Lopez Santos, Agustin et al. (2017) International Worm Meeting "Distinct genetic mechanisms underlie natural variation in left-right arrangement of the major organs in males and hermaphrodites of C. elegans."

Callander, Davon, Rothman, Joel, Lopez Santos, Agustin, Alcorn, Melissa

[International Worm Meeting, 2017]

Establishing the proper arrangement of internal organs is critically important during development, and defects in this process can lead to serious and even lethal birth defects. While the left-right (L/R) anatomical handedness in C. elegans was originally found to be essentially invariant in the laboratory N2 strain, we have observed that the L/R arrangement of the gonad and gut is frequently reversed under some environmental conditions in N2 and in many of the ~100 wild isolates examined. In some strains, up to ~11% of males show complete reversal in L/R gut/gonad arrangement, while in others, reversals were never seen. GWAS analysis revealed at least two regions of the genome responsible for these reversals in males. We also found that hermaphrodites of some strains show partial L/R reversals ("heterotaxy") of only the anterior or only the posterior gonad arm, with a small fraction of these animals showing complete reversals involving both arms. Variation in this phenotype across the wild isolates ranged from 0% to ~10% total reversals of all three types in hermaphrodites. Surprisingly, we found that the propensity for reversals in males and hermaphrodites is not significantly correlated across the isolates, indicating that the genetic basis for L/R reversals is independent in the two sexes. Analysis of 99 RILs generated from two strains at the extreme ends of the distribution of the hermaphrodite phenotypes (N2, 0% reversals, MY16, 10% reversals) revealed that the phenotype is multigenic and provided evidence of transgressive segregation, with one RIL showing ~20% reversals. QTL analysis is being performed to assess the genetic complexity and causal basis for this variation in handedness. Preliminary evidence suggests that the reversals in hermaphrodites may in part be traced back to an embryonic event, during which the developing gut undergoes a L/R asymmetric twist in comma-stage embryos (*Hermann et al., 2000), consistent with our finding that lin-12 mutants, which do not undergo normal gut twist, show frequent L/R gut/gonad reversals. Thus, variations in the anatomical handedness among wild isolates may reflect varying fidelity in the mechanism controlling the rotation of gut cells during mid-embryogenesis.

Alcorn, Melissa et al. (2015) International Worm Meeting "The propensity for stochastic PCD during male tail development varies across C. elegans isotypes."

Alcorn, Melissa, Rothman, Joel, Birsoy, Bilge, Cieslak, Matthew, Lopez Santos, Agustin, Callander, Davon

[International Worm Meeting, 2015]

While somatic programmed cell death (PCD) occurs in a largely invariant pattern in C. elegans, we have found that it arises sporadically and inappropriately during the formation of the 18 bilaterally symmetric sensory structures (rays) of the adult male tail. The rays, each consisting of the dendritic ends from two neurons and a structural support cell, arise during post-embryonic development. Wild-type N2 males frequently lack one or more rays. Males defective for components required for PCD contain few or no missing rays, suggesting that ray loss is the result of stochastic, inappropriate PCD. We found that the propensity for ray loss varies widely between 97 unique isotypes: while some isotypes virtually never show missing rays, in others, up to ~35% of males lack rays. As with N2, the loss of rays in these other strains appears to be the result of PCD, as they are restored by egl-1(RNAi). EMMA analysis applied to the full dataset of the 97 strains revealed that the variation is associated with a region on Chromosome I. We have also developed a complementary computational approach to analyzing this type of data by using a predictive regression model based on machine learning. This method applied to this and other datasets has identified regions of significance on Chromosomes II, III, V and X that were not predicted from standard EMMA analysis. Both approaches indicate that the sporadic PCD in the male is a polygenic trait. Ongoing analysis of RILs between two strains that represent phenotypic extremes is being used to identify the specific causal genes that control stochastic PCD during formation of the male rays. .

Vincent Galy et al. (2006) European Worm Meeting "MEL-28, a Novel Nuclear Envelope and Kinetochore Protein Essential for Zygotic Nuclear Envelope Assembly"

Carmen Lopez-Iglesias, Vincent Galy, Iain W. Mattaj, Cerstin Franz, Peter Askjaer

[European Worm Meeting, 2006]

Vincent Galy, Peter Askjaer, Cerstin Franz1, Carmen Lopez-Iglesias5, and Iain W. Mattaj1. The nuclear envelope (NE) of eukaryotic cells mediates nucleo-cytoplasmic transport and contributes to control of gene expression. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. Assembly of nuclear pore complexes (NPCs) and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here we report the identification and characterization of MEL-28, a large conserved protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. Light microscopy and transmission electron microscopy analysis demonstrate that MEL-28 localizes at NPCs during interphase, at kinetochores in early to mid mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early assembling, stable NE component required for all aspects of NE assembly. Based on its dynamic localization, we suggest that MEL-28 may link chromatin to the assembling NE.

Schiffer, Jodie et al. (2017) International Worm Meeting "How do worms determine their readiness to cope with environmental stress?"

Stumbur, Stephanie, Heath, William, Tam, Hannah, McGowan, Natalie, Vogelaar, Abigail, Schiffer, Jodie, Apfeld, Javier, Stanley, Julian

[International Worm Meeting, 2017]

At any time in their life, worms may encounter harmful environmental conditions such as high concentrations of oxidants or high temperature. We are interested in understanding how worms prepare for these potentially lethal events. We want to know: what establishes how prepared they are? To determine the mechanisms that control survival under harmful conditions, we are examining the effects of strong loss-of-function and null mutants in a collection of intercellular signaling receptors and transcription factors regulated by these receptors. We measure survival under oxidative stress and at high temperature using a "Lifespan Machine" cluster of flat-bed scanners [1]. This automated technology presents substantial advancements in throughput and sensitivity compared to manual methods. Using this approach, we have identified several signaling receptors and transcription factors that regulate survival under oxidative conditions. Interestingly, we identified both receptors and transcription factors that function to either confer or limit oxidative-stress resistance. This indicates that the combined level of activity of these genes sets the worm's readiness to cope with oxidative stress. We are currently investigating whether these genes act together or independently to determine the worm's normal level of resistance to various stressors, and the mechanisms that establish the normal activity levels of each of these genetic determinants of stress resistance. Reference: 1. Stroustrup N, Ulmschneider BE, Nash ZM, Lopez-Moyado IF, Apfeld J, Fontana W. The Caenorhabditis elegans Lifespan Machine. Nature Methods. 2013;10(7):665-70.

Mansfield, R. et al. (2015) International Worm Meeting "Disruption of cellular proteostasis drives sleep behavior in C. elegans."

Mansfield, R., Van Buskirk, C.

[International Worm Meeting, 2015]

Sleep is widely regarded to be a restorative state, and the physiological perturbations associated with sleep deprivation are extensive. Despite intensive study, none of these perturbations has in turn been shown to drive sleep. Recently our lab has shown that in C. elegans, environmental stressors such as heat, cold and toxins induce a sleep-like state (Hill et al., 2014). As noxious environmental stressors are known to interrupt normal proteostasis, the existence of a subsequent sleep state suggests that sleep serves to assist in the restoration of normal proteostasis. This idea is supported by our observation that sleepless animals have impaired survival following severe stress (Hill et al., 2014). Further, we have found that chaperone response defective mutants display exaggerated sleep responses. These mutants include animals lacking the stress-induced transcription factors hsf-1/HSF-1, daf-16/FOXO and a component of the endoplasmic reticulum stress-response pathway hsp-4/BiP. We are testing site-of-action for this effect by examining strains with tissue-specific rescue of HSF-1. In addition, we are using pharmacological inhibitors of the proteasome to test whether direct inhibition of the proteostasis machinery can trigger sleep. Last, we are performing RNAi against both positive and negative regulators of the heat shock transcriptional response, and screening for sleep phenotypes. The results of these efforts will be presented.Hill AJ, Mansfield R, Lopez JMG, Raizen DM, Van Buskirk C (2014) Cellular stress induces a protective sleep-like state in C. elegans. Curr Bio 24:1-7.Zimmerman JE, Naidoo N, Raizen DM, Pack AI (2008) Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 31:371-6.

Mutlu, Beste et al. (2015) International Worm Meeting "Increasing histone H3K9 methylation and nucleosome stability promote higher-order nuclear re-organization during embryogenesis."

Mango, Susan, Mutlu, Beste, Wang, Jie, Chen, Huei-Mei, Hall, David, Liu, Tao

[International Worm Meeting, 2015]

Open chromatin is a conserved feature of pluripotent cells and is lost upon differentiation. As cells differentiate, the nucleus shrinks and chromatin undergoes global re-organization to generate domains of euchromatin and higher-order heterochromatin1,2. While much is known about chromatin organization at the local level, much less is known about the factors that govern large-scale nuclear organization and their regulation during embryogenesis. We have used ultrastructural and genomic approaches to track nuclear organization in C. elegans embryos as they transition from a developmentally plastic state towards differentiation. As embryos mature, higher-order heterochromatin emerges, visible as electron dense regions (EDRs) by electron microscopy. Concomitant with the appearance of EDRs, methylated histone H3 accumulates, and nucleosomes become more stable, as monitored by FAIRE (formaldehyde-assisted identification of regulatory elements)3. We find that the H3K9 methyltransferase MET-2 is required to build EDRs and promote loss of plasticity, but not to stabilize nucleosomes. These results highlight two apparently independent processes that generate a differentiated nucleus: i) generation of higher-order heterochromatin by MET-2, and ii) increased nucleosome stability. These changes may help focus the transcriptional machinery to discrete regions of open chromatin within differentiated cells.References1. Yuzyuk, T., Fakhouri, T.H.I., Kiefer, J., and Mango, S.E. (2009). The polycomb complex protein mes-2/E(z) promotes the transition from developmental plasticity to differentiation in C. elegans embryos. Dev. Cell 16, 699-710.2. Gaspar-Maia, A., Alajem, A., Meshorer, E. & Ramalho-Santos, M. Open chromatin in pluripotency and reprogramming. Nat. Rev. Mol. Cell Biol. 12, 36-47 (2011).3. Giresi, P.G., Kim, J., McDaniell, R.M., Iyer, V.R., and Lieb, J.D. (2007). FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) isolates active regulatory elements from human chromatin. Genome Res. 17, 877-885.

Braukmann, Fabian et al. (2015) International Worm Meeting "Extracellular vesicle (ecv)RNA in C. elegans."

Braukmann, Fabian, Miska, Eric

[International Worm Meeting, 2015]

In all domains of life, non-coding RNAs regulate gene expression1. This mechanism called RNA interference has been a powerful technique in basic research over the last two decades. Today, RNAi enters the hospital and RNAi therapeutics have the potential to revolutionize drug development 2. One of the key challenges for RNAi therapeutics remains efficient RNA delivery. RNAi effectors move from cell to cell and spread through tissues in invertebrates, plants and fungi 3. In the mammalian system, extracellular vesicles (ECVs) shuttle RNA between cells 4. Recently, Wang et al. showed that C. elegans secrets ECVs in to the environment, possibly delivering RNA from animal to animal 5. However, the presents of RNA in ECVs and the molecular mechanism of mobile RNA between individuals is not known in any system. Here, I propose a purification protocol for extracellular vesicle (ecv)RNA released by the model organism C. elegans. Using this protocol, we sequenced small RNAs and RNAs from males, hermaphrodites and males deficient in vesicle release. A subset of small non-coding RNAs and messenger RNAs were enriched in male ECVs. This work characterizes the ecvRNA profile of C. elegans and may help to elucidate the social role of RNAs and has the potential to inform new approaches to RNAi therapy.1. Cech, T. R. & Steitz, J. A. The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94 (2014).2. Ozcan, G., Ozpolat, B., Coleman, R. L., Sood, A. K. & Lopez-Berestein, G. Preclinical and clinical development of siRNA-based therapeutics. Adv. Drug Deliv. Rev. (2015). doi:10.1016/j.addr.2015.01.0073. Sarkies, P. & Miska, E. A. Molecular biology. Is there social RNA? Science 341, 467-468 (2013).4. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654-659 (2007).5. Wang, J. et al. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr. Biol. 24, 519-25 (2014).

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Katherine A. Waters et al. (2007) International Worm Meeting "VRK-1 regulates CEP-1 in the germ line to prevent cell cycle arrest."

Baiqing Lin, Valerie Reinke, Katherine A. Waters, Carolyn S. Stankiewicz

[International Worm Meeting, 2007]

The C. elegans p53 homolog, CEP-1, has elevated expression in two regions of the germ line: distally within the germline stem cells and at the more proximal region of pachytene exit (Schumacher et al., 2005). Similar to p53 activity in mammalian systems, CEP-1 causes a cell cycle arrest in the stem cells and apoptosis of differentiated meiotic cells in response to DNA damage (Derry et al., 2001; Schumacher et al., 2001, Derry et al., 2007). However, the canonical inhibitor of p53, MDM2, is not conserved in C. elegans, suggesting that an alternative mechanism exists for CEP-1 regulation in the absence of genomic stress. We have identified vaccinia-related kinase-1 (vrk-1) as an inhibitor of CEP-1. VRK-1 is a conserved kinase with enriched expression in proliferating germ lines. Human VRK1 phosphorylates murine p53 in vitro (Lopez-Borges and Lazo, 2000) and preliminary data indicate that C. elegans VRK-1 phosphorylates CEP-1. vrk-1(ok1181) mutant germ lines are very small and have condensed chromosomal morphology. The germline proliferation and chromosomal abnormalities are largely rescued in a cep-1(gk138) background, suggesting that vrk-1 negatively regulates cep-1. The loss of DNA-checkpoint response effectors mrt-2, clk-2, and hus-1 in the vrk-1(ok1181) background failed to rescue germline development, indicating that vrk-1 regulates cep-1 either downstream of or parallel to the DNA-damage checkpoint response. Since a ced-4(n1162) background failed to rescue either the vrk-1(ok1181) or cep-1(gk138);vrk-1(ok1181) phenotypes, we believe vrk-1 causes a cep-1-dependent cell cycle arrest rather than ectopic apoptosis. Given that germline genomic stability is maintained by a rapid response to DNA damage achieved by elevated CEP-1 levels within germline stem cells, CEP-1 must be strictly regulated. We propose that VRK-1 phosphorylation of CEP-1 inhibits the DNA-damage-response pathway that causes cell cycle arrest while sustaining the germ lines ability to respond to genomic stress. The role of VRK-1 in regulating proliferation explains the paradox of elevated CEP-1 levels in the mitotic stem cells despite the lack of MDM2.

van der Linden, Alexander et al. (2017) International Worm Meeting "Global accumulation of circRNAs during aging in C. elegans."

Bauer, Matthew, Gruner, Matthew, Voda, Alexandru-Ioan, Miura, Pedro, Gruner, Hannah, Cortes-Lopez, Mariela, van der Linden, Alexander, Cooper, Daphne

[International Worm Meeting, 2017]

Circular RNAs (circRNAs) have recently been identified as a natural occurring family of wide-spread and diverse endogenous non-coding RNAs (reviewed in Cortes-Lopez and Miura, Yale J. Biol, 2016). They are remarkably stable RNA molecules mostly generated by backsplicing events from known protein-coding genes, and are abundantly expressed in many animals, from C. elegans (Ivanov et al, Cell Rep, 2015; Memczak et al, Nature 2013), to humans. Recent reports suggest functional roles for circRNAs, such as regulating transcription, binding proteins, and as microRNA sponges. CircRNAs accumulate during aging on a genome-wide level in Drosophila and mice (Westholm et al, Cell Rep, 2014; Gruner H. et al, Sci Rep, 2016), but their potential role and function in the aging process is not known. We profiled for the first time genome-wide circRNA changes during aging in C. elegans. Using total RNA-seq of whole C. elegans from L4-staged larvae, Day-1, Day-7 and Day-10 adults, we identified 1166 circRNAs including 575 novel annotations. Individual circRNAs were validated by Northern blot, qRT-PCR, and Sanger sequencing. We identified a massive and progressive accumulation of circRNAs during aging, which is independent of linear RNA changes from shared host genes. More specifically, 290 circRNAs were significantly upregulated in Day-10 compared to Day-1 adults, whereas only 6 were downregulated. This includes a >20-fold enrichment in a circRNA derived from the gene for the CREB homolog crh-1, as verified by qRT-PCR. Other genes in longevity-associated pathways produce age-accumulated circRNAs, including daf-16 and daf-2. Unlike in other species, the age-accumulation trend appears not to be confined to neuronally expressed genes. Given the progressive accumulation of circRNAs and their resistance to decay, we propose that stability in post-mitotic tissues is the main driver of the age-upregulation trend. We are currently examining whether circRNA accumulation patterns change in mutants with altered lifespan phenotypes. Taken together, our characterization of age-accumulated circRNAs lays the foundation for future investigations into the functions of circRNAs and how they might play a role in the aging process.