Lindsay MacDonald et al. (2006) Development & Evolution Meeting "Cloning and Characterization of syt(oz237), a Gene Involved in Regulating Proliferation in the Germline"

Aaron Knox, Lindsay MacDonald, Dave Hansen, Malcolm Williamson

[Development & Evolution Meeting, 2006]

The GLP-1/Notch signaling pathway controls the proliferation vs. meiotic entry decision in the germline. GLP-1/Notch signaling promotes proliferation by negatively regulating two redundant downstream pathways (GLD-1/GLD-2), which inhibit proliferation and/or promote meiotic entry. To identify genes that function in the GLD-1 pathway a mutant screen in a gld-2(0) background was performed. Syt(oz237) was identified in this screen as being synthetically tumourous with gld-2(0). The oz237 single mutant is homozygous recessive with a sterile and protruding vulva phenotype. Along with being synthetic tumourous with gld-2(0), oz237 is also synthetic tumourous with gld-3(0). When GLP-1 activity is removed from either synthetically tumourous double mutant, the animals are no longer tumourous but rather have the premature meiotic entry Glp phenotype. This suggests that syt(oz237) may not function in the GLD-1 pathway, but may actually function upstream of Notch signaling. We have mapped oz237 to a 40 kB region between lst-1 and tli-1 on Chromosome I. There are 14 genes is this region and we are currently performing RNAi and sequencing candidate genes to determine the identity of the syt(oz237) gene.

Lindsay D. MacDonald et al. (2007) International Worm Meeting "Mutation in pas-5, subunit of 20S proteosome, enhances overproliferation in the germline."

Dave Hansen, Lindsay D. MacDonald, Malcolm Williamson, Aaron Knox

[International Worm Meeting, 2007]

The GLP-1/Notch signaling pathway controls the proliferation vs. meiotic entry decision in the germline. GLP-1/Notch signaling promotes proliferation by negatively regulating two redundant downstream pathways (GLD-1/GLD-2), which inhibit proliferation and/or promote meiotic entry. To identify genes that function in the GLD-1 pathway a mutant screen in a gld-2(0) background was performed. The oz237 allele was identified in this screen as being synthetically tumourous with gld-2(0). The oz237 single mutant is sterile (Mog) and has a protruding vulva. We have mapped oz237 to a 40 kB region between lst-1 and tli-1 on Chromosome I. There are 14 genes in this region; we used RNAi and sequenced candidate genes to determine the gene corresponding to the oz237 allele. A mutation was discovered in the coding region of pas-5 (proteasome alpha subunit). An extrachromasomal array containing full length WT pas-5 can partially rescue the sterile phenotype in the oz237 mutants, suggesting the lesion in pas-5 is the oz237 allele. oz237 is likely a partial loss of function allele since RNAi to pas-5 causes embryonic lethality, and oz237/(0) causes a more severe phenotype than seen in the oz237/oz237 mutant worms. To help identify the protein or proteins that are not being degraded properly in the oz237 mutant we first determined if it interacts with other Notch regulated cell fate decisions. pas-5 may act as a general negative regulator of Notch signaling since the oz237 mutant can enhance the multi-vulva phenotype of two weak gain of function mutations of lin-12. We also performed epistasis experiments with other regulators of the proliferation vs. differentiation decision in the germline. Along with being synthetic tumourous with gld-2(0), oz237 is also synthetic tumourous with gld-3(0). When GLP-1 activity is removed from the gld-2 oz237 synthetically tumourous double mutant, the animals are no longer tumourous, but rather have the premature meiotic entry Glp phenotype. This interaction suggests pas-5 may function upstream of Notch signaling. However when GLP-1 activity is removed from the oz237; gld-3 double mutant the germline remains tumourous. This suggests that gld-2 and gld-3 have separate functions in relation to the oz237 mutation when involved in the mitosis vs. meiosis decision in the germline.

MacDonald, Lindsay D. et al. (2009) International Worm Meeting "Protein degradation in the C. elegans germ line can regulate entry into meiosis."

Hansen, Dave, MacDonald, Lindsay D., Knox, Aaron

[International Worm Meeting, 2009]

In any population of stem cells a delicate balance between proliferation and differentiation must be maintained to ensure proper stem cell function and tissue homeostasis. In the C. elegans germ line, this balance is controlled by the GLP-1/Notch signaling pathway. GLP-1/Notch signaling promotes proliferation, at least in part, by negatively regulating two redundant downstream pathways (GLD-1/NOS-3, GLD-2/GLD-3), which inhibit proliferation or promote meiotic entry. We have recently identified a weak loss of function allele of one of the proteasome alpha subunits (pas-5(oz237)), that enhances an overproliferation phenotype in the C. elegans germ line. Through genetic epistasis we have found that the proteasome has two separate functions in regulating the decision between proliferation and meiotic entry. First, the proteasome acts as a general negative regulator of Notch signaling. sel-10, an E3 ubiquitin ligase, has been implicated in GLP-1 INTRA degradation by targeting INTRA to the proteasome. However, when proteasome function is reduced and SEL-10 is no longer present in the cell, an enhancement of Notch signaling is still seen, suggesting a second E3 ubiquitin ligase is required for proper ubiquitination and subsequent degradation of INTRA. Second, the proteasome is involved in negatively regulating proliferation downstream of GLP-1/Notch signaling, preferentially in the GLD-1/NOS-3 pathway. GLD-1 is a KH domain containing protein that has been shown to bind the 3''UTRs of specific transcripts to prevent their translation to promote meiotic entry. These findings lead us to a model in which the proteasome is responsible for degrading proteins that are also regulated by GLD-1 to more efficiently remove them from the cell and promote entry into meiosis as the cells move out of the mitotic zone of the gonad. In order to identify the proteins whose degradation is necessary for proper entry into meiosis we are performing an RNAi screen of all substrate recognition subunits (SRS). These SRS''s are responsible for binding to the specific target protein to allow that protein to be ubiquitinated and subsequently degraded by the proteasome. If we knockdown the function of the SRS, its target protein will not be degraded by the proteasome, resulting in an overproliferation phenotype similar to that seen in the pas-5(oz237) mutant. If we can identify the SRS we can then use biochemical methods to identify the protein that is required for proliferation and whose degradation is required for entry into meiosis.

Lindsay MacDonald et al. (2008) Development & Evolution Meeting "Mutation in pas-5, Subunit of 20S Proteasome, Enhances Overproliferation in the C. elegans Germ Line"

Dave Hansen, Aaron Knox, Lindsay MacDonald

[Development & Evolution Meeting, 2008]

The GLP-1/Notch signaling pathway controls the proliferation vs. meiotic entry decision in the C. elegans germ line. GLP-1/Notch signaling promotes proliferation by negatively regulating two redundant downstream pathways (GLD-1, NOS-3/GLD-2, GLD-3), which inhibit proliferation and/or promote meiotic entry. To identify genes that function in the GLD-1 pathway a mutant screen in a gld-2(0) background was performed. The oz237 allele was identified in this screen as being synthetically tumourous with gld-2(0). We have mapped oz237 to a 40 kB region between lst-1 and tli-1 on Chromosome I in the C. elegans genome. There are 14 genes in this region; we used RNAi and sequenced candidate genes to determine the gene corresponding to the oz237 allele. A mutation was discovered in the coding region of pas-5 (Proteasome Alpha Subunit). An extrachromasomal array containing full length WT pas-5 can partially rescue the germ line phenotype in the oz237 mutants, suggesting the lesion in pas-5 is the oz237 allele. pas-5(oz237) is likely reducing proteasome activity since conventional RNAi to pas-5 causes embryonic lethality but low dose RNAi can phenocopy the pas-5(oz237); glp-1(gf) germline phenotype. To help identify the protein or proteins that are not being degraded properly in the pas-5(oz237) mutant we determined if it interacts with other Notch regulated cell fate decisions. pas-5 may act as a general negative regulator of Notch signaling since the oz237 mutant can enhance the multi-vulva phenotype of two weak gain-of-function mutations of lin-12(Notch). It also causes a multi-vulva phenotype with sel-10(0), an F-Box protein thought to target LIN-12(INTRA) to the proteasome. We also performed epistasis experiments with other regulators of the proliferation vs. differentiation decision in the germ line. Along with being synthetic tumourous with gld-2(0), pas-5(oz237) is also synthetic tumourous with gld-3(0). When GLP-1 activity is removed from the gld-2 pas-5(oz237) tumourous double mutant, the animals are no longer tumourous, but rather have the premature meiotic entry Glp phenotype. This interaction suggests pas-5 may function genetically upstream of Notch signaling. However when GLP-1 activity is removed from the pas-5(oz237); gld-3 double mutant the germ line remains tumourous; however the tumor is not as robust as in the double mutant. This suggests that gld-2 and gld-3 have separate functions in relation to the pas-5(oz237) mutation when involved in the mitosis vs. meiosis decision in the germ line.

Gupta, Pratyush et al. (2013) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Leahul, Lindsay, Gupta, Pratyush, Hansen, David, Jasper, Katie

[International Worm Meeting, 2013]

We previously demonstrated that the proteasome inhibits the proliferative fate in the C. elegans germ line. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald, Knox et al. 2008). Presumably, the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To identify these PPPs, we first sought to identify the substrate recognition subunits (SRS) of E3 ubiquitin ligases that may target the PPPs for degradation by the proteasome. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five that enhance over-proliferation. One of these, RFP-1, specifically enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. Large-scale 2-hybrid screens identified potential binding partners for RFP-1 (Crowe and Candido 2004; Zhong and Sternberg 2006), which could be PPPs that are targeted for proteasomal degradation by RFP-1. One of these, mrg-1, partially suppresses the over-proliferation phenotype of glp-1(ar202gf); rfp-1(ok572), suggesting that it may function as a PPP. We found that MRG-1 levels do increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition. Therefore, MRG-1 protein levels are likely regulated through proteasomal-mediated degradation. We are currently determining how mrg-1 may participate in controlling the proliferative fate in the germline.

Wender, Nora et al. (2011) International Worm Meeting "Analysis of cellular toxicity mechanisms of alpha-synuclein in C. elegans."

Wender, Nora, Hegermann, Jan, Eimer, Stefan

[International Worm Meeting, 2011]

Aggregation of a-synuclein (aS) in Lewy bodies is one of the pathological hallmarks of Parkinson's disease (PD). Mutations in aS as well as increased aS expression levels have been associated with PD. As the PD mutants of aS are more prone to forming fibrillar aggregates, these fibrils were initially considered to be causing toxicity. However, in different model systems no clear correlation between toxicity and fibril formation was found. This indicates that small oligomeric precursors rather than fibrils might be the toxic species. In order to investigate which form of aS is causing toxicity, we were making use of a designed variant of aS that stops at the stage of soluble oligomers. This variant is called TP aS (triple proline aS) as it was derived from wt aS by introduction of three proline residues. In vitro NMR studies as well as in vivo analysis of the aggregation of aS variants tagged with YFP in C. elegans muscle cells confirmed that the occurrence of aS fibrils was strongly reduced in TP aS. Next we were assessing toxicity of TP aS in comparison to wt aS as well as PD mutants of aS by investigation of neurite degeneration and impairment of dopamine-associated behavior upon expression of different aS variants in C. elegans dopaminergic neurons. We found that TP is the most toxic aS variant, exhibiting the most severe effects on dopaminergic neuron morphology and function. This suggests that indeed the small oligomers rather than aS fibrils are causing toxicity. So far, the cellular function of aS and the mechanisms causing toxicity remain unclear. It is known that aS binds to intracellular membranes including the mitochondrial membrane. Furthermore, mitochondrial impairment upon expression of wt and mutant aS was reported. Recently it has been reported that several PD-associated genes play a role in mitochondrial dynamics and that loss of function or overexpression leads to morphological changes in the mitochondrial network. Therefore we aimed in understanding how expression of different variants of aS affects mitochondrial dynamics. Thus we were analyzing mitochondrial morphology and distribution by Electron Microscopy as well as Spinning Disk Microscopy. We found that expression of aS in C. elegans muscles and neurons leads to mitochondrial fragmentation and severe changes in mitochondrial morphology. Remarkably, similar changes could be observed in aged worms not expressing aS. It is therefore tempting to suggest that increased expression levels of aS accelerate mitochondrial aging.

GUPTA, P. et al. (2015) International Worm Meeting "MRG-1 and RFP-1 regulate proliferation in the germline."

Hansen, D., Wang, X., GUPTA, P., LEAHUL, L., WANG, C.

[International Worm Meeting, 2015]

The germ line in C. elegans is organized in an assembly line fashion, with each region containing cells at a specific point in development. As cells move proximally, they progress further through the developmental stages of gametogenesis. Presumably, different factors are expressed in each region to execute each developmental stage. As cells move proximally, some factors must be turned on, while others are turned off. We previously demonstrated that proteasomal degradation of proteins is key for cells to properly exit the proliferative fate and enter into meiotic prophase. Reduced proteasomal function enhances over-proliferation in a sensitized genetic background (Macdonald et al. 2008). We hypothesized that the proteasome is involved in degrading proteins that either promote the proliferative fate, or inhibit meiotic entry. We refer to these as proliferation promoting proteins (PPPs). To understand how proteasomal degradation contributes to maintaining the balance between proliferation and differentiation, we sought to identify these PPPs. To do this, we first focused on identifying the substrate recognition subunits (SRSs) of the E3 ubiquitin ligases to find those that may target the PPPs for degradation. Since the SRSs directly bind their target proteins, we reasoned that if we genetically identify the SRSs involved, we could then biochemically identify the PPPs as interacting proteins. We screened 826 SRSs by RNAi in four sensitized genetic backgrounds and found five proteins that enhance over-proliferation when their activities are reduced. One of these, RFP-1 (putative E3 ligase), enhances glp-1(ar202gf) to form a germline tumor, albeit incomplete. To identify the target proteins of RFP-1, and potential PPPs, we screened the interacting proteins of RFP-1 and looked for suppression of the germline tumor when their activity was reduced. Through this, we identified the chromo-domain containing protein MRG-1. mrg-1(0) partially suppresses over-proliferation in glp-1(ar202gf); rfp-1(ok572) animals, suggesting that it may function as a PPP. We found that MRG-1 levels increase in an rfp-1 mutant, as well as when proteasomal function is decreased through genetic mutation or chemical inhibition, suggesting that its activity is regulated, at least in part, through its levels of accumulation that is likely controlled through proteasomal-mediated degradation (Gupta et al. 2015). Analysis in tissue culture cells further supports that RFP-1 directly targets MRG-1 for degradation by the proteasome. Therefore, we have identified MRG-1 as a key player in regulating the proliferation vs. differentiation decision and determined that its activity is controlled, at least in part, through proteasomal degradation.

Cain, S. et al. (2019) International Worm Meeting "Genome Browsing at WormBase."

Gao, S., Grove, C., Nuin, P., Stein, L., Harris, T., Wright, A., Cain, S.

[International Worm Meeting, 2019]

Here we present recent updates to the genome browser at WormBase, JBrowse. Since it's introduction three years ago, JBrowse's presence in WormBase has grown, as has its functionality. JBrowse updates include the addition of new datasets, display improvements and increased functionality. Dataset changes include the addition of common and rare predicted introns from RNAseq data as well as the ability to add modERN and other public datasets. Visualization changes include improvements to how modENCODE tracks are displayed as well as the addition of new and improved glyphs throughout the app.

Michael Han et al. (2007) International Worm Meeting "WormBase: Comparative Genomics."

Michael Han, Todd Harris, Sheldon McKay

[International Worm Meeting, 2007]

WormBase is the reference database for multiple Caenorhabditae species and provides comprehensive information on comparative Nematode genomics. The comparative genomics data in Wormbase has been extended by including more species, additional methods for comparison and by more regular updates. Data on the new Caenorhabditis briggsae assembly and the draft Caenorhabditis remanei genome has been included as well as the first draft of the Caenorhabditis sp 2801. Additional and updated ortholog predictions like EnsEMBL - Compara, TreeFam and InParanoid, supplemental experimental evidence and tighter integration with EnsEMBL provides a wider range of species for intergenome comparisons as well as a more comprehensive view of currently available data. Pairwise genome alignments from WABA as well as multiple genome alignments using MLAGAN / PECAN can be accessed through the new syntheny viewer in addition to being downloadable as flatfiles from the WormBase FTP-servers.

Queiros, Libania et al. (2021) International Worm Meeting "Evidence for the inclusion of Caenorhabditis elegans in Environmental Risk Assessment routines"

Queiros, Libania, J. M. Goncalves, Fernando, Pereira, Patricia, Pacheco, Mario, Aschner, Michael, L. Pereira, Joana

[International Worm Meeting, 2021]

Caenorhabditis elegans has long been used as a choice for a laboratory model organism, and it has been linked to several discoveries that have led to Nobel Prizes. Although C. elegans' journey as an invaluable model in developmental biology and neurobiology is long, dating to the 1960s, its application in other research fields such as environmental toxicology is more recent. As a well-known experimental organism bearing high sensitivity to different environmental contaminants, and representing important functional levels in soil and aquatic ecosystems, C. elegans has high potential to be extensively integrated within Environmental Risk Assessment (ERA) routines. The major advantages supporting the inclusion of C. elegans in lower tiers of ERA, where a first screening of potential hazardous scenarios to the biota of both environmental compartments is performed, as well as the large array of endpoints that can be tested in this context, are herein presented. In addition, its sensitivity to contaminants such as metals and pesticides, as well as specific strengths and limitations, are compared to other laboratorial model organisms commonly used in ERA, such as Daphnia magna (crustacean) and Eisenia fetida (earthworm). The inclusion of C. elegans in ERA routines is encouraged, since it may provide ecologically relevant insights on the effects of contaminants, thus improving the establishment of appropriate environmental protection benchmarks.