chen, Y. et al. (2013) International Worm Meeting "The Effect of Hydrolysable Tannins from Eucalyptus Leaves on C. elegans Lifespan."

Huang, Q., Driscoll, M., Chen, H., Cao, Y., Onken, B., Chen, Y., Xiao, S.

[International Worm Meeting, 2013]

To identify potential pharmacological compounds that delay the progression of age-related degenerative changes and illness, we monitored the effects of six hydrolysable tannins with high antioxidant activities isolated from Eucalyptus leaves (Oenothein B (OEB), 1,2,3,4,6-penta-O-galloyl-b-D-glucose (PGG), Tellimagrandin I (T1), Tellimagrandin II(T2), Pedunculagin (Ped) and Gemin D (GD) ) on C. elegans lifespan at four different concentrations. We found that of the six, OEB and PGG extended lifespan in a dose-dependent manner and increased median lifespan by up to 22%. OEB significantly prolonged youthful locomotory ability. We also found that the survival curves of T1-treated animals at all tested concentrations were significantly increased, although median lifespan was not significantly improved. OEB, PGG and T1 did not significantly affect the age-associated physiological functions of reproduction, pharyngeal pumping rate, or age pigment accumulation. Animals treated with 40 mM Ped significantly extended median lifespan by 11%, but we did not observe any significant benefits at other Ped concentrations. T2 and GD did not cause significant lifespan extension at any tested concentration. To further determine how OEB and PGG prolong C. elegans lifespan, we investigated the genetic requirements for these benefits. Strikingly, we found that lifespan extension with OEB and PGG treatment was driven by the insulin signaling, dietary restriction, and mitochondrial function pathways, indicating that these compounds target multiple longevity mechanisms to promote lifespan. Together, our results demonstrate broad-based aging benefits with these botanical compounds, which may be exploited in novel therapies to promote healthy aging.

Shih-Chieh Chien et al. (2007) International Worm Meeting "The MIG-15 kinase functions in canonical Wnt signaling."

Gian Garriga, Shih-Chieh Chien

[International Worm Meeting, 2007]

MIG-15 is an ortholog of Nck-interacting kinase (NIK) in mice and Misshapen in Drosophila. All three molecules belong to the Ste20 kinase superfamily. We have characterized the cell migration defects of mig-15 mutants, confirming and extending some of the observations made by Zhu and Hedgecock (1997)1. During embryogenesis, the HSN neurons migrate anteriorly, and we found that the HSNs often stopped prematurely in mig-15 mutants. In L1 animals, the descendents of the QR neuroblast migrate anteriorly, while QL descendents migrate posteriorly. In mig-15 mutants, the QR descendents stopped prematurely while the QL descendents migrated anteriorly in the wrong direction. These cell migration defects are similar to those found in Wnt mutants, raising the intriguing possibility that mig-15 may function in Wnt signaling. To test this hypothesis, we asked whether expression of mab-5 in QL was affected by mig-15 mutations. The Wnt EGL-20, acting through a canonical Wnt signaling pathway, turns on mab-5 expression. MAB-5, a Hox transcription factor, then specifies the posterior migration program of QL. Using a mab-5::lacZ reporter, we found that mab-5 expression was greatly attenuated in mig-15 mutants, consistent with MIG-15 playing an important role in Wnt signaling. We also found that a gain-of-function mab-5 mutation suppressed the QL defect of mig-15 mutants, providing genetic evidence that mig-15 acts upstream of mab-5 in the Wnt signaling pathway. Drosophila Misshapen has been shown to function in the Planar Cell Polarity, which is mediated by Frizzled receptors and Dishevelled, components that also function in Wnt signaling. Yet the role of MIG-15 in QL migration is novel since its homologs have not been implicated in canonical Wnt signaling. We will define where MIG-15 acts in the canonical Wnt signaling pathway and whether it acts directly in the process, or indirectly by regulating one of the Wnt pathway components. 1: Zhu, X. & Hedgecock, E.M. (1997). International C. elegans Meeting.

Maduro MF et al. (1998) West Coast Worm Meeting "GOTTA LOTTA GATAs: THE DIVERSE ROLES OF GATA FACTORS IN C. ELEGANS EMBRYOS"

Rothman JH, Koh K, Newman-Smith ED, Maduro MF, Strohmaier K

[West Coast Worm Meeting, 1998]

Zinc-finger transcription factors of the GATA type (which bind the consensus sequence WGATAR) are known in fungi, worms, flies and vertebrates. In animals, GATA factors are implicated in regulating differentiation of a range of different cell types. Six GATA factors, each with two fingers, have been identified in vertebrates, three of which are involved in hematopoesis, and three others in formation of organs such as heart and endodermal organs. To evaluate the range of developmental events that GATA factors might direct in an animal, we are analyzing the expression and function of a number of C. elegans GATA factors. From the C. elegans genomic sequence, we can now deduce how many GATA factors are required to make a metazoan. Five were known from genetic and molecular studies: ELT-1 and ELT-3 are involved in development of the embryonic epidermis (Page et al. 1997; Gilleard and Shafi, wm97e186) and ELT-2, END-1 and END-3 regulate specification of the endoderm and differentiation of the gut (Fukushige et al. wm97e169; Zhu et al. 1997; Maduro et al. this meeting). Blast searches of the C. elegans sequence database led us to identify at least four more apparent GATA factors, tentatively called ELT-4 through 7. All appear to be single-finger GATA factors. elt-4 and elt-5, adjacent genes on the left end of IV, appear to correspond to a polycistronic unit; these genes are expressed in, and appear to be involved in the development of, seam cells among other cell types (see abstract by Koh et al.). Although GFP constructs with elt-6 X (T24D3.1) have been uninformative, and no phenotype was observed by RNAi, ectopic expression of ELT-6 shows that it can promote pharynx development at the expense of other cell types: hs-elt-6 embryos often arrest with large numbers of pharynx muscles and no intestine (i.e., the reciprocal effect of the endoderm-specific GATA factors). Analysis of elt-7 V (cosmid C18G1) is in progress. Our tentative conclusion is that most, if not all GATA factors in C. elegans, function in distinct embryonic specification and/or differentiation events. References: Page et al. (1997) Genes & Dev. 11: 1651-1661. Zhu et al. (1997) Genes & Dev. 11: 2883-2896.

Fernando Gomez et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Characterizing Two coq-3 Mutants in Caenorhabditis elegans"

Adam Lunceford, Catherine F Clarke, Peter Dang, Fernando Gomez, Ryoichi Saiki

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

Coenzyme Q (referred to simply as Q) is a lipophilic component of the electron transport chain in mitochondria. The biosynthesis of Q is known to include several steps, including two O-methyltransferase reactions catalyzed by COQ-3. The coq-3 gene is well-conserved among various organisms, from Homo sapiens to E. col, where the gene is termed ubiG. Expression of C. elegans COQ-3 restores respiratory growth and Q biosynthesis in an E. coli ubiG mutant, and in a S. cerevisiae coq-3 mutant. In C. elegans, coq-3 mutants demonstrate decreased fertility, activity, survival and behavior. One mutant, coq-3(qm188), possesses a deletion of the third and fourth exons. Sequencing of the coq-3(qm188) mRNA transcript shows a spliced product containing exons 1, 2 and 5 in a complete reading frame. Another mutant, coq-3(ok506), is missing exon 3 only; however, the spliced mRNA is frameshifted and is predicted to generate an early stop codon. Interestingly, coq-3(qm188) mutants are sterile on a Q-replete bacterial diet of OP50, whereas coq-3(ok506) mutants generate approximately 100 larvae that arrest at the L1 stage. Both mutant strains are sterile and when fed the Q-deficient bacteria strain GD-1. Neither the coq-3(qm188), nor the coq-3(ok506) mutant is rescued, however, when grown on media containing NovaSol Q10, a lipid-soluble coenzyme Q10 delivery system. RT-PCR conducted on these strains shows mRNA product of the predicted sizes for both strains. Sequencing of these transcripts reveals no cryptic splice sites. The coq-3 gene is situated in a three-gene operon. Transcripts of the flanking genes in the operon from the coq-3(qm188) mutant allele are present, as evaluated via RT-PCR. Currently we are optimizing small-scale quinone analysis in lipid extracts of 50 adults worms via HPLC-MS/MS.

Sagulo, Seth et al. (2015) International Worm Meeting "Investigating the Role of Host Cell Factor 1 and Ribosomal S6 Kinase in Regulation of Longevity."

Sagulo, Seth, Lee, Siu Sylvia

[International Worm Meeting, 2015]

The Lee lab has identified Host Cell Factor 1 (HCF-1) as a transcriptional corepressor of DAF-16/FOXO, a major downstream effector of the highly conserved insulin/insulin-like (IIS) signaling pathway. C. elegans that are mutant in hcf-1 have an extended lifespan that is dependent on daf-16. Host Cell Factor 1 is a nuclear expressed protein that acts as a scaffolding protein as it does not have a clear DNA binding domain. Thus it likely plays a role in various protein-protein interactions, some of which may be important for regulating lifespan. Since many of the proteins that interact with HCF-1 have not yet been identified immunoprecipitation-mass spectrometry (IP-MS) was performed in collaboration with the Ruvkun lab and Ming Zhu, Meng-Qiu Dong of the Dong lab. This was done using a strain of C. elegans expressing HCF-1 tagged with GFP. Several chromatin related factors including the histone deacetylase SIN-3 which has been previously shown to interact with HCF-1 in addition to many metabolism proteins were found to interact with HCF-1. Surprisingly, the downstream kinase of the highly conserved target of rapamycin (TOR) pathway, ribosomal S6 kinase (RSKS-1/S6K1) was also identified which suggests it may play a role in the regulation of HCF-1 through phosphorylation. Interestingly, previously published data shows that rsks-1 mutants have an extended lifespan that is dependent on daf-16 which is similar to hcf-1 mutants. To support the IP-MS data, the genetic relationship of rsks-1 and hcf-1 was tested. The rsks-1;hcf-1 double mutant does not show an additive extension of lifespan compared to either of the rsks-1 or hcf-1 single mutants providing evidence that HCF-1 and RSKS-1 may be working through the same pathway to regulate longevity in C. elegans. Since HCF-1 and RSKS-1 are both highly conserved proteins understanding the novel biochemical and genetic connection between them will be relevant to aging in mammals.

Gomez F et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Characterizing a Long-lived Coq-3 Mutant in C. elegans"

Saiki R, Clarke C F, Srinivassan C, Chin R, Gomez F

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Coenzyme Q (referred to simply as Q) is a lipophilic component of the electron transport chain in mitochondria. The biosynthesis of Q is known to include several steps, including two O-methyltransferase reactions catalyzed by COQ-3. Expression of C.elegans COQ-3 restores respiratory growth and Q biosynthesis in an E. coli ubiG mutant. In C. elegans, coq-3 mutants demonstrate altered fertility and survival.One mutant, coq-3(qm188), is sterile. Another mutant, coq-3(ok506), is fertile. Both mutant strains are sterile when fed the Q-deficient bacteria strain GD-1. Also, both strains fail to respond to Novasol Q10, a micelle-based coenzyme Q10 delivery particle that has shown to rescue fertility in clk-1 homozygotes, which are mutant for a later step in Q biosynthesis. Western blot analyses with a COQ-3 protein antibody indicate that both mutants also lack the wild-type protein. The coq-3 gene is situated in a three-gene operon. QT-PCR analysis shows that transcript levels of the upstream gene, nuo-3, in coq-3(qm188)mutants are widely variant compared to wild-type and coq-3(ok506) mutants.This erratic expression pattern indicates that coq-3(qm188) is not a representative coq-3 mutant. Mutant coq-3(ok506)raised on the Q-less E. coli strain GD1 possess small, thin and severely underdeveloped gonads, and live 125% longer than wild-type worms raised on a this diet. Mutants raised on OP50 have a 40% shorter life span than wild type worms and an asymmetrical uterine anatomy. Intriguingly, fertility and development are partially rescued when these mutants are raised on 1100Deltabc,a bacteria strain that is mutant for one of the ATP-synthase components but is still capable of generating Q, demonstrating that it is not the respiratory status that is responsible for coq-3(ok506) mutants' failure to mature on GD1, but the absence of Q itself in the bacteria. These data suggest that gonad development requires Q, and the lack of a mature gonad in turn may affect the life span of the worm.

Shih-Chieh Chien et al. (2008) C.elegans Neuronal Development Meeting "The MIG-15 Kinase Acts with Wnts to Regulate Neuronal Migration and Polarity"

Shih-Chieh Chien, Gian Garriga

[C.elegans Neuronal Development Meeting, 2008]

MIG-15 is the C. elegans ortholog of Nck-interacting kinase (NIK) in mice and Misshapen in Drosophila. We have characterized the cell migration defects of mig-15 mutants, confirming and extending observations made by Zhu and Hedgecock (1997). In L1 animals, the descendents of the QR neuroblast migrate anteriorly, while QL descendents migrate posteriorly. In mig-15 mutants, the QR descendents stopped prematurely while the QL descendents migrated anteriorly in the wrong direction. These defects are similar to those found in Wnt mutants, suggesting that mig-15 may function in Wnt signaling. The Wnt EGL-20 controls the posterior migration of QL descendents by activating the Hox gene mab-5 through a canonical signaling pathway. We found that mab-5 expression in QL descendents was attenuated in mig-15 mutants and that a gain-of-function mab-5 mutation completely suppressed the QL migration defects of mig-15 mutants. A mutation in pry-1/Axin, a negative regulator of mab-5 expression, also suppressed the mig-15 QL defects. These observations suggest that MIG-15 acts in the EGL-20/Wnt pathway upstream of PRY-1/Axin. Wnts also control the polarity of the mechanosensory neuron ALM . We found that mig-15 mutants exhibit a low penetrance of ALM polarity defects and that these mutant phenotypes were enhanced by a mutation in cwn-1, one of five C. elegans Wnt genes. Thus, mig-15 may mediate the effects of Wnts on ALM polarity. In S. cerevisiae, Kic1p, a distant relative of MIG-15, acts in the RAM signaling pathway to regulate polarized cell growth. We asked whether C. elegans orthologs of RAM signaling molecules regulate ALM polarity. We found that sax-2, an ortholog of the scaffold protein Tao3p, interacted synergistically with cwn-1 in ALM polarity. SAX-2 and SAX-1, a kinase in the NDR family, coordinately regulate process length of the mechanosensory neuron PLM . However, we failed to detect an ALM phenotype in cwn-1; sax-1 double mutants. We are currently asking whether other NDR kinases function in ALM polarity.

AK Walker et al. (1999) International C. elegans Meeting "Role of General Transcription Factors in Early C. elegans Gene Regulation"

AK Walker, Y Shi, TK Blackwell

[International C. elegans Meeting, 1999]

Early zygotic transcription activates differentiation-specific genes necessary for blastomere specification and gene products needed for core biological functions. ama-1 (RNA polymerase II) RNAi embryos fail to initiate zygotic transcription, arresting at about 100 undifferentiated cells. We are evaluating the requirements for general transcription regulators in early transcriptional events. Biochemical and genetic analysis in yeast have identified many regulatory cofactors which function between the polymerase and gene specific activators. Some, such as the TAFIIs, have been predicted to coordinately regulate cell cycle genes or to interact with activators. However, their role in metazoan gene regulation is unclear. Our approach has been to (1) identify C. elegans homologues of regulatory cofactors and obtain corresponding clones (from Y. Kohara or the Sanger Centre), (2) evaluate the RNAi phenotype and (3) analyze the RNAi effect on reporter genes in vivo. RNAi of orthologs for human TAFII250, TAFII130, TAFII100, and TAFII31/32 produced an early embryonic arrest, similar to the ama-1 RNAi. We have also analyzed CeTAFII250 and CeTAFII100 RNAi in the end-1 ::gfp strain (obtained from J. Rothman). end-1 is expressed in the E2-E8 cells and is regulated by the maternal factor SKN-1. Ectopically expressed end-1 induces exogenous gut and adoption of the long Ea/Ep-like cell cycle (Zhu (98) G&D 12:3809). RNAi of skn-1 and ama-1 in end-1 ::gfp worms blocks gfp expression. RNAi of CeTAFII250 or CeTAFII100 does not affect gfp expression, even while the embryos show the early ama-1 like arrest. Thus, CeTAFII250 and CeTAFII100 are required for core biological functions, but not to express a differentiation specific gene. Lineage analysis of the CeTAFII250 RNAi embryos shows that the E daughters divide early, with the MS daughters. Our results show that RNAi of CeTAFII250 uncouples end-1 expression from the end-1 dependent cell cycle lengthening in the E daughters. We will continue to evaluate requirements for specific TAFIIs and other regulatory cofactors on early reporter genes in vivo , and use this as a model system to investigate how these biochemically defined factors are required for specific gene regulation in vivo .

Desse, V. et al. (2019) International Worm Meeting "SAX-7/L1CAM in the maintenance of neuronal architecture."

Perrat, P., Blanchette, C., Desse, V., Benard, C.

[International Worm Meeting, 2019]

Whereas remarkable advances have been made in understanding neuronal development, the mechanisms that maintain the nervous system architecture lifelong are poorly understood. How does the nervous system established during embryogenesis subsequently maintain its integrity throughout life, despite the animal's growth, the addition of new neurons, maturation processes, body movements, and aging? The evolutionarily conserved gene sax-7, homologous to mammalian L1CAM, is one of the factors required for maintaining the structural integrity of ganglia and fascicles in C. elegans1. Thus, sax-7 not only contributes to nervous system development (roles in guidance and fasciculation of a subset of developing neurites)2, but is also required for maintaining the organization of neural architecture after its initial development. Indeed, some neural structures that initially develop normally become disorganized in animals lacking the function of sax-7. With heat shock assays, we show that expression of sax-7S(+) during larval stages is sufficient to rescue the maintenance defects of sax-7 mutants that were devoid of functional sax-7 during nervous system development. We are characterizing a true null allele (generated using CRISPR/Cas9), as well as the expression pattern of a functional SAX-7S::sfGFP (insertion in the endogenous locus using CRISPR/Cas9), and we find that SAX-7S is present in neurons, at the plasma membrane. We are exploring the structure-function relationship of SAX-7S, a transmembrane protein harboring extracellular Ig and FnIII domains, in the context of maintenance of the nervous system architecture. A conditional knockout of L1CAM in the adult mouse brain results in behavioral and learning defects, indicating that L1CAM plays a post-developmental role in mammals as well. Our studies are expected to help understand the role of SAX-7/L1CAM in conserved molecular mechanisms ensuring neuronal maintenance, which may go awry in neurodegenerative conditions. 1 Pocock, Benard et al 2008; Sasakura et al 2005; Wang, Chen et al 2005; Zallen et al 1999; Ramirez-Suarez et al 2019 2 Diaz-Balzac et al 2015; Salzberg et al 2013; Yip and Heiman 2018, Dong et al 2013; Zhu et al 2017

Gomez, F. et al. (2009) International Worm Meeting "Characterizing Two coq-3 Mutants in Caenorhabditis elegans."

Clarke, C.F., Gomez, F., Srinivasan, C., Chin, R., Saiki, R.

[International Worm Meeting, 2009]

Coenzyme Q (referred to simply a Q) is a lipophilic component of the electron transport chain in mitochondria. The biosynthesis of Q is known to include several steps, including two O-methyltransferase reactions catalyzed by COQ-3. The coq-3 gene is well-conserved among various organisms, from Homo sapiens to E. coli, where the gene is termed ubiG. Expression of C. elegans COQ-3 restores respiratory growth and Q biosynthesis in an E. coli ubiG mutant, and in a S. cerevisiae coq-3 mutant. In C. elegans, coq-3 mutants demonstrate decreased fertility, activity, survival and behavior. One mutant, coq-3(qm188), possesses a deletion of the third and fourth exons. Sequencing of the coq-3(qm188) mRNA transcript shows a spliced product containing exons 1, 2 and 5 in a complete reading frame. Another mutant, coq-3(ok506), is missing exon 3 only; however, the spliced mRNA is frameshifted and is predicted to generate an early stop codon. Interestingly, coq-3(qm188) mutants are sterile on a Q-replete bacterial diet of OP50, whereas coq-3(ok506) mutants generate approximately 100 larvae that slowly develop into adults with limited fertility. Brood size analysis on heterozygotes show that the coq-3(qm188) allele is not dominant negative. Both mutant strains are sterile when fed the Q-deficient bacteria strain GD-1. Neither the coq-3(qm188), nor the coq-3(ok506) mutant is rescued, however, when grown on media containing NovaSol Q10, a water soluble formulation containing coenzyme Q10. Western blot using COQ-3 protein antibody indicates that both mutants also lack the wild-type protein. RT-PCR conducted on these strains shows mRNA product of the predicted sizes for both strains. Sequencing of these transcripts reveals no cryptic splice sites. The coq-3 gene is situated in a three-gene operon. Transcripts of the flanking genes in the operon from both coq-3 mutants are present; however, qT-PCR analysis shows that transcript levels of the upstream gene, nuo-3, in coq-3(qm188) mutants are widely variant compared to wild-type and coq-3(ok506) mutants. This erratic expression pattern indicates that coq-3(qm188) is not representative of a coq-3 mutant, and thus further studies are to be conducted with the coq-3(ok506) mutant exclusively.