Willis IM (2018) Biochim Biophys Acta "Maf1 phenotypes and cell physiology."

Willis IM

[Biochim Biophys Acta, 2018]

As a master regulator of transcription by RNA polymerase (Pol) III, Maf1 represses the synthesis of highly abundant non-coding RNAs as anabolic signals dissipate, as the quality or quantity of nutrients decreases, and under a wide range of cellular and environmental stress conditions. Thus, Maf1 responds to changes in cell physiology to conserve metabolic energy and to help maintain appropriate levels of tRNAs and other essential non-coding RNAs. Studies in different model organisms and cell-based systems show that perturbations of Maf1 can also impact cell physiology and metabolism. These effects are mediated by changes in Pol III transcription and/or by effects of Maf1 on the expression of select Pol II-transcribed genes. Maf1 phenotypes can vary between different systems and are sometimes conflicting as in comparisons between Maf1 KO mice and cultured mammalian cells. These studies are reviewed in an effort to better appreciate the relationship between Maf1 function and cell physiology. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.

J Willis et al. (1999) International C. elegans Meeting "K-Cl Cotransporters in C. elegans"

J Willis, E Kipreos

[International C. elegans Meeting, 1999]

In vertebrate cells the K-Cl cotransporter has the capacity to reduce cellular concentration of K and Cl. The value of this activity is unclear. The carrier is activated by hypotonic cell swelling in mammalian but not in fish red cells; in the latter it is activated only by isotonic swelling. In mammalian red cells the carrier is activated by slight warming and this insures balance of K influx and efflux at elevated temperature. Isoforms of the carrier have been implicated in downsizing of mammalian erythrocytes during differentiation and in reduction of cell chloride concentration in developing neurons. The gene sequences for two mammalian isoforms were identified in 1996 and at that time two similar sequences were recognized in the C. elegans database (found in cosmids KO2A2, "KO2", on chromosome 2, and R13A1, "R13", on chromosome 4). R13 exists as an EST, and using primers based on KO2 we have obtained a PCR product of the appropriate length using a C. elegans cDNA library as template. Hence both genes are expressed. We have made double-stranded RNA for segments of each of these genes and have injected these singly and in combination into ovaries of C. elegans. Screens for lethality, growth rate and productivity among the progeny of injected worms have not yielded differences. About a third of surviving worms injected with dsRNA for both KO2 and R13 have produced progeny that exhibit reduced survival at elevated temperature. Screens based on isosmotic and hypotonic cell swelling are being developed. These studies may help to establish the role of K-Cl cotransporters in a whole organism.

Willis, A. R. et al. (2019) International Worm Meeting "Revealing mechanisms of inherited immunity using a C. elegans model of microsporidia infection."

Zhao, W. Y., Reinke, A. W., Willis, A. R.

[International Worm Meeting, 2019]

It has recently emerged that some animals can pass on 'memory' of previous infection to their offspring, thereby enhancing immunity in their progeny. This 'inherited immunity' is an example of epigenetic inheritance, whereby the parental environment modulates the germline, without altering the DNA sequence. Though inherited immunity has been shown to provide resistance to bacterial, viral and fungal pathogens, the molecular mechanisms underlying protection are unknown. Studies in C. elegans have enabled key advances in both immunity and epigenetics. Microsporidia are poorly-characterised fungal pathogens, and Nematocida parisii is a microsporidial parasite of C. elegans. Here, we show that the progeny of N. parisii-infected worms exhibit robust immunity against microsporidia. Whilst microsporidia replicate in the intestinal cells of naive larvae and inhibit worm development, parasitism is reduced in our immune-primed larvae. These striking phenotypes make the N. parisii-C. elegans infection model an ideal system to uncover mechanisms of inherited immunity. We are now fully characterising this inherited immunity phenotype in vivo. In particular, our studies have shown that the level of immunity correlates with the strength of parental infection, lasts a single generation, and is strongest in the early larval stages of primed animals. We have also shown that immunity prevents parasite invasion of the worm intestinal cells, suggesting a protective luminal factor. We are now using gene expression analyses to reveal putative immune effectors. We are also assaying epigenetic factors to better understand the mechanism of immune transmission from parent to progeny. This study offers unique insight into the immune reactivity of the genome. Epigenetic factors mediating inherited immunity in C. elegans may have homologues in higher organisms; inherited immunity in vertebrates is unexplored and would have major implications for human health and evolution. Whilst microsporidia are important opportunistic pathogens of humans and parasitize many agriculturally important species, very little is known of their infection biology. Study of this medically and environmentally relevant pathogen will suggest new ways to treat infection.

John H Willis et al. (2003) International Worm Meeting "act-2: An essential, maternally expressed C.elegans actin gene."

John H Willis, Bruce Bowerman, Aaron Severson

[International Worm Meeting, 2003]

In the early cell divisions of the C. elegans embryo, the microfilament cytoskeleton is required for polar body extrusion, establishment of anterior-posterior (a-p) polarity, and cytokinesis. To investigate the function and regulation of the microfilament cytoskeleton we have screened for temperature-sensitive(ts), embryonic lethal mutants with cell division defects. One recessive, ts mutant, or295, appears normal with respect to polar-body extrusion, a-p polarity, and cytokinesis, but exhibits spatial and temporal abnormalities in contractile activity of the cortical microfilament cytoskeleton. In mutant embryos, starting at the one-cell stage, small, unstable, membrane ingressions form at the cell surface with some ingressions sweeping in either direction along the cortex. These ingressions are reduced during mitosis and cytokinesis is always successful. At the 2-cell stage, these ingressions increase in severity and frequency during interphase in both cells although they are more prevalent in the anterior blastomere, AB. This same pattern is observed at the 4-cell stage : ingressions are reduced during mitosis, but increase during interphase and are more prevalent in anterior blastomeres. These undulating ingressions led us to call this mutant bellydancer. The abnormal membrane ingressions resemble cleavage furrows in that reducing the function of contractile ring components such as profilin, the FH-containing protein, cyk-1,the myosin light chain, mlc-4 by RNAi, and the small GTPase, rhoA, severely reduces this hyperactivity. Microfilaments and the non-muscle myosin, NMY-2, are both present at the cell cortex in a wild-type pattern. Positional cloning suggests that or295 is an allele of act-2 (TO4C12.5). or295 animals have a mis-sense mutation in act-2 (G16R) that was not present in the strain used for mutagenesis. To confirm gene identity, we are using germ-line transformation to test act-2 for rescue. The act-2 gene is one of five actins in C. elegans. Only act-2 is known to be enriched for mRNA expression in the germline (1). Our goal is to investigate how many actins are required for cell division in early embryonic cells, and if they are entirely redundant or have any specialized functions. References 1) Piano F, Schetter A, Mangone M, Stein L, and Kemphues KJ. RNAi analysis of genes expressed in the ovary of Caenorhabditis elegans. Current Biology 2000, 10:16191622.

John Willis et al. (2002) European Worm Meeting "cow-1 is required to spatially restrict contraction of the actin cytoskeleton in the early embryo."

John Willis, Bruce Bowerman, Aaron Severson

[European Worm Meeting, 2002]

The actin cytoskeleton is essential for a number of processes of the early embryo including anterior cortical ruffling and pseudocleavage following fertilization. In order to identify gene products that regulate the actin cytoskeleton during early embryogenesis we performed a genetic screen for temperature-sensitive, embryonic lethal mutations with defects in actin dependent processes. In this screen we identified one allele (or295) of a gene we called cow-1 (contractile waves) The most prominent defect in this mutant allele is waves of extenuated contractile activity that takes place throughout the first cell divisions. In cow-1 mutant embryos the restriction of cortical ruffling to the anterior of the one-cell zygote is lost; excessive contractions are prominent throughout the embryo and become more prominent through later cell divisions. Interestingly, analyses of Nomarski movies indicate no defects in cytokinesis and embryonic polarity. These cortical waves of contraction require an intact actin cytoskeleton and associated motor protein activity; reduction of a C. elegans profilin (pfn-1), cyk-1 or mlc-4 gene activity by RNAi silences the ectopic cortical contractions. To determine the role of cow-1 in the regulation of the actin cytoskeleton we have begun staining mutant embryos for protein localization of actin, the associated motor proteins, NMY-2 and MLC-4, and CYK-1. In order to determine the molecular identity of cow-1, we have mapped it to a 0.5 map unit interval on LGV. We are currently pursuing cosmid rescue while getting more map data.

John Willis et al. (2001) International C. elegans Meeting "PHYSIOLOGICAL ROLE OF K-CL COTRANSPORTERS IN C. ELEGANS"

John Willis, Edward Kipreos

[International C. elegans Meeting, 2001]

In vertebrate cells the K-Cl cotransporter has the capacity to reduce cellular concentration of K and Cl. This plays several documented roles: regulation of cytoplasmic chloride concentration, reduction of cell size in development, defense against cell swelling under hypoosmotic and isoosmotic conditions. The last, which includes the defense against swelling during moderate warming due to hyperactivity of the Na-K pump, has received the least attention but may be the most physiologically relevant. The gene sequences for two mammalian isoforms of this cotransporter were identified in 1996 and at that time two similar sequences were recognized in the C. elegans database (KO2A2.3, "KO2", on chromosome 2, and R13A1.2, "R13", on chromosome 4). Both C. elegans isoforms are expressed,as demonstrated by their presence in EST's and cDNA libraries. K02 has been shown to have K-Cl cotransport activity when transfected into mammalian cells. We have carried out dsRNAi blockages against both of these genes, singly and in combination, and found that about a third of the injected parents produced progeny that exhibit reduced survival at elevated temperature (33degC and 37degC) and increased sensitivity (seen as immobilization) to isoosmotic challenge induced by exposure to 100 or 200 mM ammonium chloride in M9 medium. A deletion mutant of K02 obtained from Gary Moulder at U. Oklahoma has not shown a phenotype by these tests and is now being used as a target for RNAi knockout of the R13 gene. GFP's expressed from upstream sequences of the two genes indicate that both isoforms are consistently expressed in pharyngeal muscle. The extent to which either is expressed elsewhere is still being determined. These studies may lead to establishing the role of K-Cl cotransporters in the whole organism and lay the foundation for a genetic analysis of their control

Willis AR et al. (2022) J Vis Exp "Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection."

Tamim El Jarkass H, Reinke AW, Willis AR

[J Vis Exp, 2022]

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Willis CRG et al. (2020) iScience "Comparative Transcriptomics Identifies Neuronal and Metabolic Adaptations to Hypergravity and Microgravity in Caenorhabditis elegans."

Szewczyk NJ, Reinsch SS, Costes SV, Udranszky IA, Etheridge T, Conley CA, Willis CRG

[iScience, 2020]

Deep space exploration is firmly within reach, but health decline during extended spaceflight remains a key challenge. In this study, we performed comparative transcriptomic analysis of Caenorhabditis elegans responses to varying degrees of hypergravity and to two spaceflight experiments (ICE-FIRST and CERISE). We found that progressive hypergravitational load concomitantly increases the extent of differential gene regulation and that subtle changes in ∼1,000 genes are reproducibly observed during spaceflight-induced microgravity. Consequently, we deduce those genes that are concordantly regulated by altered gravity per se or that display inverted expression profiles during hypergravity versus microgravity. Through doing so, we identify several candidate targets with terrestrial roles in neuronal function and/or cellular metabolism, which are linked to regulation by daf-16/FOXO signaling. These data offer a strong foundation from which to expedite mechanistic understanding of spaceflight-induced maladaptation in higher organisms and, ultimately, promote future targeted therapeutic development.

Willis, Alexandra et al. (2021) International Worm Meeting "A parental transcriptional response to microsporidia infection induces inherited immunity in offspring"

Reinke, Aaron, Claycomb, Julie, Zhao, Winnie, Wadi, Lina, Willis, Alexandra, Tamim El Jarkass, Hala, Sukhdeo, Ronesh

[International Worm Meeting, 2021]

Inherited immunity is an emerging field with important consequences for our understanding of health and evolution. Inherited immunity describes how infected parents can transfer immunity to offspring, promoting progeny survival in the face of infection. Critically, the mechanisms underlying inherited immunity are mostly unknown. Microsporidia are intracellular parasites that infect almost all animals, including humans; Nematocida parisii is a natural microsporidian pathogen of C. elegans. Here, we show that N. parisii-infected worms produce offspring that are resistant to microsporidia infection. We find that immunity is induced in a dose dependent manner and lasts for a single generation. Intergenerational immunity prevents host cell invasion by N. parisii and also enhances survival to the bacterial pathogen Pseudomonas aeruginosa. Further, we show that inherited immunity is triggered by the parental transcriptional response to infection, which can also be induced through maternal somatic depletion of negative regulators PALS-22 and the retinoblastoma protein ortholog LIN-35. We show that other biotic and abiotic stresses, such as viral infection and cadmium exposure, that induce a similar transcriptional response to microsporidia can also induce immunity in progeny. Our results demonstrate that distinct stimuli can induce inherited immunity to provide resistance against multiple classes of pathogens. These results show that activation of an innate immune response can provide protection against pathogens not only within a generation, but also in the next generation.

Willis, John et al. (2015) International Worm Meeting "Microevolution of microRNAs: developmental profiles and target recognition within C.remanei and C.latens."

Willis, John, Fierst, Janna, Phillips, Patrick

[International Worm Meeting, 2015]

Little is known about the relationship between the evolution of microRNAs and the mRNAs they target during gene regulation. To gain insight we have taken a genomic, bioinformatic and molecular approach to identify both unique and conserved microRNAs and their targets in the hyperdiverse nematodes, C.remanei and C.latens. These obligate outcrossing worms have genomes ~20% larger then C.elegans with close to 4000 more genes and significantly greater natural genetic variation at the population scale. To identify novel microRNAs we sequenced small RNA libraries using the Illumina platform. Between both species, we have generated over 400 million reads covering multiple developmental stages including biological replicates and both sexes as young adults. Using a combination of miRDeep2 and miRdentify we have stringently identified over 100 new microRNAs between both species for which we also have developmental profiles MicroRNAs display a wide variety of developmental patterns, with specific microRNAs displaying peak expression at each stage analyzed. Overall, expression appears to group within a small number of clustered patterns (5-6) within each species. In addition to characterizing the developmental profiles of microRNAs in these species, we are endeavoring to identify the suites of mRNAs that are targeted by individual microRNAs. To this end, we are using a in-vitro pull down assay similar to the LAMP assay developed in the Tsai lab (NAR, 2009). Using biotinylated, synthetic mature microRNAs and cell extracts we have been able to sequence RNA libraries from streptavidin bead pull downs. Utilizing both wild type and Argonaute mutant extracts from C.elegans in addition to microRNAs with normal and mutated seed sequence we hope to identify mRNA targets for any microRNA. Additionally, we have made peptide derived polyclonal antiobodies against the C.remanei ALG-1 protein and will present data on the efficacy of these antibodies for pulling down the RISC complex for a more comprehensive analysis of microRNA function.