Uszkoreit, Simon et al. (2021) International Worm Meeting "Somatic Regulators of the Non-Cell-Autonomous CEP-1/p53-Mediated DNA Damage Response in Primordial Germ Cells"

Schumacher, Bjorn, Ou, Hui-Ling, Uszkoreit, Simon

[International Worm Meeting, 2021]

The integrity of heritable genomes is a prerequisite for species maintenance. For more than a century, it was thought that the genomes in the germline of an organism are isolated from somatic influences by the so-called Weismann barrier. In C. elegans, the primordial germline consists of two somatic gonad precursors (SGPs) and two primordial germ cells (PGCs). We previously established that PGCs are relying on global genome nucleotide excision repair (GG-NER) to repair UV-induced DNA lesions. When GG-NER is compromised and the DNA damage persists the C. elegans p53-like, CEP-1, protein is induced in PGCs and keeps them arrested. Using a forward genetic approach, we established an unexpected non-cell-autonomous regulation of the CEP-1-mediated DNA damage response (DDR) in PGCs via the somatic niche. The niche control of the DDR in PGCs is mediated by the translation initiation factor IFE-4 operating in the SGPs and communicated via FGF-like signalling. Moreover, we determined that the niche control mechanisms of the p53 response is highly conserved from worms to mammals. In mammals, the IFE-4 orthologue eIF4E2 regulates the p53 induction in hair follicle stem cells upon UV-induced DNA damage. Therefore, we propose that a better understanding of the non-cell-autonomous control of the p53-mediated DDR in C. elegans will be highly relevant to better understand this central tumour suppressor mechanism in humans. To elucidate the somatic regulators of the DDR in PGCs, we used a targeted genetic approach and interrogated pathways that we hypothesised to regulate the response to genotoxic stress. We determined an important regulatory role for nutritional interpretation and neuronal signalling. Impairment in these signalling processes suppressed the PGC cell cycle arrest and germline development upon persistent DNA damage. To dissect the mechanisms of this somatic influence in more detail, current emphasis is put on tissue-specific transcriptomics on fluorescence activated cell sorting (FACS)-based PGC/SGP isolations. We show for the first time a neuronal influence on the non-cell-autonomous DDR in PGCs and broaden the understanding of somatic regulations on the germline of C. elegans thus challenging the Weismann barrier.

Ou HL et al. (2021) STAR Protoc "Evaluating DNA damage response through immunofluorescence staining of primordial germ cells in Caenorhabditis elegans L1 larva."

Ou HL, Schumacher B

[STAR Protoc, 2021]

<i>C.elegans</i> L1 larvae have two well-defined primordial germ cells embedded in a niche comprising two somatic gonad precursor cells. Thus, <i>C.elegans</i> provides an ideal model for studying intercellular signaling in response to DNA damage. However, existing staining protocols are focused on worms in later developmental stages and are not optimized for the L1 larvae. Here, we present a revised protocol for assessing the DNA damage response utilizing immunofluorescence staining specifically in <i>C.elegans</i> L1 larva. For complete details on the use and execution of this protocol, please refer to Ou etal. (2019).

Ding, Siyu Serena et al. (2015) International Worm Meeting "Elucidating the role of nmy-2 in seam cell division patterns."

Ding, Siyu Serena, Woollard, Alison

[International Worm Meeting, 2015]

C. elegans seam cells are multipotent neuroectodermal cells that undergo both symmetric and asymmetric divisions during larval development, thus providing a paradigm for the stem cell mode of division. Reiterative seam cell asymmetric division typically produces a differentiative anterior daughter and a proliferative posterior daughter. Non-muscle myosin is fundamental to processes such as cellular reshaping and migration, hence it emerges as a potential regulator of seam cell asymmetric divisions. A precedent for the role of non-muscle myosin 2 (nmy-2) in C. elegans post-embryonic development has been established in the Q neuroblast lineage (Ou et al., 2010). This further sparked our interest in the role for nmy-2 in seam cells. While post-embryonically abrogating nmy-2 function either using temperature sensitive mutants or by RNAi failed to produce significant changes in seam cell numbers, combining the two treatments robustly reduced terminal seam cell number. In addition to expected cytokinesis failures, we observed progressive seam cell loss after each round of asymmetric division. We identified three classes of nmy-2 depletion phenotypes in seam cell divisions and generated a multi-color fate reporter strain in order to dissect the cell molecular basis of these phenotypes. These results will be presented. We are also in the process of investigating whether there is a relationship between nmy-2 function and asymmetric spindle positioning.Reference: Ou G. et al. "Polarized myosin produces unequal-size daughters during asymmetric cell division." Science. 2010;330(6004):677-80.

Ralph Menzel et al. (2006) European Worm Meeting "PCB52 Induced Gene Expression in the Nematode Caenorhabditis elegans"

Jana Kulas, Stephen Sturzenbaum, Shuang Li, Hui Ling Yeo, Ralph Menzel

[European Worm Meeting, 2006]

Ralph Menzel1, Jana Kulas1, Hui Ling Yeo1, Shuang Li1, and Stephen Strzenbaum2. Polychlorinated biphenyls (PCBs) are ubiquitous organic chemicals which can affect numerous biological responses. Because PCBs are toxic to man, recalcitrance to degradation and persist in the environment, they are listed in the inventory of priority pollutants compiled by numerous Environmental Protection Agencies.. Here we present an investigation of PCB52, an ortho-substituted, non-coplanar 2,2,5,5-tetrachlorbiphenyl. PCB52 affected the reproductive capacity of C. elegans in a strict concentration dependent manner, thereby highlighting its toxicological significance. Based on the calculated EC20 (5 mg/L), whole genomic DNA microarray experiments were performed to identify differentially expressed genes. Using a two-fold increase/decrease cut-off, 1158 genes were up-regulated and 560 down-regulated by PCB52. Up-regulated genes constituted family members of small heat shock proteins, chemoreceptors, nuclear hormone receptors, lipases as well as cytochromes P450 (CYP) and short-chain dehydrogenases/reductases (SDR), which are known to be involved in the biotransformation of persistent organic pollutants. Significant enriched classes of down-regulated genes are involved in organelle biogenesis, cell integrity and further general physiological processes, such as macromolecule metabolism and nucleotide binding.. The induction of five highly differentially regulated gene classes was confirmed by semi-quantitative RT-PCR experiments. Using a multiple RNAi approach the consequences of gene knockdown of single genes as well as entire gene subfamilies was determined. The efficiency of RNAi was proven by RT-PCR. Whilst GST and SDR gene expression knockdown intensify the toxicity of PCB52 (reflected in a reduced reproductive output) the knockdown of several CYP forms increased the reproductive capacity. This may indicate that the lack of CYP gene expression prevents the generation of toxic metabolites.

Osborne, Jennifer F et al. (2021) MicroPubl Biol

Hart, Anne C, Osborne, Jennifer F, Yanagi, Katherine S

[MicroPubl Biol, 2021]

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. While the mechanisms underlying motor neuron death in ALS are not well understood, mutations in over 25 genes can cause this disease (Marangi and Traynor 2015). It remains unclear which, if any, of these genes act in the same disease-associated pathway(s), or if they act in the same pathway(s) as genes associated with the related disorder, frontotemporal dementia (FTD) (Ling et al. 2013). The first ALS-causing gene to be identified was superoxide dismutase 1 (SOD1), a regulator of cytoplasmic redox homeostasis (Rosen et al. 1993). We can begin to construct a pathway for neurodegeneration through SOD1 by identifying genes whose loss of function (LOF) modifies the level of degeneration in a C. elegans SOD1 ALS model. This will contribute to our understanding of whether ALS/FTD genes act in a single or multiple pathways to cause disease.

Graber JH et al. (2007) RNA "C. elegans sequences that control trans-splicing and operon pre-mRNA processing."

Salisbury J, Hutchins LN, Graber JH, Blumenthal T

[RNA, 2007]

Many mRNAs in Caenorhabditis elegans are generated through a trans-splicing reaction that adds one of two classes of spliced leader RNA to an independently transcribed pre-mRNA. SL1 leaders are spliced mostly to pre-mRNAs from genes with outrons, intron-like sequences at the 5''-ends of the pre-mRNAs. In contrast, SL2 leaders are nearly exclusively trans-spliced to genes that occur downstream in polycistronic pre-mRNAs produced from operons. Operon pre-mRNA processing requires separation into individual transcripts, which is accomplished by 3''-processing of upstream genes and spliced leader trans-splicing to the downstream genes. We used a novel computational analysis, based on nonnegative matrix factorization, to identify and characterize significant differences in the cis-acting sequence elements that differentiate various types of functional site, including internal versus terminal 3''-processing sites, and SL1 versus SL2 trans-splicing sites. We describe several key elements, including the U-rich (Ur) element that couples 3''-processing with SL2 trans-splicing, and a novel outron (Ou) element that occurs upstream of SL1 trans-splicing sites. Finally, we present models of the distinct classes of trans-splicing reaction, including SL1 trans-splicing at the outron, SL2 trans-splicing in standard operons, competitive SL1-SL2 trans-splicing in operons with large intergenic separation, and SL1 trans-splicing in SL1-type operons, which have no intergenic separation.

Michelle S. Teng et al. (2006) European Worm Meeting "Expression of Mammalian GPCRs in C. elegans Generates Novel Behavioural Responses to Human Ligands"

John McCafferty, Martijn P.J. Dekkers, Andrew G. Fraser, Michelle S. Teng, Bee Ling Ng, Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2006]

Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.

Antebi A et al. (1996) East Coast Worm Meeting "daf-12 Coordinates Multiple Developmental Events"

Hedgecock EM, Antebi A

[East Coast Worm Meeting, 1996]

In the nematode, developmental events within or between tissues are precisely coordinated. The DAF-12 nuclear hormone receptor plays multiple roles in synchronizing development. As a heterochronic regulator, it instructs stage specific programs in both gonad and soma. In the dauer pathway, it selects dauer or continuous development in response to growth conditions. Gonadal, somatic and dauer functions are genetically separable, suggesting a complex locus, and alleles can be grouped into six idealized classes. In collaboration with Don Riddle, Pam Larsen and Wen Hui Yeh, we have begun a molecular analysis of alleles to correlate structure with function. So far, we have found a sharp clustering of alleles which selectively disrupt dauer formation within the zinc fingers of the receptor. A unifying hypothesis is that a hormone, acting through DAF-12 and related receptors, coordinates events in gonad and some, as well as dauer development. We suggest a simple model in which the commitments or starts to each developmental stage are synchronized by a hormonal pulse available to all tissues. Moreover, we propose that the starts to each developmental stage lie between the molts, as judged by new cuticle synthesis in the hypodermic and transitions in the migratory behavior of gonadal leader cells. These "parastages" may more accurately reflect the actual functional boundaries of developmental stages.

Ding, Siyu S et al. (2013) International Worm Meeting "Elucidating the role of nmy-2 in seam cell division patterns."

Ding, Siyu S, Woollard, Alison, Appleford, Peter J

[International Worm Meeting, 2013]

C. elegans seam cells are multipotent neuroectodermal cells that undergo both symmetric and asymmetric divisions during larval development, thus providing a paradigm for the stem cell mode of division. Reiterative seam cell asymmetric division typically produces an anterior daughter that rounds up and a posterior daughter that elongates after division. The anterior daughter then moves out of the seam line and differentiates whereas the posterior daughter further elongates to re-form the seam line and maintains the stem fate. Non-muscle myosin is fundamental to processes such as cellular reshaping and migration, hence it emerges as a potential regulator of seam cell asymmetric divisions. A precedent for the role of non-muscle myosin 2 (NMY-2) in C. elegans post-embryonic development has been established in the Q neuroblast lineage (Ou et al., 2010). This further sparked our interest in the role for NMY-2 in seam cells. While post-embryonically abrogating nmy-2 function either using temperature sensitive mutants or by RNA interference failed to produce significant changes in seam cell numbers, combining the two treatments robustly reduced terminal seam cell number from 16 in wild-type animals to 11.6 in treated animals. Progressive cell loss was observed in each round of asymmetric division. Our current focus is to understand the cell molecular processes by which nmy-2 influences seam cell asymmetric divisions using live imaging and video lineage analysis.

Yu YB et al. (2010) PLoS One "Cinnamomum cassia bark in two herbal formulas increases life span in Caenorhabditis elegans via insulin signaling and stress response pathways."

Dosanjh L, Shim BS, Yu YB, Luo Y, Tan M, Lao L

[PLoS One, 2010]

BACKGROUND: Proving the efficacy and corresponding mode of action of herbal supplements is a difficult challenge for evidence-based herbal therapy. A major hurdle is the complexity of herbal preparations, many of which combine multiple herbs, particularly when the combination is assumed to be vitally important to the effectiveness of the herbal therapy. This issue may be addressed through the use of contemporary methodology and validated animal models. METHODS AND PRINCIPAL FINDINGS: In this study, two commonly used traditional herbal formulas, Shi Quan Da Bu Tang (SQDB) and Huo Luo Xiao Ling Dan (HLXL) were evaluated using a survival assay and oxidative stress biomarkers in a well-established C. elegans model of aging. HLXL is an eleven herb formula modified from a top-selling traditional herbal formula for the treatment of arthritic joint pain. SQDB consists of ten herbs often used for fatigue and energy, particularly in the aged. We demonstrate here that SQDB significantly extend life span in a C. elegans model of aging. Among all individual herbs tested, two herbs Cinnamomum cassia bark (Chinese pharmaceutical name: Cinnamomi Cortex, CIN) and Panax ginseng root (Chinese pharmaceutical name: Ginseng Radix, GS) significantly extended life span in C. elegans. CIN in both SQDB and HLXL formula extended life span via modulation of multiple longevity assurance genes, including genes involved in insulin signaling and stress response pathways. All the life-span-extending herbs (SQDB, CIN and GS) also attenuated levels of H2O2 and enhanced small heat shock protein expression. Furthermore, the life span-extending herbs significantly delayed human amyloid beta (Abeta)-induced toxicity in transgenic C. elegans expressing human Abeta. CONCLUSION/SIGNIFICANCE: These results validate an invertebrate model for rapid, systematic evaluation of commonly used Chinese herbal formulations and may provide insight for designing future evidence-based herbal therapy(s).