Jiang, Yanwen et al. (2021) International Worm Meeting "Post-transcriptional regulation of egl-1BH3-only, the key activator of apoptosis during C. elegans development"

Jiang, Yanwen, Conradt, Barbara

[International Worm Meeting, 2021]

The highly reproducible pattern of programmed cell death (apoptosis) during C. elegans development is dependent on the central apoptosis pathway and its most upstream component egl-1, which encodes a pro-apoptotic member of the Bcl-2 family of apoptosis regulators. Unlike other components of the central pathway (ced-9, ced-4, and ced-3), the egl-1 gene is predominantly transcribed in cell death lineages (i.e. lineages in which a cell death occurs), and this specificity is mediated by lineage-specific transcription factors. Within a particular cell death lineage, egl-1 mRNA copy number is dynamic. Using single-molecule RNA FISH, we previously found a low number of egl-1 mRNAs in mothers of cells programmed to die, a high number in daughters programmed to die and essentially zero in daughters programmed to survive. Although the mechanisms underlying this dynamic pattern are not fully understood, we have evidence that it is at least partially due to microRNA-dependent control of egl-1 mRNA turn-over. Specifically, we reported that the miR-35 and miR-58 families of microRNAs act through respective binding sites in the egl-1 3' UTR to increase egl-1 mRNA turn-over in mothers, thereby preventing their precocious deaths (Sherrard et al, 2017). We have now identified additional elements in the egl-1 3' UTR that are required for egl-1 3' UTR-mediated repression of reporter gene expression. These elements include a conserved 3' terminal element (TPTE) that is necessary and partially sufficient to repress reporter gene expression. We hypothesize that RNA-binding proteins (RBPs) also play important roles in post-transcriptional control of egl-1 expression. To identify functionally relevant RBPs, we individually knocked down 660 genes predicted to encode RBPs and analyzed the resulting animals with respect to egl-1 3' UTR-dependent reporter gene expression. Using this approach, we identified 37 repressor candidates and 26 activator candidates. We are currently in the process of screening these candidates for potential cell death phenotypes and plan to verify egl-1 mRNA-RBP interactions using biochemical approaches. We propose that regulation at the post-transcriptional level through miRNAs and potentially RBPs is crucial for spatio-temporal control of the level of egl-1 expression in cell death lineages.

Mondol V et al. (2012) Curr Top Dev Biol "Let's make it happen: the role of let-7 microRNA in development."

Mondol V, Pasquinelli AE

[Curr Top Dev Biol, 2012]

Noncoding RNAs have emerged as an integral part of posttranscriptional gene regulation. Among that class of RNAs are the microRNAs (miRNAs), which posttranscriptionally regulate target mRNAs containing complementary sequences. The broad presence of miRNAs in lower eukaryotes, plants, and mammals highlights their importance throughout evolution. MiRNAs have been shown to regulate many pathways, including development, and disruption of miRNA function can lead to disease (Ivey and Srivastava, 2010; Jiang et al., 2009). Although the first miRNA genes were discovered in the nematode, Caenorhabditis elegans, almost 20 years ago, the field of miRNA research began when they were found in multiple organisms a little over a decade ago (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001; Lee et al., 1993; Pasquinelli et al., 2000; Wightman et al., 1993). Here, we review one of the first characterized miRNAs, let-7, and describe its role in development and the intricacies of its biogenesis and function.

Mukhopadhyay A et al. (2006) Exp Gerontol "Worming pathways to and from DAF-16/FOXO."

Tissenbaum HA, Oh SW, Mukhopadhyay A

[Exp Gerontol, 2006]

In Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response . This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO). DAF-16 also receives input from several other pathways that regulate life span such as the germline and the JNK pathway [Hsin, H., Kenyon, C., 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399, 362-366; Oh, S.W., Mukhopadhyay, A., Svrzikapa, N., Jiang, F., Davis, R.J., Tissenbaum, H.A., 2005. JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16. Proc. Natl. Acad. Sci. USA 102, 4494-4499]. Therefore, DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse processes. Here, we discuss the signals to and from DAF-16, with a focus on life span regulation.

Dave Markwardt et al. (2001) International C. elegans Meeting "A screen for natural targets of NMD using C. elegans cDNA microarrays"

Dave Markwardt, Philip Anderson

[International C. elegans Meeting, 2001]

mRNA transcripts that contain premature termination codons are degraded in a regulated manner by a specific decay pathway termed mRNA surveillance or nonsense mediated mRNA decay (NMD). mRNA surveillance has been described in all eukaryotes tested including plants, flies, yeast, nematodes, and mammals. Loss-of-function mutations affecting any of seven smg genes eliminates mRNA surveillance in C. elegans . While much progress has been made in defining the cis -acting elements and trans -acting factors necessary for mRNA surveillance, less is know about the mRNAs produced during the normal course of gene expression that serve as substrates of NMD. In order to systematically investigate these natural targets of NMD, I have probed high density microarrays of C. elegans cDNA clones. These arrays, made in Stuart Kim's lab at Stanford University, consist of PCR fragments from 17,871 genes currently annotated in the C. elegans genome (Jiang et al., 2001). We performed nine different hybridizations, each comparing expression profiles of poly(A) + mRNA from smg(-) and smg(+) samples. The microarray screen identified 402 mRNAs whose abundance is 2-fold or greater in smg(-) mutants. Several observations suggest that microarrays are a reliable method for identifying natural targets of mRNA surveillance: 1. Control mRNAs known previously to be natural targets of mRNA surveillance ( rpl-12 and srp-20 ) were consistently elevated on the microarray screen. 2. Both northern blots and quantitative PCR analyses confirm the microarray results. These results indicate that the microarray screen will be useful for the systematic investigation of the C. elegans natural targets of NMD. We are presently analyzing smg(-) affected mRNAs to establish whether they are direct or indirect targets of NMD, and understand why their abundance is increased in smg(-) mutants. We anticipate that analysis of the ~400 candidate genes will identify broad categories of mRNAs produced in wild-type animals yet targeted for degradation by NMD and, possibly, new principles of post-transcriptional regulation of gene expression. Jiang M, Ryu J, Kiraly M, Duke K, Reinke V, Kim SK. Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans . Proc. Nat. Acad. Sci. USA. (2001) 98 (1):218-23.

Delattre M et al. (2001) Dev Biol "Development and evolution of a variable left-right asymmetry in nematodes: the handedness of P11/P12 migration."

Felix MA, Delattre M

[Dev Biol, 2001]

In Caenorhabditis elegans, two lateral blast cells called P(11/12)L and P(11/12)R are symmetric left-right homologs at hatching, migrate subsequently in opposite anteroposterior directions during the first larval stage, and adopt two different fates, thus breaking the symmetry between them. Our results show that, unlike most other cell fate decisions in C. elegans, the orientation of P(11/12)L/R migration is highly biased, but not fixed. The handedness of their migration is linked to whole body handedness and is randomized in lin-12/Notch mutants and by ablation of the Y cell. Migration handedness is independent of P11 and P12 fate determination, previously shown to require the LIN-44/Wnt and the LIN-3/EGF pathways (L. I. Jiang and P. W. Sternberg, 1998, Development 125, 2337-2347). We further show that several changes in P(11/12)L/R asymmetry have occurred during nematode evolution: loss of asymmetry or reversals in orientation of migration. Strikingly, for most species studied, handedness of migration is highly biased but not fixed. Thus, whereas the final cell fate pattern of P11/12 is invariant, the developmental route leading to it is subject both to developmental indeterminacy and to evolutionary variations.

Zhiyong Shao et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Dual Functions of EGL-9: Defining the Molecular Mechanisms by which EGL-9 inhibits HIF-1 Transcriptional Activity"

Zhiyong Shao, Jo Anne Powell-Coffman

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In mammals and in C. elegans, the hypoxia inducible transcription factor (HIF-1) mediates most of the gene expression changes induced by hypoxia (Jiang H et al., 2001 PNAS 98: 7916; Shen et al., 2005 JBC 280: 20580). EGL-9 has a central role in HIF regulatory networks. EGL-9 hydroxylates the proline in the LXXLAP motif of HIF-1, using oxygen as a co-substrate. This promotes HIF-1 degradation through a ubiquitination/proteasome pathway that requires the VHL-1 E3 ligase. When oxygen levels are low, EGL-9 hydroxylase activity is inhibited, and HIF-1 is stabilized (Epistein et al. 2001 Cell 107: 43). In prior studies, we have shown that EGL-9 functions through at least one additional pathway to inhibit HIF-1. In animals carrying a deletion mutation in vhl-1, loss-of-function mutations in egl-9 have no effect on HIF-1 protein levels but dramatically up-regulate expression of HIF-1 target genes (Shen et al., 2006 Genetics 174: 1205). In the past year, we have conducted a series of molecular genetic experiments to distinguish between alternative hypotheses for EGL-9 function. Our data support a model in which EGL-9 has two distinct functions: (1) hydroxylation and destabilization of HIF-1 via the well-characterized VHL-1 pathway; and (2) repression of HIF-1 transcriptional activity by a novel pathway that does not require HIF-1 hydroxylation. We gratefully acknowledge funding from the NIH (GM078424).

Powell-Coffman, Jo Anne et al. (2009) International Worm Meeting "Dose-dependent roles for the C. elegans HIF-1 hypoxia-inducible factor in stress resistance and aging."

Feng, Dingxia, Zhang, Yi, Shao, Zhiyong, Powell-Coffman, Jo Anne, Zhai, Zhiwei

[International Worm Meeting, 2009]

Oxygen homeostasis is essential to metazoan life, and animals have evolutionarily conserved strategies for adapting to changing levels of environmental oxygen levels during development, homeostasis and disease. The hypoxia-inducible factor (HIF) heterodimeric transcription factors are the central regulators of oxygen-sensitive gene expression in animals as diverse as humans and C. elegans. HIF stability and activity are regulated by environmental oxygen levels and by reactive oxygen species, and HIF complexes control the expression of batteries of genes that enable adaptation to hypoxic stress. We and others have shown that C. elegans HIF-1 and its regulators have important roles in adaptation to hypoxia, neuronal development, pathogen resistance, aerotaxis behavior, and egg laying [Trent et al., Genetics 1983; Darby et al. PNAS 1999; Jiang et al., PNAS 2001; Shen et al., JBC 2005; Pocock and Hobert, Nature Neuro 2008; Chang and Bargmann PNAS 2008]. We have begun to elucidate the roles of HIF-1 in the C. elegans stress response network, and we have discovered that HIF-1 overexpression results in dose-dependent extension of average lifespan. Interestingly, we find that hif-1 deletion mutations also increase C. elegans longevity, but via different genetic pathways. We have investigated interactions between HIF-1 and other stress responsive transcription factors and have discovered feedback loops in which SKN-1 modulates HIF-1-dependent gene expression. Collectively, these findings provide a greater understanding of how HIF-1 is integrated into the C. elegans stress response network. These studies have been supported by grants from NIGMS (GM078424) and by the ISU Center for Integrated Animal Genomics.

Todd Lamitina et al. (2001) International C. elegans Meeting "Progress towards the cloning of fer-14, a gene required for fertilization in C. elegans sperm"

Todd Lamitina, Steven W L'Hernault

[International C. elegans Meeting, 2001]

In the last act of spermatogenesis, competent sperm recognize, bind, and fuse to the oocyte. fer-14 is a mutant that is defective in this final stage of spermatogenesis. Like the EGF-repeat containing spe-9 gene, fer-14 mutant sperm exhibit wild type motility, form a wild type pseudopod, localize to the spermatheca upon insemination into the female reproductive tract, and participate in sperm competition (Singson et al, Genetics 152;201-208, 1999). fer-14 has been mapped to a .5 map unit interval on the right arm of chromosome I, between lev-11 and sur-2 . Various triply marked lines have been contructed for use in polymorphism mapping and this approach has narrowed the fer-14 containing physical interval down to 40Kb. Utilizing the male/female microarray data from Jiang et al., a candidate gene that is expressed in a strongly sperm-enriched manner has been identified. Efforts are currently underway to generate transgenic lines containing this candidate gene and rescue fer-14 . fer-14 shows a unique deficiency complementation pattern to LGI deficiencies. eDf4 and eDf15 appear to have the same left breakpoint. However, eDf4 complements fer-14 whereas eDf4 fails to complement. Physical mapping of these breakpoints using PCR should further confirm and narrow the physical region containing fer-14 . fer-14 is currently defined by one slightly temperature sensitive allele, which when put over a non-complementing deficiency, also has a spe phenotype. We are engaged in a screen for new alleles to unambigously determine the null phenotype for fer-14 .

Steven Jones et al. (2005) International Worm Meeting "Transcription profiling of C. elegans developmental stages: A comparison of different platforms."

Nathan Pofahl, Peter Huang, Steven Jones, Sheldon J McKay, Kim Wong, Courtney Mills, Peter Ruzanov, Michael Hogan, Roland Green, David L Baillie

[International Worm Meeting, 2005]

We are using serial analysis of gene expression (SAGE), Affymetrix GeneChip arrays and Nimblegen chips to profile C. elegans developmental gene expression. SAGE, Affymetrix and Nimblegen microarray experiments were performed in parallel on the same pools of RNA. With 6 SAGE libraries totaling more than 650,000 SAGE tags and 19 chip experiments the scale of this comparion is unprecedented. Expression of genes across developmental stages shows the best agreement for genes that are highly or moderately expressed or for those with large changes in expression during development. Comparison of expression patterns from the SAGE and Affymetrix platforms with spotted microarray data from Jiang et al. (Science 2001 293:2087-2092) show a similar trend.Probes on NimbleGen arrays are produced using in situ using maskless photolithography to produce a high-density oligonucleotide array. Our custom array contains 24-mer probes targeting over 19,000 genes annotated. There is good correlation of expression profiles from Affymetrix and NimbleGen arrays, but both correlate more modestly with SAGE. We are designing a second chip containing 60-mer probes in hopes of seeing improved agreement between observed expression profiles from SAGE and the NimbleGen array. We have identified developmentally regulated genes by looking for genes showing at least 2-fold enrichment at various developmental landmarks. In order to find the most reliable subset of developmentally regulated gene candidates, we have adopted the strategy of requiring agreement between at least two of the three platforms on stage-enriched genes. Many genes already known to be expressed in a life-stage specific manner were identified, in addition to many transcription factors, signal transducers and genes with unknown function.

K Wong et al. (2004) West Coast Worm Meeting "TRANSCRIPTION PROFILING OF C. ELEGANS DEVELOPMENTAL STAGES USING SERIAL ANALYSIS OF GENES EXPRESSION AND MICROARRAYS"

K Wong, D L Baillie, P Ruzanov, C Mills, P Huang, S J McKay, M A Marra, DG Moerman, S J M Jones

[West Coast Worm Meeting, 2004]

We are using serial analysis of gene expression (SAGE) and the Affymetrix GeneChip array to profile gene transcription through the course of C. elegans development. Synchronized populations of embryos, each of the larval stages, and young adult worms were sampled. SAGE and Affymetrix microarray experiments were performed in parallel on the same pools of RNA. Direct comparison of the expression profiles was facilitated by mapping observed SAGE tags and the GeneChip probes to a virtual transcriptome. The virtual transcriptome was assembled from wormbase gene models, including confirmed untranslated regions (UTRs) identified by EST analysis and predicted UTRs where EST evidence was lacking. Overall, expression of genes across developmental stages shows the best agreement for genes that are highly or moderately expressed or for those with large changes in expression during development. Comparison of expression patterns from the SAGE and Affymetrix platforms with spotted microarray data from Jiang et al. (Science 2001 293:2087-2092) showed a similar trend. To identify developmentally regulated genes, we have used SAGE and microarray expression patterns for genes showing at least 2-fold enrichment at various developmental landmarks. SAGE identifies almost 5000 stage-enriched genes. The Affymetrix and spotted microarrays identify almost 6500 and 2300 stage-enriched genes, respectively. In order to find the most reliable subset of developmentally regulated gene candidates, we have adopted a conservative strategy of requiring agreement between at least two of the three platforms on stage-enriched genes. As expected, many genes already known to be expressed in a life-stage specific manner were identified including collagens, genes involved in spermatogenesis, oogenesis, and embryogenesis. In addition, many transcription factors, signal transducers, and genes with unknown function were identified as being developmentally regulated.