Gaeta, Anthony et al. (2021) International Worm Meeting "Dietary Composition Modulates Neurodegeneration in a C. elegans Parkinson's Disease Model"

Caldwell, Kim, Caldwell, Guy, Keogh, Candice, McKay, Luke, Gaeta, Anthony

[International Worm Meeting, 2021]

The diet of any organism is crucial in providing the energy necessary to not only exist and persist in the environment, but to overcome stress when challenged. One such type of stress comes in the form of Parkinson's disease (PD), which is characterized by the progressive loss of dopaminergic (DA) neuron function and integrity. Although it has been reported and observed that diet type has wide ranging effects on phenotypes in both humans and animal models, the effect that diet has on the progression of DA neuron loss in PD is largely unknown. However, evidence supporting a connection between the brain and the gut microbiome has become increasingly relevant to neurodegenerative diseases. Furthermore, the effect of parental diet on attributes of PD in subsequent generations has not been adequately investigated. The findings of this study indicate that a non-standard diet for Caenorhabditis elegans (C. elegans) has an impact on PD-associated pathology in this animal, in a transgenerational manner. Specifically, feeding worms an alternative bacterial diet of HB101 E. coli, as opposed to the laboratory standard OP50 E. coli, promotes significant, transgenerational protection of DA neurons in response to the stressor and pathological hallmark of PD, a-synuclein (a-syn) overexpression and accumulation. This protection is shown to be dependent on the normal function of Systemic RNA Interference Defective (SID) proteins, including SID-1, SID-2, and SID-3. These proteins are involved in the ability of double-stranded RNAs (dsRNAs) to traverse cellular boundaries, systemically, and silence target genes in C. elegans to varying degrees. Comparative transcriptomic analysis between worms reared on OP50 vs. HB101 E. coli in an a-syn model background has revealed genes and pathways associated with a wide range of processes. These included genes encoding proteins involved in metabolism, cell signaling, development, transcription, stress responses, and transmembrane transport, all of which were observed to be differentially expressed in worms reared on HB101 E. coli. Subsequent functional analysis of select targets by RNA interference revealed the contribution of individual genes to neuroprotection against a-syn-mediated neurodegeneration. This research lays a foundation for future investigation of the subtle distinctions in diet that can impact conserved pathways modulating neuron survival in response to stress.

Stephen C Pak et al. (2005) International Worm Meeting "Protein aggregate formation and disruption of post-embryonic development in worms expressing SRP-2::GFP"

Clifford J Luke, Gary A Silverman, Stephen C Pak

[International Worm Meeting, 2005]

Serpins are high molecular weight serine proteinase inhibitors that irreversibly inactivate target proteinases using a suicide substrate-like mechanism. Although in vitro biochemical characterizations have provided some clues as to the identity of the target proteinases, the pathways in which serpins play a role are currently unknown. To determine whether serpins plays a role in C. elegans development, growth and longevity, we generated mutants that lack or over-express serpin genes. In general, with the exception of srp-6 (see poster by Luke et al.,), null mutants displayed no overt phenotypes. In contrast, transgenic animals over-expressing serpins displayed a variety of developmental and growth defects. In particular, over-expression of SRP-2::GFP resulted in distruption of post-embryonic development characterized by early larval arrest, slow growth and molting defects. Over-expression of SRP-2 mutants harboring amino-acid substitutions that diminish the serpins ability to inhibit proteinases failed to suppress the phenotype. This result suggests that the phenotype is independent of the proteinase-inhibitory function of SRP-2. Preliminary structure/function mapping results suggest that a short, N-terminal portion of SRP-2 is sufficient for the phenotype. Interestingly, we observed large, insoluble SRP-2::GFP aggregates in the cytosol of anterior hypodermal cells. The relationship between aggregate formation and the developmental defects is currently unclear. We are currently conducting a genetic screen to identify modifiers of the SRP-2 developmental and aggregation phenotypes. We hope to present the results of the genetic screen at the conference.

Robert Barstead et al. (2005) International Worm Meeting "The C. elegans Gene Knockout Consortium"

Anna Rankin, Candice Navaroli, Owen Dadivas, Joanne Lau, Angela Fisher, Nadereh Rezania, Lucy Liu, Mark Edgley, Robert Barstead, Allison Hay

[International Worm Meeting, 2005]

The C. elegans Gene Knockout Consortium (http://www.celeganskoconsortium.omrf.org/) pursues systematic targeted gene disruption in the nematode, both upon request for the research community and in the larger context of the whole set of genes for which human orthologs exist (about 7,000 genes). This work is currently based on a reverse-genetics PCR method, generating single-gene deletions. All deletions are stabilized as homozygous or balanced heterozygous strains, the deletion breakpoints are sequenced, and the strains and data are placed immediately into the public domain through our collaborators at the Caenorhabditis Genetics Center (CGC: http://biosci.umn.edu/CGC/CGChomepage.htm) and WormBase (http://www.wormbase.org/). To date the Consortium has generated deletions in more than 1800 genes, and more than 1300 of these have been stabilized and archived at the CGC. Distribution of Consortium strains accounts for an increasing proportion of distributions from the CGC every year, and stood at 20% for the calendar year 2004.The reverse genetics PCR method using UV/TMP as the mutagen has evolved in our hands into a robust system for efficient generation of deletion mutations. However, some target genes have been refractory to this method, and we therefore continue to evaluate other isolation/detection methods (for example, see abstracts on array CGH and resequencing at this meeting). We will report on our latest progress using all methods.Additional authors for whom inadequate space was allowed by abstract submission system: Peter Huang (UBC and BC Genome Sciences Centre), Jason Maydan (UBC), Sheldon McKay (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Adam Warner et al. (2006) Development & Evolution Meeting "The LIM Domain Protein C28H8.6 is Required for Viability and is Localized to Dense Bodies in Caenorhabditis elegans Body Wall Muscle Cells"

Adam Warner, Donald Moerman, Barbara Meissner

[Development & Evolution Meeting, 2006]

Proper conversion of the contractile force generated by sliding myofilaments within muscle into movement of a single nematode requires attachment of actin to structures known as dense bodies. These structures in C. elegans muscle are both homologous and analogous to Z-lines found in mammalian muscle cells. The dense body projects inward from the sarcolemma towards the cytoplasm and anchors thin filaments that participate in an organized lattice, the sarcomere. This lattice also contains myosin thick filaments, which are anchored to the sarcolemma by the M-line. Dense bodies play a pivotal role in the development of muscle cells. Evidence supporting this conclusion comes from examination of mutants with loss of core structural proteins. Loss of these proteins results in either lethality, or animals with reduced movement. While most muscle mutants have been uncovered through mutational screens focusing on these phenotypes, we propose that additional sarcomeric proteins exist for which there is a less severe or entirely different mutant phenotype. In order to search for these proteins, we used a combination of SAGE expression data (McKay et al. 2003) and predicted protein information to uncover a small number of potential candidates. One of these, the LIM domain protein C28H8.6, has a high level of homology to the C-terminal half of human paxillin, a core component of focal adhesions. This multi-LIM domain protein appears to have an important role in C. elegans muscle. First, a full-length C28H8.6::GFP functional fusion localizes to dense bodies. Secondly, the homozygous gene knockout of C28H8.6 results in uncoordinated animals arrested at the L1 stage of development. Further analysis is underway to characterize this newly discovered muscle protein.

Ana Vaz Gomes et al. (2003) International Worm Meeting "Gene expression profiles in cells, tissues and development of C. elegans"

Heide Kai, Peter Huang, Allan Mah, Ana Vaz Gomes, Kim Wong, Adam Warner, Lily Fang, David Baillie, Nicholas Dube, Claes Wahlestedt

[International Worm Meeting, 2003]

A new consortium of labs in Canada and Sweden has been established to complement other Caenorhabditis elegans functional genomics efforts by using additional high-throughput approaches to gene expression profiling through the course of development. We are examining expression profiles for 2000 genes, with an emphasis on human orthologs and genes that are currently being knocked out by the C. elegans gene knockout consortium. We are interested in profiling temporal and spatial patterns of gene expression and furthering our understanding of cellular protein network organization in a three dimensional, organismal context. The advanced state of genome science and well-defined developmental program in C. elegans make it an ideal candidate organism. We are using serial analysis of gene expression (SAGE) and high resolution image analysis of expression patterns detected with green fluorescent protein (GFP)-promoter fusion constructs. The initial focus is on developmental SAGE of various life stages from egg to adult and the generation of GFP-promoter fusion strains suitable for high-resolution image analysis of in vivo gene expression patterns. Thus far, we have generated promoter-GFP transgenic strains for 400 genes (see Johnson et al, "Expression of promoter-GFP constructs in C. elegans") and SAGE libraries for various stages of C. elegans development including embryo, the four larval stages and young adult (see McKay et al, "Evaluation of SAGE for the study of developmental gene expression profiles in C. elegans"). To generate tissue-specific SAGE libraries, we are refining techniques to use selected GFP constructs in conjunction with fluorescence activated cell sorting to isolate samples enriched for particular cell types from disrupted embryos. This project is funded by Genome British Columbia and Genome Canada

David Baillie et al. (2003) International Worm Meeting "Evaluation of SAGE for the study of developmental gene expression profiles in C. elegans"

Helen McDonald, Greg Vatcher, Richard Varhol, Peter Huang, Pawan Pandoh, David Baillie, Kim Wong, Adam Warner, Heide Kai, Scott Zuyderduyn

[International Worm Meeting, 2003]

As part of a consortium of laboratories in Canada and Sweden (see McKay, et al, "Gene expression profiles in cells, tissues and development of C. elegans"), we have developed computational and biological approaches to assist in high throughput analysis of gene expression. Biological source material for SAGE libraries was generated using synchronized populations as well as micro-dissected gut tissue. We are also refining GFP-mediated cell sorting techniques to isolate other cell types from disrupted embryos. SAGE data are generated and tracked in an automated pipeline from sequencing of cloned concatamers through to annotation, quality control and mapping of individual tags. Tags are associated with attributes such as cumulative phred scores, source clone, parent ditag and tag-to-gene mapping data to aid in downstream quality filtering and analysis steps. To facilitate tag-to-gene mapping and comparison of SAGE and Affymetrix GeneChip technologies, a virtual C. elegans transcriptome with both confirmed and estimated untranslated regions (UTRs) for all known and predicted transcripts was constructed using the current gene models and expressed sequence tag (EST) data annotated in wormbase. We are also refining tag-to-gene mapping methods in order to detect previously undocumented transcripts and alternative splice variants. Comparison of the Affymetrix C. elegans chip with the current virtual transcriptome showed that about 97% of the confirmed or predicted transcripts are detected by all or part of at least one probe set. At the time of writing, four developmentally staged SAGE libraries to be used in developmental gene expression profiling have been completed. Preliminary SAGE studies and results of a direct comparison of gene expression levels measured from the same biological source material with SAGE, long-SAGE and Affymetrix GeneChip technologies will be presented. This project is funded by Genome British Columbia and Genome Canada.

Johnsen RC et al. (1996) West Coast Worm Meeting "WHAT'S NEW WITH sDp2: MAPPING ESSENTIALS ON LGI(LEFT) GENETICALLY AND PHYSICALLY."

Johnsen RC, Rose AM

[West Coast Worm Meeting, 1996]

With the physical map and the genomic sequencing nearing completion the challenge is to correlate biological information with the predicted genes. The approach we have taken is to do: 1) Large scale screening for lethal mutants in balanced regions of the genome; 2) Mapping the mutants to smaller sub-regions; and 3) Rescuing of mutant phenotypes using transgenic strains containing sequenced cosmids. In collaboration with D. Janke and D. Baillie at SFU, and the C. elegans sequencing labs, we are using transgenic strains to cross to the lethals. The Rose lab is working on the large genomic region of LG I (Howell et al. 1987; Howell and Rose 1990; McKim et al. 1992; J. McDowell 1996) balanced by sDp2. This region comprises the left third of the chromosome, at least 17 mu. Irradiation-induced derivatives of sDp2 (McKim and Rose 1989) and other rearrangement breakpoints have been used to subdivide the region into relatively small zones. From the 31,600 chromosomes screened, 406 lethal mutants that map into the sDp2 balanced region are being analyzed. The Rose lab has published the position of 93 essential genes derived from the 406 lethal mutations. We estimate that the lethal mutants will define approximately 150 essential genes which is about 2/3 of all the essential genes balanced by sDp2. We report here an additional 23 bringing the total to 116 essential genes. So far 20 essential genes in the sDp2 region have been complementation rescued by transgenic worms (S. McKay 1994; J. McDowall 1996; A. Tinker pers. comm.). As a result of the CGAT project, D. Janke of the Baillie lab has provided us with a set of transgenics. Initially, we are using the following cosmids: C04F1, K02F2, C27A12, F33D11, R12E2, and C18E3. C04F1 is near let-378, let-393, and let-513. K02F2 is near let-394, let-534 and dpy-14. C27A12 is near let-384, let-391 and let- 512. F33D11 is near let-388 and let-514. R12E2 and C18E3 are predicted to be within hDf6. Their positions relative to the genetic map will be confirmed by using PCR and complementation mapping. A number of collaborations with other labs working on specific genes that correspond to mutants in the sDp2 region have been established.

Barbara Meissner et al. (2005) International Worm Meeting "A Comprehensive RNAi Screen for Novel Muscle Mutants in Caenorhabditis elegans."

Adam Warner, Barbara Meissner, Don Moerman, Nicholas Dube

[International Worm Meeting, 2005]

One of the fundamental features of metazoan development is myogenesis. A crucial step during myogenesis is the assembly and anchorage of the sarcomere, the essential repeat unit responsible for muscle contraction. In Caenorhabditis elegans, three phenotypic classes of muscle mutants defective in some aspect of muscle structure and function have been identified in mutagenesis screens: the uncoordinated (unc) class, typified by uncoordinated, slow or no movement, the late embryonic lethal class known as paralyzed and arrested at two-fold stage (pat) mutants, and the class where animals are capable of wildtype development and movement but have disorganized muscle (dim). Recent work from our lab and others has begun to describe the complement of genes expressed in developing body wall muscle (McKay et al., 2004; Roy et al., 2002, R. Fox and D. Miller, personal communication). Using SAGE and microarray chip analysis we have identified 3,500 genes, excluding ribosomal genes, which are expressed in muscle. Using an RNAi feeding library (Kamath & Ahringer, 2003), we are screening this muscle expressome for genes affecting sarcomere assembly, stability and/or function. Worms harboring an extrachromosomal array containing a myosin heavy chain gene, myo-3, fused in frame to green fluorescent protein (GFP; a gift of P. Hoppe) are fed bacteria expressing dsRNA corresponding to each gene within the muscle expressome. The progeny of these worms are examined for mislocalization of myo-3::GFP using a compound microscope. Progeny are also examined to see if any exhibit a pat or unc phenotype. This approach is proving to be a rapid and sensitive means to identify genes that are required to organize sarcomeric proteins into a highly ordered myofilament lattice. To date, we have screened ~500 genes, successfully identifying 85 genes with defects in myo-3::GFP localization (41 moderate and 44 severe). These RNAi treated animals display an array of myofilament disruptions ranging from small aggregations of myo-3::GFP to large deposits, often accompanied by disorganization of the myofilaments. The high percentage of tested genes affecting muscle sarcomeres, 17% , we suspect reflects the fact that we have already enriched for genes expressed in this tissue. Many of the genes affecting sarcomere integrity have human homologs for which little or nothing is known.

Don Moerman et al. (2003) International Worm Meeting "Expression of Promoter-GFP Constructs in C. elegans"

Steve Jones, Marco Marra, Erik Sonnhammer, Allan Mah, David L Baillie, Francis Ouellette, Don Moerman, Domena Tu, Zhongying Zhao, Sheldon McKay

[International Worm Meeting, 2003]

We are examining expression profiles for 2000 C. elegans genes with human orthologs (see McKay et al. abstract) or which have been requested from the CE Knockout Project. Our aim is to develop understanding of human genes of unknown function. Analyzing human orthologs in C. elegans will enable us to quickly determine temporal, spatial and level of gene expression. We have generated promoter-GFP transgenic strains for 400 human orthologs which we are analyzing by green fluorescent protein (GFP) microscopy. We use PCR stitching to fuse potential promoter-containing regions of genomic DNA with a promoterless GFP expression cassette. Identifying target regions and designing suitable PCR primers were automated using custom software developed to integrate the C. elegans AceDB genomic database (wormbase) and other data sources with the primer design and validation programs primer3 and e-PCR to facilitate high throughput generation of constructs. Primers were designed for more than 15,000 genes, with most of the remainder having unsuitable upstream regions. Simple arrays of promoter-GFP construct and dpy-5(+) DNA was injected into Dpy-5 hermaphrodites. Wild-type progeny were analyzed for their GFP-expression patterns. About 73% of the 400 strains show expression and about 49% of these express only in one tissue type. Gut is the most common tissue for expressed genes with 38% of the GFP expressing constructs. 47% of the constructs that express in gut do not express in any other tissue. Effectively all tissues express more than one construct and most tissues have at least one construct that does not express in any other tissue. For example: muscle (17% expression and 7% only in muscle); pharynx (22% and 1.3%); and nerve cord (9% and 1.3%). 39% of the expressing transgenics show embryonic expression (7% do not show expression at any developmental stage other than embryonic). Usually promoter-GFP constructs show mosaic expression patterns which makes analysis more difficult especially in embryo; therefore, we are using X-irradiation to induce stably expressing lines. So far, we have stable lines for 30 promoter-GFP constructs. Data will be made publicly available at a website. Funded by Genome BC and Genome Canada.

Michelle Kudron et al. (2006) Development & Evolution Meeting "Functional investigation of nst-1, a conserved nucleolar protein, in germline stem cell proliferation"

Valerie Reinke, Zhang Yang, Michelle Kudron

[Development & Evolution Meeting, 2006]

A mitotic population of germ cells at the distal tip of the gonad bears several hallmarks of stem cells: they self-renew, differentiate into different cell types, and require a “niche”. In order to identify transcripts enriched in germline stem cells (GSC) relative to meiotic germ cells, we performed whole genome microarray analysis comparing wild-type to glp-1(gf) mutants bearing excess stem cells (Berry et al. Development 124:925, 1997). Approximately 10% of the GSC-enriched transcripts encode proteins that are likely to function or localize to the nucleolus. The nucleolus has been shown to play a direct role in modulating cell proliferation by sequestering cell cycle factors (Visintin and Amon, Curr. Opin. Cell Biol. 12:372, 2000). One of the GSC-enriched transcripts found in the microarray is orthologous to nucleostemin, a nucleolar GTPase recently found to be upregulated in mammalian neural stem cells and cancer cells (Tsai and McKay, Genes Dev. 16:2991, 2002). The C. elegans ortholog of nucleostemin is highly conserved, especially in the two GTPase domains; thus we have named it nst-1. Because of the link to mammalian cells, we have studied this gene further. We isolated a deletion mutant in nst-1 that removes 75% of the protein, including both GTPase domains. The nst-1(vr6) homozygous mutants arrest as young larvae possibly due to the requirement for NST-1 in larval somatic tissues. We utilized RNAi and transgene rescue to bypass the somatic defect and investigate the effects of nst-1 loss in the germline. We performed nst-1(RNAi) in an rrf-1(pk1417) mutant background (defective for RNAi in soma (Sijen et al., Cell. 107:465, 2001)) which results in sterility with severe defects in proliferation and differentiation in the germline. When an nst-1 rescuing transgenic line is crossed to nst-1(vr6), the animals are rescued in the soma, but display germline defects. These experiments demonstrate that nst-1 acts in the germline to modulate germ cell proliferation. We also examined the localization of NST-1 by generating a translational fusion strain. NST-1 is localized to the nucleolus and nucleoplasm of numerous somatic tissues, including cells in the pharynx, vulva, and intestine. We plan to investigate germline localization of NST-1 and we are currently elucidating roles for nst-1 in somatic cell proliferation and growth.