Figure 1: C. elegans l ife cycle and the molecular pathways which control dauer formation

1. K12 Bacterial metabolites and their influence on dauer formation in C. elegans K12 Rebecca Gerrity, Sharon Epstein, Sven Lang, Pankaj Kapahi. Buck Institute, Novato, CA. LysR LysR * * * ZntA Introduction:C. elegans are microscopic nematodes, or worms, which are ubiquitous in nature but are cultured on agar plates and fed with E. coli when studied in the lab [9]. There has been great success using C. elegans as a model organism and they continue to serve as an instrumental tool in microbiology. There are four developmental larval stages, which the worms pass through before becoming mature adults [cf. Fig 1]. There is also an alternate state that the worms may enter, after the L1 stage, known as dauer, if environmental conditions are harsh [9]. In the dauer state worms can stall development for a few months, until conditions improve [9]. Three distinct molecular signaling pathways regulate dauerformation: the Guanylylcyclase pathway, the TGFβ-like pathway, and the Insulin-like pathway [9]. Testing development in the presence of genetic mutations in these pathways allow scientists to better understand the molecular triggers of dauerformation [3]. This study used mutant strains in the daf-2/insulin-like pathway and the daf-7/TGFβ-like pathway. We tested whether feedinggenetically altered bacteria alters dauerformation. Follow up assays included, western blots, qRT-PCR, and microscopy. Abstract: The food an organism consumes must contain a multitude of various nutrients at proper concentrations in order to maintain a healthy metabolism. Perturbations to the levels of individual essential nutrients of food could have sweeping consequences for the organism consuming it [3]. Regulating the nutrient intake of the worms by genetically altering the bacteria on which they feed, allows for the metabolic processes of the different worm strains, in response to altering single nutrients, to be monitored [3]. Our project researched the influence of diet on development in C. elegans. Three mutant bacteria (LysR, ZntA, ZntR) were tested against the control strain (K12) to assess the formation of an alternative larval stage called dauer. Using two strains of C. elegans, daf-2 (e1368) and daf-7 (e1372), we examined if and how the food sources affected dauer formation in mutants for both the daf-2/insulin-like pathway and the daf-7/TGFβ-like pathway.Significant increases in dauer formation were seen in daf-2 (e1368) worms fed with LysR and ZntR mutant bacteria relative to the control strain. QRT-PCR results indicated that Sod-3 mRNA levels, a gene located downstream of the daf-2/insulin-like pathway [4], was elevated for all the mutant-fed worms. In addition, we looked at proteinlevels using western blots. Significant decreases in protein expression of myo-3::Mit::GFP, a mitochondrial GFP fusion protein under a muscle promoter [8], were observed in both ZntA and ZntR fed worms. However, these differences in expression were not seen using fluorescence microscopy. Further studies should investigate the role of mitochondriain dauer formation and repeat the dauer assays to verify results and test for significance. ZntA * Figure 2: Different bacterial food sources affect dauerformation ZntR B A ZntR * * C D Figure 1: C. eleganslife cycle and the molecular pathways which control dauer formation A B (A) The life cycle of the C. elegans nematode involves four larval stages and concludes with a mature, egg laying, adult. There is an alternate state, known as dauer, which the worms may enter after the L1 stage if environmental conditions are perceived as harsh [1]. (B) The pathways involved in dauer formation used in this project are the daf-7/TGFβ pathway, and the daf-2/Insulin-like pathway [2]. Western blotting was used to compare protein expression levels of two genes downstream of the daf-2/insulin-like pathway: Daf-16 and Sod-3, and a mitochondrial marker, myo-3::Mit::GFP. These genes were tagged with a GFP translational fusion marker in order to measure protein synthesis. (A) Protein levels for Daf-16::GFP, located in the daf-2/insulin-signaling pathway and the primary activator of dauer formation [7], was not altered in worms fed mutant bacteria as compared to those fed the control strain. (B) Similarly, Sod-3::GFP protein levels were not significantly altered. (C) Protein levels in worms fed with ZntA and ZntRmutant bacteria were elevated for Myo-3::Mit::GFP, a mitochondrial GFP fusion protein under a muscle promoter [8] (* p<0.05; **p<0.01). Figure 4: Analysis of the protein expression levels of genes downstream of the Insulin-like signaling pathway and of a mitochondrial marker Figure 3: mRNA expression levelsof genes involved in the regulation of dauer pathway and dietary restriction for worms grown on different bacterial food sources Bright field GFP A B A Figure 5: Differences in protein levels observed via microscopy of myo-3::Mit::GFP C. elegans daf-16 tubulin To examine their influence on dauer formation LysR, ZntA, and ZntR E. coli knockout strains were fed to daf-2 (e1368) and daf-7 (e1372) mutant worms. For the dauer assay, the number of dauers and adults in each plate were counted after eggs were allowed to hatch and develop to adulthood. (A) daf-2 (e1368) worms fed two mutant bacterial strains, one lacking a gene in the lysine biosynthetic pathway and the other lacking a gene in metal (Pb, Cd, Zn, Hg) export pathway, significantly increased dauer formation (*p<0.05; **p<0.005). (B) daf-7 (e1372) plated on the ZntA bacterial mutant tended to decrease dauer formation. (C) In an attempt to recover the dauer phenotype in worms fed LysR bacteria, different concentrations of lysine were added to the agar. Contrary to our hypothesis, that adding lysine would revert the phenotype, the lysine fed worms had increased dauer formation. (D) Dauer formation in daf-7 (e1372) worms decreased with increasing lysine concentration. Bright field GFP K12 LysRZntAZntR B sod-3 Microscopy was performed to analyze differences in GFP protein levels of the Myo-3::Mit::GFP worm strain fed mutant bacteria in comparison those fed control bacteria (K12).  (A) Unlike western blotting (Fig. 4) quantification of microscopy images did not show any significant differences in the expression of the Myo-3::Mit::GFP gene in the worm body.  (B) Similarly, images taken focusing specifically on the vulva, a muscle known for high expression of Myo-3::Mit::GFP, did not show any noticeable changes in expression. tubulin K12 LysRZntAZntR C Conclusions • Dauer formation in daf-2 (e1368) worms is significantly increased when fed with E. coli mutant bacteria strains LysR and ZntR. • The attempt to recover the LysRdauer phenotype by adding lysine to the agar did not produce significant results indicating that this method of rescue was unsuccessful. • qRT-PCR results show that the mRNA levels of the Sod-3 gene for worms fed with bacterial mutants were elevated when compared to the control indicating that the daf-2/insulin-like pathway was activated when worms were fed mutant E. coli. • Protein levels for the Myo-3 gene were significantly lower for worms fed with ZntA and ZntR mutant bacteria as evaluated by Western Blot, however these results were not confirmed via GFP fluorescence microscopy. myo-3 tubulin K12 LysRZntAZntR Quantitative real time polymerase chain reaction (qRT-PCR) was used to examine the transcriptional levels of three genes. mRNA levels increased relative to the levels of the control for the sod-3 gene, a downstream reporter of the daf-2/insulin-like dauerpathway [4], when worms were fed all three mutants. However, Daf-8, a gene located in the TGFβpathway [5], did not show a significant difference from control. Similarly, the mRNA levels of sams-1, a marker for dietary restriction [6], were also not effected. References 1. Strange, K., From genes to integrative physiology: ion channel and transporter biology in Caenorhabditiselegans. Physiological reviews, 2003. 83(2): p. 377-415. 2. Liu, T., K.K. Zimmerman, and G.I. Patterson, Regulation of signaling genes by TGFbeta during entry into dauer diapause in C. elegans. BMC developmental biology, 2004. 4: p. 11. 3. Vargas,M., Nutrient Response and Aging in Invertebrate Models. PhD Thesis submitted to the University of Manchester, 2012. 4. WormBase: http://www.wormbase.org/db/get?name=WBGene00004932;class=gene 5. WormBase: http://www.wormbase.org/db/get?name=WBGene00000904;class=gene 6. WormBase: http://www.wormbase.org/db/get?name=WBGene00008205;class=gene 7. WormBase: http://www.wormbase.org/db/get?name=WBGene00000912;class=gene 8. WormBase: http://www.wormbase.org/db/get?name=WBGene00003515;class=gene 9. Hu, P.J. Dauer (August 08, 2007), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.144.1, http://www.wormbook.org. I would like to acknowledge the Buck Institute and the Kapahi Lab, as well as the REU Fellowship Program for providing me with this opportunity. My sincerest appreciation is extended to Dr. PankajKapahi for welcoming me into his lab this summer, Dr. Matthew Laye and Dr. Nuno Luis for their assistance in poster editing, and Dr. Sharon Epstein, Dr.Sven Lang and Dr. Julie Mangada for their remarkable mentorship. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).