orientation, targeting them for silencing and the loss of the piRNA pathway results in reactivation of transposons. In addition, some endogenous protein-coding genes are under control of the piRNA pathway.Many aspects of the C. elegans piRNA pathway remain poorly understood and elucidating the biogenesis of 21U RNAs is of particular interest. Importantly, the fundamental questions of how the synthesis of piRNA precursors is initiated and directed to specific loci, and how the maturation of the precursor molecules is executed, remain unanswered. Furthermore, the developmental regulation of piRNA expression is unknown.In a forward genetic screen our lab has identified prde-1 (piRNA defective) as an essential germline-expressed factor for piRNA biogenesis (Weick et al. 2014). prde-1 is involved in the initial steps of piRNA biogenesis and is indispensable for piRNA precursor synthesis and/or stability. Interestingly, PRDE-1 protein localises specifically to parts of chromosome IV coinciding with the large piRNA cluster. Although the molecular mechanisms of prde-1 function are currently unknown, these findings provide an exciting starting point to elucidate the mechanisms of piRNA cluster transcription and regulation.Here we will present our most recent progress on understanding the molecular machinery required for the biogenesis of piRNAs. We used a range of biochemical approaches to identify and characterise PRDE-1-interacting proteins and their role in piRNA biogenesis.