Don Fox et al. (2001) International C. elegans Meeting "Securin and"

Don Fox, Barry Chestnut, Andy Golden

[International C. elegans Meeting, 2001]

Cell division results in the precise halving of genetic material. In yeast, such partitioning requires the protease separin 1 . This protease acts at the metaphase to anaphase transition to degrade sister chromatid cohesion, which in turn permits chromosomes to segregate to opposite poles of a dividing cell . In addition to facilitating chromosome segregation, separin promotes spindle elongation in S. cerevisiae through the activity of a calcium-binding domain 2 . Separin activity also depends heavily on the anaphase inhibitor securin, which localizes separin to both the nucleus and spindle mid-zone, and inhibits its protease activity until anaphase. Here, we report a requirement for the C. elegans homologue of yeast separin during oocyte meiosis. RNAi of this homologue, which contains both the protease and calcium binding domains of S. cerevisiae separin, results in multi-nucleated one-cell embryos with multiple spindles. In these embryos, the chromosomes are disorganized and the embryos fail to produce polar bodies. Taken together, these results suggest a role for C.elegans separin in chromosome segregation and cytokinesis in the early embryo. Currently, we are investigating if C. elegans separin also plays a role in exit from M-phase of the cell cycle. Additionally, we hope to identify protein partners of C.elegans separin by using the protein as bait in a yeast 2-hybrid screen. This screen may prove the most effective way of identifying a C. elegans securin homologue, as all of the known securins from other organisms demonstrate no amino acid conservation. Also, to date, no separin interacting proteins other than securin have been identified. Results of the screen will be discussed at the meeting. 1.Uhlman, F., Wernic, D., Poupart, M., Koonin, E., and K. Nasmyth. 2000. Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast. Cell 103:375-386. 2.Jensen, S., Segal, M., Clarke, D., and S. Reed. 2001. A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation: Evidence that Proper Spindle Associatioin of Esp1 is Regulated by Pds1. J.Cell Biol. 152:27-40.

Fox BW et al. (2020) Nat Chem Biol "Toward spatially resolved metabolomics."

Fox BW, Schroeder FC

[Nat Chem Biol, 2020]

Fox RM et al. (2007) Genome Biol "The embryonic muscle transcriptome of Caenorhabditis elegans."

Brodigan TM, Fukushige T, Fox RM, McDermott J, Kraus M, Watson JD, Miller DM, Von Stetina SE

[Genome Biol, 2007]

ABSTRACT: BACKGROUND: The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also found in animals with more complex body plans. To this end, we have applied Micro-Array Profiling of C. elegans Cells (MAPCeL) to muscle cell populations extracted from developing C. elegans embryos. RESULTS: Fluorescence Activated Cell Sorting (FACS) was used to isolate myo-3::GFP-positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos. Microarray analysis identified 7,070 expressed genes, 1,312 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell. The muscle-enriched gene set was validated by comparisons to known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. These results confirm the utility of MAPCeL for cell type-specific expression profiling and reveal that 60% of these transcripts have human homologs. CONCLUSIONS: This study provides a comprehensive description of gene expression in developing C. elegans embryonic muscle cells. The finding that over half of these muscle-enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in C. elegans could reveal evolutionarily conserved models of muscle gene function with ready application to human muscle pathologies.

Fox A et al. (2009) Pac Symp Biocomput "High throughput interaction data reveals degree conservation of hub proteins."

Fox A, Slonim DK, Taylor D

[Pac Symp Biocomput, 2009]

Research in model organisms relies on unspoken assumptions about the conservation of protein-protein interactions across species, yet several analyses suggest such conservation is limited. Fortunately, for many purposes the crucial issue is not global conservation of interactions, but preferential conservation of functionally important ones. An observed bias towards essentiality in highly-connected proteins implies the functional importance of such "hubs". We therefore define the notion of degree-conservation and demonstrate that hubs are preferentially degree-conserved. We show that a protein is more likely to be a hub if it has a high-degree ortholog, and that once a protein becomes a hub, it tends to remain so. We also identify a positive correlation between the average degree of a protein and the conservation of its interaction partners, and we find that the conservation of individual hub interactions is surprisingly high. Our work has important implications for prediction of protein function, computational inference of PPIs, and interpretation of data from model organisms.

Fox BW et al. (2022) Nature "C. elegans as a model for inter-individual variation in metabolism."

Walker M, Lee YU, Zhang G, Kolodziej AR, Andersen EC, Crombie TA, Giese GE, Yilmaz LS, Curtis BJ, Schroeder FC, Fox BW, Roberto NM, Ponomarova O, Na H, Zdraljevic S, Walhout AJM, Rodrigues PR

[Nature, 2022]

Individuals can exhibit differences in metabolism that are caused by the interplay of genetic background, nutritional input, microbiota and other environmental factors^1-4. It is difficult to connect differences in metabolism to genomic variation and derive underlying molecular mechanisms in humans, owing to differences in diet and lifestyle, among others. Here we use the nematode Caenorhabditis elegans as a model to study inter-individual variation in metabolism. By comparing three wild strains and the commonly used N2 laboratory strain, we find differences in the abundances of both known metabolites and those that have not to our knowledge been previously described. The latter metabolites include conjugates between 3-hydroxypropionate (3HP) and several amino acids (3HP-AAs), which are much higher in abundance in one of the wild strains. 3HP is an intermediate in the propionate shunt pathway, which is activated when flux through the canonical, vitamin-B₁₂-dependent propionate breakdown pathway is perturbed⁵. We show that increased accumulation of 3HP-AAs is caused by genetic variation in HPHD-1, for which 3HP is a substrate. Our results suggest that the production of 3HP-AAs represents a 'shunt-within-a-shunt' pathway to accommodate a reduction-of-function allele in hphd-1. This study provides a step towards the development of metabolic network models that capture individual-specific differences of metabolism and more closely represent the diversity that is found in entire species.

Alic N (2016) Cell Metab "Of FOXes and Forgetful Worms."

Alic N

[Cell Metab, 2016]

Age-related cognitive decline is one of the most haunting aspects of human aging. In a recent publication, Coleen Murphy and colleagues (Kaletsky et al., 2016) describe the transcriptional program that maintains youthful function of aging neurons in the nematode worm.

Rebecca M Fox et al. (2004) West Coast Worm Meeting "Finding unc-4 Target Genes"

Kathie Watkins, Denis Dupuy, Clay Spencer, Stephen E Von Stetina, Rebecca M Fox, Marc Vidal, David M Miller III, Joseph D Watson, Kellen Olszewski

[West Coast Worm Meeting, 2004]

The UNC-4 homeodomain protein regulates the specificity of synaptic input as well as the strength of synaptic output in the C. elegans motor circuit. UNC-4 is expressed in A class motor neurons; embryonically derived DA motor neurons and larval VA motor neurons. In unc-4 mutants, VAs are miswired with inputs normally reserved for their lineal sister cells, the VBs. Both DAs and VAs show decreased numbers of synaptic vesicles in unc-4 mutants. These effects also depend on UNC-37, a Groucho homolog, that functions as a transcriptional co-repressor with UNC-4 in these neurons. The identification of UNC-4/UNC-37 regulated target genes is a major goal of this laboratory. We have adopted two microarray-based approaches to define the full complement of unc-4 target genes. One strategy employs the mRNA tagging method to identify unc-4 regulated genes in VA motor neurons (see Von Stetina et al., this meeting). The second approach uses FACS to isolate unc-4 ::GFP labeled motor neurons from primary embryonic cell culture. unc-4 ::GFP is expressed in 13 embryonic motor neurons (9DA, 3SAB, 1I5). Although these neurons are not miswired in unc-4 mutants they do display the reduction in synaptic vesicle number found in all unc-4 motor neurons. unc-4 ::GFP cells can be enriched to 90% by FACS. Following two rounds of amplification, cRNA from wildtype unc-4 ::GFP neurons was hybridized to the Affymetrix C. elegans array and compared to cRNA from unc-4(e120) and unc-37(e262) mutants. We have identified ~90 genes that are significantly enriched in these mutants. To date, we have generated 15 GFP strains from this list using conventional cloning and overlap PCR. We have also created 14 GFP lines with constructs from the Promoterome project. We are now crossing these lines into unc-4(e120) and unc-37(e262) mutant backgrounds to confirm regulation by UNC-4/UNC-37.

Fox, Nicola et al. (2015) International Worm Meeting "Modulation of aging characteristics with an anti-aging compound."

Fox, Nicola, Mellor, EJC

[International Worm Meeting, 2015]

Aging and age-related disorders pose huge social and economic challenges to our society as the world's population ages. Investigating the cellular processes anti-aging compounds interact with can identify genes and pathways involved in the general aging process. Chronos Therapeutics compound RDC5 extends lifespan in yeast and C. elegans, and ameliorates osteoporosis, neurodegeneration and age-related weight gain in rodents. The anti-aging mechanism of RDC5 is unknown, although it is known to bind members of the FKBP (FK506-binding protein) family.As the general mechanisms of aging are well conserved between C. elegans and mammals, drug-sensitive C. elegans strains have been used to understand how RDC5 acts on aging. RDC5 treatment was shown to not inhibit E. coli OP50 growth or bus-5(br19) and bus-8 pharyngeal pumping at days 3 and 6 of adulthood, indicating RDC5 does not extend C. elegans lifespan by inducing dietary restriction. Instead, RDC5-treated bus-5(br19) had an increased rate of pharyngeal pumping at day 3 of adulthood, suggesting an extension of healthspan and not just lifespan. Differential transcriptome analysis of RDC5-treated and vehicle-treated bus-8 has been used to suggest potential targets of RDC5 for RNAi knock-down to identify the genes required for RDC5 anti-aging action in C. elegans.

Paul Fox et al. (2008) Development & Evolution Meeting "Analysis of GLD-1 targets in the proliferation/meiotic entry decision"

Eleanor Maine, Tim Schedl, Valerie Reinke, Paul Fox, Valarie Vought, Min-Ho Lee

[Development & Evolution Meeting, 2008]

Regulating the decision of stem cells to either proliferate or differentiate forms a key regulatory event for normal germline development. In the C. elegans germline, multiple pathways work together to regulate this process. The GLP-1 notch signaling pathway promotes the proliferative fate, while the redundant GLD-1 and GLD-2 pathways promote the meiotic fate. Work from our lab identified GLD-1 as an RNA binding protein that represses the translation of target mRNA's. A central hypothesis that thus emerges is that certain key GLD-1 target genes have important functions in either promoting the proliferative fate or inhibiting the meiotic fate. In order to dissect the role that these target genes may play in regulating the decision of germ cells to either proliferate or differentiate, we performed an RNAi based screen. As a first step, we conducted the RNAi screen of molecularly identified GLD-1 target genes into wild type and a glp-1 sensitized genetic background (rrf-1 and rrf-1;glp-1(bn18) respectively). We assayed for loss of proliferative cells on RNAi of the GLD-1 target genes to uncover genes that may function in promoting the proliferative fate of germ cells. However loss of genes that function in cell cycle progression would also result in loss of the proliferative cells. Thus, we screened for genes that specifically enhance the glp-1(bn18) sensitized genetic background to uncover genes with specific roles in the regulatory decision. From this analysis we identified numerous genes that affect the size of the proliferative zone. We performed a secondary screen wherein we quantified the number of proliferative cells to determine the nature of the interaction between GLD-1 target genes and partial loss of GLP-1 signal. On one of our best candidates from this screening, cye-1, we then performed detailed time course analysis of germ cells poised for entry into meiosis using various mitotic and meiotic cell cycle markers. Results from our analysis support the hypothesis that cye-1 functions, at least in part, downstream of GLD-1 to regulate the proliferation / meiotic entry cell fate decision.

Rebecca M Fox et al. (2005) International Worm Meeting "MAPCeL: Microarray Profiling C. elegans Cells"

Thomas Brodigan, Rebecca M Fox, Michael Krause, Kellen Olszewski, Susan J Barlow, Joan McDermott, W Clay Spencer, David M Miller, Stephen E Von Stetina

[International Worm Meeting, 2005]

Individual C. elegans cells can be easily visualized in vivo with GFP reporters but, owing to their small size, they are generally inaccessible for molecular analysis. We have now developed new methods for the isolation and gene expression profiling of GFP marked C. elegans cells (MAPCeL Microarray Profiling C. elegans Cells). In this strategy, GFP cells are isolated by FACS from in vitro cultures and RNA extracted for application to the C. elegans Affymetrix array. To demonstrate the utility of our approach, we have profiled cells in the embryonic motor circuit; we have identified genes that are differentially expressed in body muscle cells and in each of the three classes of embryonic ventral cord motor neurons (DA, DB, DD). Microarray data in each case were validated by strong correlation with genes known to be expressed in these cells as well as with GFP reporters that we constructed (~80%). For example, our MAPCeL data from inhibitory DD motor neurons confirms enrichment of transcripts required for the GABA phenotype (i.e. unc-25, unc-30, unc-47, snf-11). Conversely, the excitatory DA and DB motor neurons express cholinergic genes (unc-17, cha-1) as well as specific transcription factors that regulate cholinergic signaling (unc-3, unc-4). These data can now be used to identify novel genes with key roles in the locomotory circuit. For example, we have shown that DA motor neurons express a surprisingly diverse array of G-protein coupled receptors with the potential to modulate excitatory activity in response to neuropeptides as well as classical neurotransmitters. MAPCeL data from body muscle cells lead to the discovery that ACR-16 is a component of the levamisole-insensitive nicotinic ACh receptor (See Touroutine et al., this meeting). Finally, by identifying cohorts of genes with common expression patterns we can now search for cis-regulatory elements necessary for cell-specific expression. For example, we have identified enhancer element candidate motifs in the body wall muscle and DA motor neuron datasets (see Olszewski et al, this meeting). We conclude that MAPCeL can be used to generate reliable profiles of cell specific gene expression in the motor circuit and that these data will be valuable resources for correlating gene expression with function.1.Fox, RM, Von Stetina, SE, Barlow, SJ, Shaffer, C, Olszewski, KL, Moore, JH, Dupuy, D, Vidal, M, Miller, DM III. A gene expression fingerprint of C. elegans Embryonic motor neurons. BMC Genomics 2005, 6:42.