Cheng, Eric et al. (2021) International Worm Meeting "The role of the Insulin Signaling Pathway in C. elegans Germline Stem Cell Mitosis"

Gerhold, Abigail, Cheng, Eric

[International Worm Meeting, 2021]

The spindle assembly checkpoint (SAC) monitors kinetochore-microtubule attachments and halts mitotic progression in the presence of unattached or incorrectly attached chromosomes. The SAC ensures the fidelity of chromosome segregation during mitosis by guarding against chromosome mis-segregation and aneuploidy. While the events and mechanisms that control the SAC are relatively well understood, how they are influenced by developmental and environmental signaling networks remains unclear. The insulin signaling (IIS) pathway is a universally important and highly conserved pathway that integrates cell proliferation and development in many organisms. We are using C. elegans germline stem cells (GSCs) to ask how the IIS pathway affects mitotic duration and fidelity in vivo via the in situ live imaging of these mitotically dividing cells. This pathway has a known role in cell cycle progression, with daf-2 mutants having fewer germ cells and a lower mitotic index, compared to stage-matched N2 animals, due primarily to a delay in the G2 phase of the cell cycle. Removal of daf-18 or daf-16 restores normal germ cell numbers suggesting canonical signaling from DAF-2 to DAF-16 affecting the rate of cell cycle progression. While clear links have been established between IIS and GSC proliferation, IIS involvement in events during mitosis, including the SAC, remains unclear. Evidence from cell culture models suggests that IIS may play a role during mitosis; however, it is not clear whether this occurs under in vivo physiological conditions. We have found that daf-2 mutants have an increased duration of mitosis and decreased number of cells entering mitosis. Both effects can be rescued by subsequent deletion of daf-18 or daf-16. In addition, daf-2 mutants have an increased incidence of chromosome segregation errors when the SAC is compromised, suggesting impairment of spindle assembly. Ongoing work will assess whether these effects are cell autonomous and will determine the underlying cause of the observed increase in chromosome segregation errors.

Irene Zelepuhin et al. (2005) International Worm Meeting "Genetics of sex Reversal of C. briggsae::C. remanei XO hybrids."

Scott Baird, Shannon Romer, Irene Zelepuhin

[International Worm Meeting, 2005]

Sterile F1 adult hybrids can be obtained from crosses of C. briggsae males to C. remanei females. Depending on the strains used, all hybrids are females. The absence of male hybrids results from sexual transformation, not male-specific lethality, as both XX and XO females are observed. Variant strains of C. briggsae and C. remanei suppress this hybrid sexual transformation, demonstrating genetic variation within both species the affects sex determination in F1 hybrids. This variation has been used to map hybrid sex reversal loci. In C. remanei, an ill-defined region of the X chromosome is implicated. In C. briggsae, three loci linked to hybrid sex reversal have been identified. The strongest linkage is to allelic variants in Cb-tra-2. A null mutation in Cb-tra-2 (thanks Eric) completely suppresses the transformation of XO hybrids. Other C. briggsae loci linked to sex reversal variants include Cb-daf-5 and Cb-mab-9.

Sawh, Ahilya et al. (2015) International Worm Meeting "Regulation of DCR-1-dependent RNAi pathways through DCR-1 phosphorylation."

Duchaine, Thomas, Wohlschlegel, James, Lewis, Alexandra, Sawh, Ahilya

[International Worm Meeting, 2015]

RNA interference (RNAi) pathways are directed by small (18-22nt) non-coding RNAs and orchestrate a variety of evolutionarily-conserved gene-silencing programs. The type III RNase protein Dicer is at the center of most of these pathways, including responses triggered by exogenous sources of long double-stranded RNA (exoRNAi). Other pathways, such as microRNA-mediated silencing and endoRNAi are triggered by endogenous loci, and control developmental and homeotic events. Dicer's role is crucial and three-fold: it cleaves long dsRNA precursors into smaller RNAs that direct silencing events, it loads the small RNAs into Argonaute proteins to form an active RNA-induced silencing complex (RISC), and it acts as a scaffold for several protein co-factors required for substrate recognition and processing. In C. elegans, Dicer (DCR-1) is essential in three small-RNA mediated silencing mechanisms including exoRNAi and ERI endoRNAi, two RNAi pathways that compete for downstream effectors. DCR-1 is part of distinct multi-protein complexes in each of these RNAi pathways: RDE complex in exoRNAi and ERIC in ERI endoRNAi.We discovered and validated several phosphorylated determinants of DCR-1 in the C. elegans. Particular attention was placed on a cluster of phosphorylated sites between the PAZ and RNase III domains, one of which could implicate AMPK signaling. Recent crystal structures of human Dicer have shown that this region could be involved in recognition of dsRNA substrates. We thus hypothesized that phosphorylation in this cluster could impair substrate recognition, or alter its specificity.To study the effect of DCR-1 phosphorylation on the three DCR-1-dependent RNAi pathways in C. elegans, transgenic strains were engineered expressing phosphorylation-deficient, and phosphorylation-mimetic mutants of DCR-1 in this cluster of sites. Functional analysis on these strains revealed that phosphorylation-deficient mutants display an impaired exoRNAi response, and severe developmental defects. Phosphorylation-deficient DCR-1 mutants also enhance interaction with ERI-endoRNAi DCR-1 cofactors. Taken together, these results support a model wherein DCR-1 phosphorylation may promote its shift from the ERIC to the RDE complex. Post-translational regulation of Dicer and RNAi pathways are largely unexplored and could potentially be incredibly impactful in light of their crucial roles in development and disease. The present study will shed light on how RNAi pathways integrate signals through phosphorylation signaling cascades.

G Kao et al. (1999) International C. elegans Meeting "Characterization of Laminin beta and gamma Genes."

G Kao, C Huang, WG Wadsworth

[International C. elegans Meeting, 1999]

We have characterized laminin b (lam-1) and laminin g (lam-2) genes. The sequencing project has uncovered genes for one isoform each of the two subunits and two isoforms of laminin a , epi-1 and lam-3. (See abstract by Cheng et al.). This indicates that two types of trimers likely are form in basement membranes : a A bg and a B bg . A hypomorphic mutation (rh219) exists in lam-1, whose phenotypes we have described in previous meetings (IWM 1997). Both lam-1(RNAi) and lam-2(RNAi) animals arrest development during morphogenesis. Significantly, this phenotype is identical to the arrest seen in double knockout of epi-1 and lam-3, and is more severe than either knockout alone. We examined the expression patterns of lam-1 and lam-2 using promoter:GFP fusions. Both genes are expressed in body wall muscles, pharynx, vulval muscles, the rectal muscle group and the distal tip cells. The expression patterns of b and g subunit genes overlap with and are more widespread than either epi-1 or lam-3 alone. Taken together these results suggest that lam-1 and lam-2 are found in all basement membranes and indicate that trimers a A bg and a B bg are formed.

Cho, Jihye et al. (2021) International Worm Meeting "Identify the function of mechanosensitive channel PEZO-1 in C. elegans males"

Cho, Jihye, Kim, Kyuhyung

[International Worm Meeting, 2021]

The conversion of mechanical force to biological signals is crucial for the survival of animals. Mechanosensitive ion channels play a direct role in sensing physical stimuli and are evolutionarily conserved from bacteria to mammals. In mammals, PIEZO channels have been identified as ion channels that directly detect mechanical stimuli (Coste et al., 2010, Gnanasambandam et al., 2015, Syeda et al., 2015). C. elegans genome has a single PIEZO gene, pezo-1, which encodes 14 isoforms (Bai X et al., 2020). However, the function of PEZO-1 in C. elegans has not been fully understood yet. To investigate its function, we first grouped 14 isoforms into short or long isoforms depending on the mRNA length and observed their expression patterns. We found that the promoter region of short isoforms is expressed in several tail neurons, including the 6th ray neurons of C. elegans males. Nine pairs of male ray neurons appear to have functional specialization and have been implicated to be involved in mating behavior (Zhang H, Yue X, Cheng H, et al., 2018). Interestingly, sensory endings of ray 6 neurons, but not other ray neurons, are not exposed to the external environment (Sulston and Horvitz, 1977), suggesting a distinct role in mechanotransduction during C. elegans mating. We are currently examining the contribution of PEZO-1 to mating behavior in males.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Hamill, Danielle R. et al. (2009) International Worm Meeting "Analysis of a spindle assembly mutant in C. elegans."

Hamill, Danielle R., Everett, Molly, Bowerman, Bruce, Price, Meredith, McNeeley, Marie, Mazhar, Sahar, Gearica, Amy

[International Worm Meeting, 2009]

Cell division is a highly conserved and complex process that must be carefully orchestrated. or452ts was isolated in a screen for temperature sensitive embryonic lethal cell division mutants. In or452ts mutant embryos, bipolar spindles often fail to form. In these mutants DNA localizes around microtubules emanating from a single, often misshapen, centrosome. Staining with centriole markers suggests that a centriole duplication defect may underlie the spindle assembly defect. Using live cell imaging and fluorescence transgenes (including GFP::gamma tubulin), we have noticed centrosome streaking and splitting that is not seen in wild-type embryos. We also have observed the formation of asymmetric spindles. Towards the goal of identifying the gene that is mutated in or452ts, we have done meiotic mapping using phenotypic markers and snip-SNPs. We have narrowed the genetic location down to about 0.5 cM around +2.5 on LGIII. This region contains less than 500 kbp of DNA, and there are about 40 predicted genes in this region. In collaboration with Doug Turnbull and Eric Johnson (University of Oregon), we are sequencing the DNA from this region to try to identify the mutation responsible for the or452ts mutant phenotype. We believe that characterization of this mutant, and identification of the gene responsible, will provide new insights into the process of mitotic spindle assembly and cell division.

Riga, Amalia et al. (2019) International Worm Meeting "The role of basolateral polarity regulators in epithelial tissue homeostasis."

Boxem, Mike, Pires, Helena, Riga, Amalia

[International Worm Meeting, 2019]

Polarization of epithelial cells into apical and basolateral domains is essential for the functioning of epithelia as selectively permeable barriers. The basolateral Scribble group proteins (Scrib, Lgl, Dlg) were originally identified as tumor suppressors in D. melanogaster and were later shown to play key roles in the establishment and maintenance of polarity in a broad range of cell types. In addition to promoting basolateral identity, Scribble group proteins regulate diverse processes including tissue growth, differentiation and directed cell migration, and therefore are major regulators of tissue development and homeostasis. In C. elegans, loss of LET-413/Scrib and DLG-1/Dlg results in defects in epithelial polarization and junction formation during embryonic development, resulting in embryonic lethality. However, their importance in larval epithelia is less well understood. To study the role of LET-413 in larval epithelia, we generated an allele that allows inducible protein degradation using the auxin-inducible system recently developed for C. elegans (1). We find that ubiquitous degradation of LET-413 in larval tissues results in a developmental arrest. Tissue-specific degradation of LET-413 in the larval intestine does not cause defects in development. In contrast, degradation of LET-413 in the seam cells and hypodermis mimics the developmental arrest seen upon ubiquitous LET-413 degradation. Live-cell imaging of seam cells shows a severe effect on outgrowth and reattachment following the L1 asymmetric division. In addition, we observed that loss of LET-413 results in punctate and discontinuous localization pattern of DLG-1, similar to previous observations in C. elegans embryos. We are currently following the effects of LET-413 inactivation on cell recognition and migration pathways to understand the outgrowth defects of the seam cells. (1) Zhang L, Ward JD, Cheng Z, Dernburg AF. Development. 2015 Dec 15;142(24):4374-84.

Michael Stitzel et al. (2007) International Worm Meeting "The meiotic cell cycle regulates the EGG-3/MBK-2 cortical complex essential for the oocyte-to-zygote transition."

Geraldine Seydoux, Michael Stitzel

[International Worm Meeting, 2007]

The oocyte-to-zygote transition transforms a differentiated germ cell into a totipotent zygote. The signals that activate this transition remain poorly understood. Here we report that regulation by the meiotic cell cycle of the MBK-2 kinase and its cortical anchor EGG-3 is one of the signals that initiates the oocyte-to-zygote transition in C. elegans. The DYRK kinase MBK-2 marks oocyte proteins for degradation shortly after fertilization during the second meiotic division (Nishi & Lin, 2005; Stitzel et al., 2006). GFP:MBK-2 accumulates on the cortex of oocytes and relocalizes to the cytoplasm during the second meiotic division. Cortical localization of MBK-2 depends on EGG-3, a cortical oocyte protein also essential for the oocyte-to-zygote transition (Maruyama and Singson, pers. comm). MBK-2 and EGG-3 interact directly in vitro, suggesting that EGG-3 may function as a transient cortical anchor for MBK-2 (see abstract by Cheng & Seydoux). Using cell cycle and egg-3 mutants, we found that MBK-2 is regulated both positively by regulators of meiotic M phase and negatively by EGG-3. Activators of meiotic M phase activate MBK-2 kinase activity upon oocyte maturation. EGG-3 then sequesters active MBK-2 at the cortex until the second meiotic division. This system ensures that oocyte proteins become marked for degradation only at the end of the meiotic divisions, when they are no longer needed and the egg is ready to embark on embryonic development. Remarkably, the meiotic cell cycle appears to be driving these changes independently of fertilization. We propose that, in addition to regulating chromosome segregation, the meiotic cell cycle controls egg-wide developmental changes essential for the initiation of embryonic development.

P Appleford et al. (2004) European Worm Meeting "MUTAGENESIS OF CONSERVED ELEMENTS IN THE PROMOTER OF THE C. ELEGANS T-BOX GENE MAB-9 WITH A VIEW TO IDENTIFYING UPSTREAM REGULATORS."

P Appleford, A Woollard

[European Worm Meeting, 2004]

The Caenorhabditis elegans T-box gene mab-9 has a well-defined role in the cell fate specification of posterior structures. MAB-9 is required in order for two cells in the hindgut to assume their correct fates; in mab-9 mutants these cells adopt the fates of their anterior neighbours. Adult male mutants develop tail abnormalities and hermaphrodite worms have hindgut defects. Both sexes also display a weak uncoordinated phenotype during backwards movement and mab-9 has recently been shown to have a role in axon migration of a subset of motor neurons (Huang et al, 2002). We are interested in identifying cis-regulatory elements upstream of mab-9 . A comparison of the 5' regulatory sequence and introns of mab-9 with that of its closest homologue in the related nematode C. briggsae has been used to identify five regions of close homology, each containing at least one putative T-box binding site. The conserved nature of these regions suggests that they might be of importance in directing cell-specific expression of mab-9. The fact that all five homology regions contain possible T-box binding sites may indicate that mab-9 is autoregulatory or regulated by other T-box genes. To establish the importance or otherwise of each homology region in directing MAB-9 expression, we have used a GFP-tagged rescuing construct with each of the homology regions removed by site-directed mutagenesis. The ability of mutated constructs to rescue mab-9 mutants and to be properly expressed is being investigated. Regulatory sequences which appear important in the control of MAB-9 expression will be chosen for a yeast 1-hybrid screen to identify upstream regulators. Huang X, Cheng H-J, Tessier-Lavigne M, Jin Y (2002); Neuron, 34, 563-576.