Melissa Richardson et al. (2008) Development & Evolution Meeting "Protein Synthesis in the Germline: The Role of IFE-1"

Elizabeth Cronland, Susan Strome, Brett Keiper, Melissa Richardson

[Development & Evolution Meeting, 2008]

Fertility and embryonic viability are measures of efficient germ cell growth and differentiation. During oogenesis, spermatogenesis and embryogenesis cells initially proliferate then differentiate into specific tissues. New proteins are required for both cell growth and differentiation, requiring qualitative and quantitative changes in protein synthesis. During late gametogenesis and early embryogenesis the expression of the appropriate proteins is a primary function of translational control. Translational control of mRNAs is mediated in part by eukaryotic initiation factor 4E (eIF4E). eIF4E binds the methylated 5' cap of mRNA and recruits it to the ribosome. C.elegans encodes five isoforms of eIF4E (termed IFE-1-5). IFE-1 is expressed primarily in the germline and is the only isoform that associates with P granules by binding directly to PGL-1. P granules also contain stored mRNAs needed for oogenesis and early embryogenesis. Using a strain that lacks IFE-1, we assessed the translational efficiency of maternal mRNAs bound and not bound to P granules by polysome fractionation. Translation of pos-1, pal-1, mex-1, oma-1 and glp-1 mRNA was inefficient in the ife-1(-/-) strain relative to wild type worms. GAPDH mRNA translation was not effected. We also observed differences in the expression of their products, in particular, MEX-1 during oogenesis. In males, secondary spermatocytes fail to complete late stages of maturation in absence of IFE-1. Preliminary evidence shows these immature spermatocytes induce CED-4/Apaf-1 expression, suggesting they undergo apoptosis. In ife-1(-/-) worms fertility was decreased by 80% due to decreased viability of both oocytes and spermatocytes. Our data indicates an essential role for eIF4E (IFE-1 isoform) in late oogenesis and spermatogenesis. We suggest IFE-1 preferentially recruits regulated mRNAs at critical times during germ cell development (Supported by NSF Grant MCB-0321017 to BDK).

Richardson, Claire E. et al. (2011) International Worm Meeting "Physiological IRE-1-XBP-1 and PERK/PEK-1 Signaling in C. elegans Intestinal Homeostasis and Immunity."

Kim, Dennis H., Kinkel, Stephenie, Richardson, Claire E.

[International Worm Meeting, 2011]

Endoplasmic reticulum (ER) stress activates the Unfolded Protein Response (UPR), a compensatory signaling response mediated by the IRE-1, PERK/PEK-1, and ATF-6 transmembrane proteins. Genetic studies have implicated roles for UPR signaling in animal development, but the function of the UPR under physiological conditions, in the absence of chemical agents administered to induce ER stress, is not well understood. Here, we show that in Caenorhabditis elegans, XBP-1 deficiency results in dramatically increased activities of IRE-1 and PEK-1, reflecting constitutive ER stress under basal physiological conditions. Innate immune activation and elevated physiological temperatures exacerbate this basal ER stress and necessitate IRE-1- XBP-1 and PEK-1 signaling for development and survival. Our data suggest that the negative feedback loops involving the activation of the IRE-1-XBP-1 and PERK/PEK-1 pathways serve essential roles not only at the extremes of exogenously imposed ER stress, but also in the maintenance of ER homeostasis under physiological conditions.

Richardson, Claire E. et al. (2009) International Worm Meeting "The IRE-1 Branch of the Unfolded Protein Response Functions in C. elegans Pathogen Resistance During Larval Development."

Kim, Dennis H., Kooistra, Tristan, Richardson, Claire E.

[International Worm Meeting, 2009]

The Unfolded Protein Response (UPR), a mechanism to sense and respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), has an important role in development and stress resistance from C. elegans to mammals. We asked if the UPR functions in C. elegans immune defense against the bacterial pathogen Pseudomonas aeruginosa. We found that the conserved IRE-1-XBP-1 branch of the UPR is required specifically for immunity during larval development. IRE-1 is an endoribonuclease that activates the transcription factor XBP-1 by splicing a short exon from its mRNA. Using quantitative PCR, we show that the relative amount of IRE-1-spliced xbp-1 transcript increases after pathogen exposure. Furthermore, a fluorescent reporter for IRE-1 activation is induced after exposure to Pseudomonas specifically at the site of infection - the intestine. We analyzed the relationship between the IRE-1 branch of the UPR and the PMK-1 pathway, which is known to promote C. elegans immunity, and found that IRE-1 activation in response to Pseudomonas is downstream of the PMK-1 pathway. The IRE-1-XBP-1 branch of the UPR has been recently shown to be required for cellular defense against pore-forming toxins, also functioning downstream of PMK-1 (Bischoff et al, 2008). Our data point to a more general role for the UPR in innate immunity against infection. Since PMK-1 pathway activates transcription of putative pathogen resistance proteins, we suggest that the IRE-1 branch of the UPR promotes pathogen resistance by accommodating a PMK-1-driven influx of pathogen resistance proteins to the secretory pathway. Bischof, L.J., Kao, C.-Y., Los, F.C.O., Gonzalez, M.R., Shen, Z., Briggs, S.P., van der Goot, F.G., Aroian, R.V. (2008) Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLOS pathogens, 4, 1-11.

Richardson C E et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "An Essential Role for the Unfolded Protein Response in Protection against Immune Activation in C. elegans Development"

Richardson C E, Kim D H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The Unfolded Protein Response (UPR) represents a conserved cellular homeostatic mechanism that detects and counteracts the accumulation ofunfolded proteins in the endoplasmic reticulum (ER). In metazoan animals, the UPR is mediated by three branches -- IRE1-XBP1, PERK/PEK, and ATF6 -- each of which has important roles in normal development and in the pathogenesis of disease. The IRE-1-XBP-1 branch in particular functions in mammalian immunity, as XBP-1 is required for the differentiation of the highly secretory plasma cells of the adaptive immune system, and XBP-1 also acts in inflammation and innate immunity in the intestine. We have been studying the interaction between the UPR and innate immunity in Caenorhabditis elegans. We have found that XBP-1 has an essential role in protecting the C. elegans during activation of innate immunity, which occurs in response to exposure to pathogenic bacteria through a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway. Activation of the PMK-1-mediated response to infection with Pseudomonas aeruginosa induces the XBP-1-dependent UPR. Whereas a loss-of-function xbp-1 mutant develops normally in the presence of the relatively non-pathogenic bacteria, infection of the xbp-1 mutant with P. aeruginosa leads to a distortion of ER morphology with severely attenuated development and larval lethality. The larval lethality phenotype on pathogenic P. aeruginosa is suppressed by loss of PMK-1-mediated immunity. Additionally, hyperactivation of PMK-1 causes larval lethality in the xbp-1 mutant even in the absence of pathogenic bacteria. Furthermore, by combining loss-of-function mutations in xbp-1 and pek-1, we have observed that the partially redundant activities of these separate branches of the UPR protect C. elegans against even basal levels of immune activity. Our data establish innate immunity as a physiologically relevant and developmentally critical initiator of ER stress in C. elegans and suggest that an ancient, conserved role for the UPR may be to protect the host organism from the detrimental effects of mounting an innate immune response to microbes.

Tillman, Erik et al. (2015) International Worm Meeting "Genetic Analysis of Endoplasmic Reticulum Homeostasis and Tolerance to Infection in C. elegans."

Tillman, Erik, Reddy, Kirthi, Kim, Dennis, Richardson, Claire, Cattie, Douglas

[International Worm Meeting, 2015]

We have previously identified an essential role for the conserved XBP-1 transcription factor in the maintenance of endoplasmic reticulum (ER) homeostasis during infection of Caenorhabditis elegans with pathogenic bacteria (Richardson, CE et al., Nature 2010). We conducted a genetic screen for mutations that suppress the lethality of xbp-1 loss during infection with Pseudomonas aeruginosa. We are currently carrying out the molecular characterization of suppressors that point to novel mechanisms that promote ER homeostasis independent of previously described pathways including the unfolded protein response (UPR).

Jagan Srinivasan et al. (2007) International Worm Meeting "Evolution of a sensory response network."

Jagan Srinivasan, Paul W. Sternberg

[International Worm Meeting, 2007]

We are trying to understand how neural circuits evolve to mediate ethologically relevant behaviors. While model systems help elucidate how sensory information from diverse environmental conditions is represented and processed in the neural, remarkably little is known about the evolution of neural circuits, and how selective forces shape the final architecture of neural circuits and processing circuits (Dumont & Richardson, 1986, Science). Nematodes are an ancient phylum comprising millions of species from diverse habitats. They have molecularly diverse nervous systems which in principle can help them sense several types of environmental stimuli. To trace the evolutionary history of such a sensory repertoire, we tested three different avoidance behaviors; osmotic avoidance, response to nose touch, and volatile chemical repellence in six diverse species of free-living nematodes, a) Caenorhabditis elegans (N2), b) Caenorhabditis briggsae (AF16), c) Caenorhabditis sp. 3 (PS1010), d) Pristionchus pacificus (PS312) e) Cruznema tripartitum (PS1351) and f) Panagrellus redivivus (PS2298). We found that all species tested exhibit the three avoidance behaviors. However, we also find that sensory sensitivity to the different stimuli differs among the tested nematode species. In C. elegans , response to these stimuli are mediated by the ‘polymodal ASH neurons (Kaplan and Horvitz PNAS, 1993, Hart et al. Nature, 1995, Hilliard et al Curr Biol. 2002). We identified the pairs of putative ASH neurons in different nematode species by their anatomical positions and ablated them. Ablation of the ASH neurons in these species resulted in an inability to avoid these stimuli. By further ablation experiments, we find that there is increase or decrease in the set of sensory neurons mediating osmosensation and mechanosensation. In P. pacificus , osmosensation involves the ADL neuron. In Caenorhabditis sp. 3 , mechanosensation is solely mediated by the ASH neuron as compared to 3 neurons in C. elegans . The overall conservation of ASH mediated behaviors suggests that polymodality is an ancestral feature and is evolutionarily stable, but can evolve by alterations in the level of sensitivity and the relative contributions of sensory neurons.

Grooms, N. et al. (2019) International Worm Meeting "Fluorescent screen for identifying axon regeneration genes."

Fitzgerald, M., Chung, S., Grooms, N., Urena, S.

[International Worm Meeting, 2019]

Statement of purpose: Central nervous system (CNS) injuries are prevalent and currently lack effective therapies. A conditioning lesion of the peripheral sensory axon triggers robust central axon regeneration in mammals (Richardson and Issa. Nature. 1984). Lesion conditioning could drive powerful therapies for CNS injuries. We established a model for lesion-conditioned regeneration in the invertebrate roundworm C. elegans. This approach allows for a much more comprehensive and faster search for neuronal regeneration genes compared to mammalian models. Methods: The C. elegans model permits unbiased screening. Ethyl methanesulfonate was used to stochastically introduce mutations and disrupt the regeneration pathway (Brenner. Genetics. 1974). In our screen, green fluorescence protein intensity is used as a proxy for neuronal regeneration. We selected candidate worms based on reduced intensity. Disruptions to sensory pathways in C. elegans has been shown to produce ectopic outgrowths under physiological stress. Therefore, studying ectopic outgrowth could identify genes involved in regeneration pathways (Chung, et al. PNAS. 2016). Ectopic outgrowth of dim strains is assessed via fluorescent microscopy by counting axonal outgrowths after culturing at 25 deg C. We are also quantifying the fluorescent intensity. The fluorescence of each strain is quantified using InSpeck Image Intensity Calibration Kits as a standard. Maximum intensity projections of 3D images were taken of worms next to beads for quantification. Laser surgery will be performed to assess regeneration in strains with reduced ectopic outgrowth. One-step whole genome sequencing will identify the affected gene in strains with reduced regeneration (Doitsidou, et al. PLOS ONE. 2010). Summary of results: Following mutagenesis, we isolated twelve strains, originating from six distinct F1s. These mutations have varying penetrance. In each selected strain, around 30-50% of the worms have reduced fluorescence. Ectopic outgrowth studies are expected to show decreased axonal outgrowths. The quantification of fluorescent intensities allows us to correlate intensity with regeneration on an individual and on an overall population basis. The identified genes will provide a roadmap for accelerating vertebrate lesion conditioning research. Affiliations: Funding provided by Morton Cure Paralysis Fund.

Tillman, E.J. et al. (2017) International Worm Meeting "FKH-9 modulates stress physiology and endoplasmic reticulum homeostasis during pathogen infection in C. elegans."

Kim, D.H., Reddy, K.C., Richardson, C.E., Tillman, E.J., Cattie, D.J.

[International Worm Meeting, 2017]

Animals maintain cellular homeostasis in response to environmental challenges, including microbial pathogens. Distinct stress response signaling pathways promote protein folding homeostasis in the cytoplasm, endoplasmic reticulum (ER), and the mitochondria, and a role for each has been implicated during infection of C. elegans with pathogenic bacteria. We previously demonstrated that the ER Unfolded Protein Response (UPR) is induced during infection of C. elegans with Pseudomonas aeruginosa, and that the UPR regulator XBP-1 has an essential role during infection and immune activation (1). To identify signaling mechanisms that can compensate for XBP-1 deficiency during pathogen infection, we conducted a forward genetic screen for suppressors of xbp-1 mutant lethality on the pathogenic bacteria Pesuodomonas aeruginosa. We recently reported that mutations in the eIF3k and eIF3l translation initiation factor subunits can suppress the lethality of the xbp-1 mutant in the presence of P. aeruginosa (2). Here, we show that mutations in the Forkhead family transcription factor FKH-9 also suppress the lethality of the xbp-1 mutant on P. aeruginosa. We observe that mutations in fkh-9 confer improved intestinal ER morphology and organismal tunicamycin resistance, independent of previously defined UPR signaling pathways. To define how FKH-9 activity modulates ER homeostasis, we have conducted ChIP-seq analysis to define direct transcriptional targets of FKH-9, along with functional genetic analysis, which suggest a role for FKH-9 in integrative stress signaling. Our data suggest that XBP-1-independent pathways contribute to maintenance of ER homeostasis and survival during infection and innate immune activation in C. elegans. References: 1. Richardson, C. E., Kooistra, T. & Kim, D. H. An essential role for XBP-1 in host protection against immune activation in C. elegans. Nature 463, 1092-1095 (2010). 2. Cattie, D. J. et al. Mutations in Nonessential eIF3k and eIF3l Genes Confer Lifespan Extension and Enhanced Resistance to ER Stress in Caenorhabditis elegans. PLoS Genet 12, e1006326 (2016).

Edgley ML et al. (1991) International C. elegans Meeting "Caenorhabditis genetics center."

Riddle DL, Edgley ML, Estevez AOZ

[International C. elegans Meeting, 1991]

The CGC has been in operation for 12 years, supplying Caenorhabditis strains and information to researchers. It is responsible for coordination of C. elegans genetic nomenclature, and acts as the central clearing house for naming strains, genes and alleles. Information distributed includes the genetic map, genetic map data, strain information, a bibliography of 1300 research articles and book chapters, and the C. elegans research newsletter, the Worm Breeder's Gazette, which is distributed to 450 subscribers worldwide. A collection of 1550 strains has been established, representing 30 wildtype isolates of C. elegans, wild-type strains of C. briggsae, C. remanei and C. vulgariensis, as well as alleles of 773 mutant genes and 254 chromosome rearrangements. Stocks are stored permanently in liquid nitrogen. Approximately 1350 different strains have been distributed to research laboratories in 7800 separate mailings. Investigators in 210 laboratories in 21 countries have either received strains from the CGC or contributed strains to the CGC collection. The current version of the C. elegans genetic map includes 26 pages, and contains 920 genes and 320 rearrangements. It is produced using the computer drawing program Designer (Micrografx, Inc., Richardson TX), which runs under Microsoft Windows on IBM-compatible microcomputers. The map is published biannually in Genetic Maps (S.J. O'Brien, editor; Cold Spring Harbor Laboratory) and semi-annually in the Worm Breeder's Gazette. Caenorhabditis strains and information are available upon request. All strain, bibliographic and genetic information is maintained on a microcomputer database system, and the various files are available as printouts or data on diskette. Operation of the CGC will be transferred to other investigators at the end of the current CGC contract (September 29,1992). One possible plan emerged at the last C. elegans meeting, in which Bob Herman will assume responsibility for the strain collection, and Jonathan Hodgkin will assume responsibility for the genetic map. Plans for continuation of other CGC services remain to be made. New developments in the use of computers to integrate all information on the worm will enhance the usefulness of data collected by the CGC (see Thierry-Mieg, Durbin, Schatz and Ward, this meeting). The CGC is supported by the NIH National Center for Research Resources.

Mongan NP et al. (1997) International C. elegans Meeting "AN EXTENSIVE NICOTINIC ACETYLCHOLINE RECEPTOR GENE FAMILY IN CAENORHABDITIS ELEGANS."

Baylis HA, Mongan NP, Sattelle DB

[International C. elegans Meeting, 1997]

Although the nicotinic acetylcholine receptors (nAChRs) are the best characterized ionotropic neurotransmitter receptors, the extent of the molecular and functional diversity of the nAChR gene family is not known for a single organism. A number of C. elegans nAChR subunit genes have been identified previously using genetic approaches [lev-1(non-a),unc-38(a),unc-29(non-a),deg-3(a)] and cross-species probing of C. elegans cDNA libraries [acr-2(non-a),acr-3(non-a),and Ce-21(a)]. nAChR a-subunits may be distinguished from all other ionotropic receptor subunits by the presence of adjacent cysteines at positions equivalent to 192,193 in the Torpedo a-subunit. We have applied an RT-PCR strategy to confirm the expression of 8 novel putative nAChR a-subunits predicted by GENEFINDER. Analysis of the regions of the a-subunit considered to (a) contribute to the acetylcholine binding site and (b) the channel lining, has revealed a diversity which may underlie distinct functional roles for members of this multi-gene family.