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Novel components of the dauer larva developmental signaling pathway in Caenorhabditis elegans Jensen, Victor L 2010

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Novel components of the dauer larva developmental signaling pathway in Caenorhabditis elegans by Victor L. Jensen B.Sc., Simon Fraser University, 2004 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2010  © Victor L. Jensen, 2010  Abstract In harsh conditions Caenorhabditis elegans arrests development to enter a nonaging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-! and insulin-like signaling pathways to control this developmental decision. The dauer pheromone is required for formation of dauer larvae. This thesis shows that bacterial pathogenesis is an input into dauer formation in addition to the known inputs of starvation, overcrowding and high temperature. This was first suggested by the overabundance of innate immunity genes found in a screen for novel synthetic dauer formation mutants. A total of 21 genes were identified in this screen, only one of which, srh-100, was previously identified to influence dauer formation. This work also characterizes new genes and functions that define early, middle, and late steps in the dauer pathway. Epistasis analysis and a GFP-tagged reporter places DAF-25 (abnormal DAuer Formation) function in the sensory cilia. DAF-25 was shown to be required for DAF-11 cilia localization as well as proper olfaction to various cues. daf-25 and daf-11 mutants have similar phenotypes and epistatic order. Intraflagellar transport is not defective in daf-25 mutants although the ciliary localization of DAF-25 itself is altered in a mutant that is defective in retrograde IFT. A Daf-c enhancing mutant originally isolated as a double mutant with a novel daf-2 allele was also identified. m708 is allelic with sdf-9 (Synthetic Dauer Formation). Epistasis analysis and allele-specific interactions place it in the daf-2 pathway as a possible DAF-2 interactor. The molecular lesion in m708 was identified to be an insertion of the mariner transposon Cemar1. This is the first identified hopping event described for this transposon family, the largest in C. elegans. Finally, zip5 (initially identified as a candidate DAF-16/FOXO target by serial analysis of gene expression) was shown to be a basic-leucine zipper transcription factor (bZIP) that is down regulated in a daf-2 background. zip-5 mutants show an extension of life span that is dependent on SKN-1, a bZIP-like transcription factor. ZIP-5 expression is dependant on the longevity-associated DAF-16 transcription factor and the zip-5 GATA binding sites. !  !  ""!  TABLE OF CONTENTS Abstract.............................................................................................................................. ii Table of contents .............................................................................................................. iii List of tables...................................................................................................................... vi List of figures................................................................................................................... vii List of abbreviations ...................................................................................................... viii Acknowledgements ........................................................................................................ xiii Dedication ....................................................................................................................... xiv Co-authorship statement .................................................................................................xv CHAPTER 1: Introduction...............................................................................................1 1.1 The dauer larva ..........................................................................................................1 1.1.1 Inputs into the dauer decision .............................................................................3 1.1.2 Characteristics of the dauer larva........................................................................4 1.1.3 The dauer pheromone .........................................................................................5 1.2 Pathways controlling the dauer developmental decision ...........................................7 1.2.1 cGMP signaling ..................................................................................................7 1.2.2 Amphid cilia and dauer larvae ..........................................................................11 1.2.3 Insulin/Insulin-like signaling ............................................................................13 1.2.4 TGF-! signaling ................................................................................................16 1.2.5 TOR signaling...................................................................................................18 1.2.6 Steroid hormone pathway .................................................................................19 1.2.7 Enhancers of dauer larva formation..................................................................21 1.3 Dauer signaling pathway and aging .........................................................................22 1.3.1 Life span and Daf mutants ................................................................................22 1.3.2 DAF-16 target analysis .....................................................................................24 1.3.3 SKN-1 and aging...............................................................................................26 1.4 Thesis objectives ......................................................................................................28 1.5 Bibliography.............................................................................................................29 CHAPTER 2: Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25 ...............................................................41 2.1 Introduction..............................................................................................................41 2.2 Results ......................................................................................................................43 2.2.1 Genetic epistasis analysis places DAF-25 function in the amphid cilia ...........43 2.2.2 daf-25 mutants exhibit chemosensory phenotypes independent of ciliary ultrastructure defects..................................................................................................45 2.2.3 Molecular identification of daf-25 ....................................................................48 2.2.4 DAF-25 functions in cilia .................................................................................50 2.2.5 DAF-25 is required for DAF-11 localization to cilia........................................53 2.2.6 Conservation of function for DAF-25/Ankmy2 ...............................................56 2.3 Discussion ................................................................................................................57  !  """!  2.4 Methods....................................................................................................................60 2.4.1 Mapping, epistasis and phenotyping daf-25 .....................................................60 2.4.2 Intraflagellar transport and ciliary protein localization analyses .....................62 2.4.3 Electron microscopy .........................................................................................63 2.4.4 Co-expression and co-immunoprecipitation of Ankmy2 and guanylate cyclase 1 (GC1) ......................................................................................................................64 2.5 Bibliography.............................................................................................................66 CHAPTER 3: Caenorhabditis elegans SDF-9 enhances insulin/insulin-like signaling through interaction with DAF-2 .....................................................................................71 3.1 Introduction..............................................................................................................71 3.2 Results and methods.................................................................................................73 3.2.1 Isolation and mapping of m708.........................................................................73 3.2.2 Cemar1 transposition ........................................................................................74 3.2.3 Epistatic analysis of sdf-9 and Daf-c mutants...................................................76 3.3 Discussion ................................................................................................................81 3.4 Bibliography.............................................................................................................84 CHAPTER 4: RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation .................................................................................88 4.1 Introduction..............................................................................................................88 4.2 Results ......................................................................................................................90 4.2.1 RNAi screen for enhanced dauer formation .....................................................90 4.2.2 21 SynDaf genes ...............................................................................................91 4.2.3 Genes known to affect dauer formation or insulin secretion ............................93 4.2.4 Genes with unknown function ..........................................................................94 4.2.5 Innate immunity genes......................................................................................95 4.2.6 Dauer formation on pathogenic bacteria...........................................................96 4.3 Discussion ..............................................................................................................100 4.3.1 CCB-1 and possible feedback regulation of insulin secretion ........................101 4.3.2 Germ line and dct genes..................................................................................102 4.3.3 Immunity related genes...................................................................................103 4.3.4 Mechanism for pathogenic input into dauer formation...................................104 4.4 Methods..................................................................................................................107 4.4.1 Gene target selection and RNAi screen ..........................................................107 4.4.2 Classification and comparison of positives.....................................................108 4.4.3 Dauer formation daf-7 expression, and adult survival on pathogenic bacteria ..................................................................................................................................109 4.5 Bibliography...........................................................................................................111 CHAPTER 5: ZIP-5 acts downstream of the insulin signaling pathway to regulate longevity dependent on skn-1........................................................................................118 5.1 Introduction............................................................................................................118 5.2 Results and discussion ...........................................................................................119 5.3 Experimental procedures........................................................................................123 5.3.1 Life span analysis............................................................................................123  !  "#!  5.3.2 Translational ZIP-5::GFP reporter and promoter truncation analysis ............123 5.4 Bibliography...........................................................................................................125 CHAPTER 6: General discussion ................................................................................128 6.1 DAF-25/Ankmy2 as a novel cilia protein..............................................................128 6.2 SDF-9 interacts with DAF-2 ..................................................................................129 6.3 Novel SynDaf genes...............................................................................................131 6.4 Pathogenesis as a novel input into the dauer larva decision ..................................131 6.5 ZIP-5 as a SKN-1 opposing longevity inhibitor ....................................................132 6.3 Bibliography...........................................................................................................134 Appendices......................................................................................................................138 Appendix A - Supplementary information for Chapter 2 ............................................138 Appendix B - Supplementary information for Chapter 4.............................................151 Appendix C - Targets of DAF-16 involved in Caenorhabditis elegans adult longevity and dauer formation .....................................................................................................157  !  #!  LIST OF TABLES Table 1.1 List of genes with descriptions and associated pathway or function...................9 Table 3.1 Interaction between sdf-9 and daf-2...................................................................75 Table 3.2 Effect of sdf-9 on mutants in other dauer pathways ..........................................77 Table 3.3 Maturation time..................................................................................................81 Table 4.1 Set of 21 SynDaf genes......................................................................................92 Table 4.2 Percent dauer formation of daf-8(m85) on pathogenic bacteria at 22 °C ..........97 Table 4.3 Percent dauer formation of sdf-9(m708) on pathogenic bacteria at 26 °C ........98 Table 4.4 Percent dauer formation of N2 and daf-22(m130) on pathogenic bacteria at 27 °C ..................................................................................................................................98 Table 4.5 Percent dauer formation of daf-8(m85) ; daf-6(e1377) on pathogenic bacteria at 22 °C ..................................................................................................................................99 Table 4.6 Percent dauer formation of daf-8(e1393) unc-13(e51) and daf-8(e1393) unc13(e51) ; daf-22(m130) on pathogenic bacteria at 20 °C ................................................100 Table 5.1 Life span analysis at 25 °C ..............................................................................119  !  "#!  LIST OF FIGURES Figure 1.1 Major components of the dauer signaling pathway............................................2 Figure 2.1 daf-25 mutants have chemosensory defects .....................................................46 Figure 2.2 daf-25 alleles encode the ortholog of mammalian Ankmy2 ............................48 Figure 2.3 daf-25 is expressed in many ciliated cells and encodes a novel ciliary protein ................................................................................................................................51 Figure 2.4 DAF-25 depends on IFT for proper localization within cilia but is not essential for the IFT process. ............................................................................................................53 Figure 2.5 DAF-25 is required for the localization of a guanylyl cyclase (DAF-11) to cilia.....................................................................................................................................55 Figure 2.6 Ankmy2 and GC1 can be co-precipitated in HEK293 cells.............................58 Figure 3.1 Response of sdf-9(m708) to dauer pheromone.................................................79 Figure 4.1 Survival of RNAi treated adults on S. aureus ..................................................96 Figure 5.1 zip-5 expression analysis ................................................................................120  !  "##!  LIST OF ABBREVIATIONS 1D4: Epitope from Rhodopsin ABCA4: ATP-binding cassette, sub-family A, member 4 AGE: Ageing alteration Ankmy2: Ankyrin and MYND-containing protein 2 ascr: Ascaroside ASNA: ArSeNite-translocating ATPase family BLAST: Basic local alignment search tool bp: Base pair, DNA BRA: BMP Receptor Associated protein family bZIP: Basic leucine zipper CAPS: Ca2+-dependent activator protein for secretion CCB: Calcium channel, beta subunit cDNA: Coding DNA Cemar1: C. elegans mariner transposon 1 CGH: Comparative genomic hybridization cGMP: Cyclic guanosine monophosphate CHB: che-2 body size suppressor CHE: Abnormal chemotaxis chIP: Chromatin immunoprecipitation cil: Cilia Cl: Chlorine CLC: Claudin-like in Caenorhabditis CNGA1: Cyclic nucleotide-gated channel alpha 1 co-IP: Co-immunoprecipitation CPR: Cysteine protease CUB: Complement subcomponent C1r/C1s, Uegf and bone morphogenetic protein-1 D2LIC: Dynein 2 light intermediate chain DAF: Abnormal dauer formation DBE: DAF-16 binding element DCT: DAF-16 controlled tumor suppressor  !  "###!  den: Dendrite DHS: DeHydrogenases, Short chain DNA: Deoxyribonucleic acid DPY: Dumpy DS: Distal segment DYF: Dye filling defective EAK: Enhancer of akt-1 EDTA: Ethylenediaminetetraacetic acid EGFR: Epidermal growth factor receptor EMS: Ethane methyl sulfonate ERI: Enhanced RNAi FOG: Feminization of germline FOXO: Forkhead box-o FTT: 14-3-3 GC1: Guanylate cyclase 1 GCY: Guanylyl cyclase GFP: Green fluorescent protein GLD: Germline defective GPA: G-protein alpha GPCR: G-protein coupled receptor GUCY2D: Guanylate cyclase 2d HAD: Haloacid dehalogenase HEK293: Human embryonic kidney cell line HID: High temperature induced dauer HLPC-MS/MS: High-performance liquid chromatography followed by tandem mass spectrometry HSD: Hydroxysteroid dehydrogenase homolog HSF: Heat shock factor HSP: Heat shock protein IDA: related to Islet cell Diabetes Autoantigen IFT: Intraflagellar transport  !  "#!  IIS: Insulin/Insulin-like signaling INS: Insulin InsR: Insulin receptor IRE: Insulin response element L1-L4: Larval stage 1-4 L2d: Pre-dauer L2 larva LCA: Leber’s congenital amaurosis LET: Lethal LYS: Lysozyme Mg: Magnesium mRNA: Messenger RNA MS: Middle segment MUT: Mutator MYND: Myeloid translocation protein 8, Nervy, and DEAF-1 NCR: NPC1 (human Niemann Pick C disease) related NGM: Non-glucose minimal Nr3E3: Nuclear hormone receptor Nrf2: NF-E2-related factor-2 OSM: Osmotic avoidance defective PAR: Abnormal embryonic partitioning of cytoplasm PCR: Polymerase chain reaction PDK: Pyruvate dehydrogenase kinase PHA: Defective pharynx development PI3K: Phosphoinositide 3-kinase PP2A: Protein phosphatase 2A PPTR: PP2A regulatory subunit PTCHD: Patched PTEN: Phosphatase and tensin homolog RFX: Regulatory factor X Rheb: Ras homolog enriched in brain RICT: Rictor  !  "!  RLE: Regulation of longevity by E3 ubiquitin ligase RNA: Ribonucleic acid RNA-Seq: RNA sequencing RNAi: RNA interference ROL: Roller RP: Retinitis pigmentosa RRF: RNA-dependent RNA polymerase family SAGE: Serial analysis of gene expression Sbf1: SET binding factor 1 SCD: Suppressor of constitutive dauer formation SCPx: Sterol carrier protein X SDF: Synthetic dauer formation SDS: Sodium dodecyl sulfate SGK: Serum and glucocorticoid-inducible kinase homolog SKN: Skinhead Skp1: S-phase kinase-associated protein 1 SKR: Skp1-related SL1: Splice leader 1 SL2: Splice leader 2 SNP: Single nucleotide polymorphism SRBC: Serpentine receptor class BC SRH: Serpentine receptor class H SRRM: Serine/arginine repetitive matrix 1 SUV39H1: Su(var)3-9 homolog 1 SynDaf: Synthetic dauer formation TAX: Abnormal chemotaxis TBS: Tris buffered saline TD50: Time to death 50 TEM: Transmitting electron micrograph TGF-!: Transforming growth factor beta TLDc: TBC and LysM domain containing  !  "#!  TOR: Target of rapamycin TORC1: TOR complex 1 TORC2: TOR complex 2 TRPV4: Transient receptor potential cation channel subfamily V member 4 ts: Temperature sensitive TTC8: Tetratricopeptide repeat domain 8 TZ/BB: Transition zone/basal body UNC: Uncoordinated UTR: Untranslated region UV: Ultraviolet XBX: X-box promoter element regulated ZIP: Basic leucine zipper transcription factor  !  "##!  ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Donald Riddle. I have very much enjoyed working for him in his laboratory. I am truly grateful for the guidance he has provided and for his allowance of intellectual independence. He has taught me a lot about how to conduct science in a thoughtful manner. He has always been available to discuss new and old ideas. I am proud to be his last graduate student. I would also like to thank Dr. Ann Rose for taking me in for the first 8 months of my PhD studies, for her role on supervisory committee and for helpful advice. I would also like to thank Dr. Robert Molday, Dr. Donald Moerman and Dr. Stephan Taubert for providing great advice while serving on my advisory committee. I would like to thank my co-workers in the lab who have been a great resource. I am also grateful to the VanWorm community for great discussions and advice, especially Nigel O’Neil with whom I’ve had many productive discussions. I would like to thank my Grandfather who first stimulated my love for science. I’d also like to thank Dr. David Baillie who introduced me to C. elegans research and for being a superb mentor during my undergraduate degree. I would like to acknowledge my funding including two awards from the Michael Smith Foundation for Health research and two awards from the Natural Sciences and Engineering Council of Canada. I am indebted to my wife, Jennifer, who has made up for the long hours sometimes required and for being supportive. Thanks go to my children Austin, Maren and Annika who remind me of what is important. Research undertaken in the Michael Smith Laboratories  !  "###!  DEDICATION For my children Austin, Maren and Annika, and my wife Jennifer.  !  "#$!  CO-AUTHORSHIP STATEMENT I have written all of the chapters contained in this thesis. My supervisor, Dr. Riddle provided many editorial changes. All of the collaborators listed in each chapter have also provided editorial changes. I performed the majority of the work described in this thesis with the following exceptions: Chapter 2: NJ, Bialas performed the work behind figures 2.4 and S6. PT, Nguyen performed part of the work behind figures 2.5E and S6C. SL, Bishop-Hurley perform half of the epistasis, partial mapping to half of chromosome I and survival replicate not shown. K, Kida and OE, Blacque performed the TEM work in figure S3 as well as writing the figure legend and methods for that experiment. LL, Molday and RS, Molday performed the work for figure 2.6 as well as writing the figure legend and methods for that experiment. Chapter 3: PS, Albert performed half of the epistasis, partial mapping, pheromone assays and half of the dauer counting. Chapter 4: KT, Simonsen performed the killing assay (figure 4.1) as well as confirmation of pathogenesis for each of the pathogens. Y, Lee produced the media as well as setting up the weekly experiments. D, Park did the work with P. aeruginosa including part of table 4.2 as well as figure S1. Appendix C: I wrote half of the review article. Dr. Riddle made editorial changes. Dr. Marco Gallo wrote the other half as well as making editorial changes.  !  "#!  Chapter 1: Introduction In nature, the small 1.2 mm long nematode Caenorhabditis elegans must face a variable environment. When conditions are suitable, C. elegans will progress through four larval stages (L1-L4), molting at each stage, before molting to become a reproductive adult (Cassada and Russell, 1975). When conditions are harsh C. elegans has evolved an escape strategy. Dauer (German for enduring) larvae are an alternate third larval stage. Dauer larvae are non-aging, do not eat or defecate, can survive for months while reproductive adults only survive a couple of weeks and are behaviorally adapted for dispersal to find new environments (Cassada and Russell, 1975). Once the surrounding conditions improve the dauer larva quickly recovers and molts to a fourth stage larva to resume the reproductive life plan. Post-dauer adults lay an equal number of eggs and live a life span comparable those that did not undergo dauer diapause for days to months (Klass and Hirsh, 1976). A schematic of the major components of the dauer signaling pathway can be found in Figure 1.1.  1.1 The dauer larva The characteristics of dauer larvae are remarkably different from those of larvae developing in replete environments (Cassada and Russell, 1975). The initial descriptions of the dauer larva spurred a large body of work, some ongoing, to describe the environmental inputs, physiology, signaling and genetics of the dauer larva and dauer developmental decision. More than 25 genes that cause a Daf (abnormal DAuer Formation) phenotype have been cloned and characterized, many  !  "!  Figure 1.1 Major components of the dauer signaling pathway. A schematic of the major components of the dauer signaling pathway. The pathway starts at pheromone biosynthesis with the proteins DAF-22 and DAF-28. The dauer pheromone is detected by olfactory sensation or cGMP pathway (in red). This signal results in reduced neuropeptide secretion of the TGF-! ligand DAF-7 and the insulin-like ligand DAF-28. The TGF-! ligand signals through the receptor (DAF-1/DAF-4) to the Smad proteins. The TGF-! pathway is highlighted in green. DAF-28 signals through the Insulin/Insulinlike signaling pathway highlighted in red. This includes the Insulin receptor DAF-2 as well as the downstream kinases and the output transcription factor DAF-16. Both the TGF-! and IIS pathways activate the DAF-12 nuclear hormone receptor. DAF-9, DAF36 and HSD-1 contribute to the biosynthesis of the DAF-12 ligands, the dafachronic acids. These also contribute to activation of DAF-12. The asterisk denotes SDF-9, part of the IIS pathway specific to the XXX cells. of which fall into canonical signaling pathways. Mutations that result in constitutive dauer formation are referred to as Daf-c (abnormal DAuer Formation-Constitutive) whereas those that result in the inability to form dauer larvae are Daf-d (abnormal DAuer Formation-Defective). Indeed, study of the dauer larva has contributed much knowledge to these signaling pathways including the IIS (Insulin/Insulin-like Signaling), TGF-! (Transforming Growth Factor – Beta) and TOR (Target Of Rapamycin) pathways as well as contributing to the study of the olfactory cilia in C. elegans.  !  "!  1.1.1 Inputs into the dauer decision C. elegans makes the decision to either develop through the four larval stages or to arrest as a dauer larva during the L1 or first larval stage. This larval stage lasts for approximately 10 hours post hatch until they molt into either an L2 (in favorable conditions) or L2d (in unfavorable conditions, the pre-dauer larva) (Byerly et al., 1976; Cassada and Russell, 1975; Golden and Riddle, 1984b). If conditions improve L2d larvae can molt to the L3, but if conditions remain harsh they molt into dauer larva (Golden and Riddle, 1984b). Dauer larva formation is an “all or none” decision, in that you do not see ‘partial’ dauer larvae in the wild type. Population density is one of the most important influences in dauer larva development. The L1 larva detects density by the local concentration of dauer pheromone, which is constitutively synthesized by all stages of C. elegans (Golden and Riddle, 1984b). The chemical identity and structure of the components of the dauer pheromone have been identified (Butcher et al., 2007; Jeong et al., 2005). The pheromone is balanced by a heat-stable, hydrophilic “food signal” which indicates food availability and promotes molting into the L2 instead of L2d (or L3 instead of the dauer larva). It is the ration of “food signal” to pheromone that provides the cue, not the absolute amount of either signal. High temperature is also an input into the dauer decision (Golden and Riddle, 1984b, 1984a; Ailion and Thomas, 2000, 2003). At 27 °C C. elegans adults are sterile and L1 larvae have a higher propensity to develop through L2d into dauer larvae.  !  "!  1.1.2 Characteristics of the dauer larva Dauer larvae are easily distinguishable from L3 larvae grown ad libitum. Dauer larvae are closer in length to L2 larvae. They are also radially constricted with a comparatively reduced pharynx that does not pump, as is the case with other postembryonic stages (Cassada and Russell, 1975; Vowels and Thomas, 1992). Dauer larvae do not pump because they do not eat; they have an internal cuticle based plug covering their mouths, as well as their anuses (Riddle et al., 1981). Having the stronger cuticle and closure of the major openings confers resistance to many environmental assaults as well as treatment with detergent 1% SDS (Cassada and Russell, 1975). This detergent resistance makes for a convenient method to select dauer larvae from a mixed population. Dauer larvae are not completely isolated from the environment. Many of the ciliated neurons remain in contact with the environment through pores in the cuticle at the anterior end of the amphid channel in the dauer stage (Albert and Riddle, 1988). A set of amphid neurons remains in contact with the environment allowing for olfactory assessment of when the conditions have improved (Golden and Riddle, 1984b, 1984a). The remaining ciliated neurons, which dye-fill in all other stages, have cilia that do not reach the tip of the amphid channel. This happens by a process that is still uncharacterized (Albert and Riddle, 1988). This can be seen in TEM images that show ten cilia per amphid channel in L2 larva and eight in the most anterior sections of the dauer larva.  !  "!  1.1.3 Dauer pheromone Presence of dauer pheromone not only promotes entry into the dauer stage it can also prevent recovery of dauer larvae even in the presence of food (Golden and Riddle, 1984b, 1984a). Dauer pheromone was most often produced as an organic layer extraction from a starved, over-populated liquid culture of C. elegans. At least three separate active components of the dauer pheromone were found when this lipid extraction was separated using chromatography (Golden and Riddle, 1984c). More recently, many of the different components of the pheromone have been elucidated. First identified was daumone (ascr#1), an ascarose sugar with a fatty acid side chain of 6 carbons (Jeong et al., 2005). Later, another study identified three ascarosides, including ascr#1, ascr#2 and ascr#3 (Butcher et al., 2007). It was shown that #2 and #3 are orders of magnitude more potent dauer larva inducing species. These components of the dauer pheromone function in dauer formation, and also in attraction of male C. elegans and repulsion of hermaphrodites (Srinivasan et al., 2008). Hermaphrodites will preferentially use a male’s sperm and when mated will produce more progeny (Ward and Carrel, 1979). The fact that the pheromone is also repulsive to hermaphrodites indicates that it also plays a role in dispersal and demarking less favorable environments. Not a lot is known about the synthesis of the dauer pheromone in C. elegans. One mutant that is unable to produce dauer pheromone is daf-22 (Golden and Riddle, 1985). Recently, the molecular identity of daf-22 was discovered (Butcher et al., 2009). daf-22 encodes the homolog of human sterol carrier protein SCPx, which catalyzes the last step in fatty acid !-oxidation in the peroxisome (Wanders et al., 1997). That study also identified another component in the pheromone production pathway, DHS-28 (Butcher et  !  "!  al., 2009). dhs-28 encodes the homolog of the human d-bifunctional protein that acts just upstream of SCPx in !-oxidation (Jiang et al., 1997). dhs-28 is also required for production of the dauer pheromone. It has been proposed that there are multiple receptors for the dauer pheromone because there are multiple components (Golden and Riddle, 1984c). Two serpentine GPCR (G-protein Coupled Receptor) proteins have been recently identified as dauer pheromone receptors, srbc-64 and srbc-66 (Kim et al., 2009). Knockouts of both of these proteins reduced the response to the dauer pheromone components. It is also known that the dauer pheromone signals through G-protein "-subunits, gpa-2 and gpa-3 (Zwaal et al., 1997; Lans et al., 2004). The proteins encoded by these two genes promote dauer formation when activated. They are required for response to dauer pheromone. Both SRBC and both GPA proteins show subcellular localization to the cilia. Many of the downstream signaling proteins including DAF-11 and TAX-4 are Daf-c and do not respond to the dauer pheromone as well as localizing to the amphid cilia (Kim et al., 2009; Zwaal et al., 1997; Birnby et al., 2000; Komatsu et al., 1996). Dauer larvae induced by pheromone or by mutation of daf-11 (but not tax-4) can be suppressed by 8-bromo-cGMP (a more stable, higher activity cGMP analog) in a dosedependent manner (Birnby et al., 2000). This indicates that cGMP lies downstream of DAF-11 but not TAX-4. Also, mutation of daf-6 (which encodes a patched-related protein and results blockage of the amphid channel) and daf-10 (which encodes IFT122, a component of intraflagellar transport and is required for cilia formation) are both required for sensation of the pheromone (Albert et al., 1981; Perkins et al., 1986; Perens and Shaham, 2005; Bell et al., 2006). All other tested Dyf (DYe Filling defective)  !  "!  mutants (most are ciliogenesis related) are also required for sensing the pheromone, indicating that pheromone detection requires the cilia (Ailion and Thomas, 2000).  1.2 Pathways controlling the dauer developmental decision Genetic analysis of the dauer decision has resulted in the identification of several pathways within the entire dauer-signaling network (Riddle et al., 1981). The cloning of many components of these pathways has not only impacted dauer larva genetics but has had an impact on many biological fields. The components and order of these pathways are conserved across many species and many of the players in these pathways are major players in human development and disease, including cancer and diabetes mellitus (Hu, 2007). A list of genes mentioned in the introduction can be found in Table 1.1. A schematic of the major components of the dauer signaling pathway can be found in Figure 1.1.  1.2.1 cGMP Signaling Just downstream of the pheromone is the cGMP signaling pathway or olfactory signaling pathway. This pathway functions in the sensory cilia at the afferent end of the amphid olfactory cilia (Birnby et al., 2000). These cilia are in contact with the environment and express GPCR proteins that receive the dauer pheromone signal (Bargmann, 2006). Two of these GPCRs, srbc-64 and srbc-66, have been elucidated though they do not completely abrogate response to the pheromone and are therefore only two of an unknown number of pheromone receptors (Kim et al., 2009). Upon activation, the GPCRs activate G-proteins, GPA-2 and GPA-3, which are then thought to  !  "!  bind to the receptor guanylyl cyclase DAF-11 to inhibit it. cGMP is then produced which can bind to and activate the cyclic-nucleotide gated calcium channel TAX-2/TAX-4 (Zwaal et al., 1997; Lans et al., 2004; Coburn and Bargmann, 1996; Komatsu et al., 1996). This causes depolarization of the neuron and secretion of the ligands for the IIS and TGF-! pathways (Speese et al., 2007; Hammarlund et al., 2008; Li et al., 2003; Ren et al., 1996). daf-11 mutants are Daf-c, and are also defective in chemosensation for some but not all odors (Vowels and Thomas, 1994; Birnby et al., 2000). DAF-11 is expressed in a subset of the amphid neurons, ASI, ASJ, ASK, AWB, and AWC. Of these neurons, the ASI neurons have been shown to regulate dauer entry (as well as longevity) and the ASJ neurons regulate dauer exit as determined by laser ablation studies (Bargmann and Horvitz, 1991; Alcedo and Kenyon, 2004). The mutant phenotype of daf-11 can be suppressed by treatment with exogenous cGMP in the form of 8-bromo-cGMP, which is 10 times more potent, more lipophylic and more stable (Birnby et al., 2000; Schwede et al., 2000). This is also true for another Daf-c mutant in this pathway daf-21(p673), an allele specific recessive gain-of-function mutant that appears to inhibit DAF-11 function. daf-21 encodes an Hsp90 chaperone protein. The dauer arrest phenotype of tax-4 is not suppressed by cGMP indicating that it is downstream of cGMP in this pathway (Birnby et al., 2000).  !  "!  Table 1.1 - List of genes with descriptions and associated pathway or function.  !  Gene  Description  age-1 akt-1 akt-2  Phosphoinositide 3-kinase Serine/threonine protein kinase Serine/threonine protein kinase  asna-1  Arsenite-translocating ATPase  bra-1 daf-1 daf-2 daf-3 daf-4 daf-5  BMP receptor-associated molecule TGF-! type I receptor Insulin/IGF1-like receptor Co-Smad transcription factor TGF-! type II receptor Sno/Ski-like  daf-6  Patched related protein  daf-7 daf-8 daf-9  TGF-! ligand Receptor Smad transcription factor Cytochrome P450  daf-10  WD and WAA domain protein IFT122  daf-11  Transmembrane guanylyl cyclase  daf-12  Nuclear hormone receptor  daf-14 daf-15 daf-16 daf-18  Smad Regulatory associated protein of TOR Forkhead box O transcription factor Phophatase and tensin homolog  daf-19  RFX transcription factor  daf-21  Heat shock protein chaperone  daf-22 daf-28 daf-36 dhs-28 eak-4 eak-6 eak-7 ftt-2  Sterol carrier protein Insulin-like ligand Rieske-like oxygenase 17-beta-hydroxysteroid dehydrogenase Novel protein with N-myristoylation site Phosphatase-dead phosphatase Novel protein with TLDc domain 14-3-3 chaperone  gpa-2  G-protein alpha subunit  gpa-3  G-protein alpha subunit  Pathway or Function IIS IIS IIS Neuropeptide Secretion TGF-! TGF-! IIS TGF-! TGF-! TGF-! Olfactory Signaling TGF-! TGF-! Dafachronic Acid Olfactory Signaling Olfactory Signaling Downstream Dauer Effector TGF-! TOR IIS IIS Olfactory Signaling Olfactory Signaling Dauer Pheromone IIS Dafachronic Acid Dauer Pheromone XXX Cell XXX Cell IIS IIS Olfactory Signaling Olfactory Signaling  "!  !  Gene  Description  hsd-1  Hydroxysteroid dehydrogenase  hsf-1  Heat shock transcription factor  ins-1 ins-9 ins-18 ins-19 ins-22 ins-31 let-363 ncr-1 ncr-2 par-5 pdk-1  Insulin-like ligand Insulin-like ligand Insulin-like ligand Insulin-like ligand Insulin-like ligand Insulin-like ligand Target of rapamycin Niemann-Pick type C related transmembrane glycoprotein Niemann-Pick type C related transmembrane glycoprotein 14-3-3 chaperone 3-phosphoinositide-dependent kinase  pha-4  Forkhead box A transcription factor  pptr-1 rict-1 rle-1 scd-1 scd-2 scd-3 sdf-9  Serine/threonine protein phosphatase Rapamycin-Insensitive Companion of TOR E3 ubiquitin ligase Glutamine rich novel protein Anaplastic lymphoma kinase Uncloned Phosphatase-dead phosphatase  sdf-13  T-box transcription factor  sgk-1  Serine/threonine protein kinase  skn-1  Basic leucine zipper-like transcription factor  srbc-64  G-protein coupled receptor  srbc-66  G-protein coupled receptor  tax-2  Cyclic nucleotide-gated channel beta subunit  tax-4  Cyclic nucleotide-gated channel alpha subunit  unc-31  Calcium-dependent activator protein for secretion  Pathway or Function Dafachronic Acid Longevity and Stress IIS IIS IIS IIS IIS IIS TOR Cholesterol Cholesterol IIS IIS Longevity and Stress IIS TOR IIS TGF-! TGF-! TGF-! IIS Olfactory Signaling IIS Longevity and Stress Olfactory Signaling Olfactory Signaling Olfactory Signaling Olfactory Signaling Neuropeptide secretion  "#!  TAX-2 and TAX-4 have overlapping expression with DAF-11 (Birnby et al., 2000; Coburn and Bargmann, 1996; Komatsu et al., 1996). daf-11, daf-21, and tax-4 mutants do not respond to dauer pheromone, indicating that they are all downstream of the pheromone. SRBC-64, SRBC-66, GPA-2, GPA-3, DAF-11, TAX-2, and TAX-4 all localize to the sensory cilia (Kim et al., 2009; Lans et al., 2004; Coburn and Bargmann, 1996; Komatsu et al., 1996; Birnby et al., 2000). The signaling in this pathway, leading to depolarization of the cell, is contained entirely in the sensory cilia, indicating a strong role for cilia function in dauer biology.  1.2.2 Amphid cilia and dauer larvae The sensory cilia play a key role in the dauer signaling pathway (Vowels and Thomas, 1992). Cilia allow for compartmentalization and contact of the cGMP pathway GPCRs with the environment and pheromone. Three mutants that were isolated based on their dauer phenotypes are in genes required for proper cilia development and structure. One of these, daf-6, is not expressed in the ciliated neurons but in the glial socket cells that allow for the cilia to contact the environment (Albert et al., 1981; Perkins et al., 1986; Perens and Shaham, 2005). daf-6 mutants do not form dauer larvae because they cannot sense any dauer pheromone. They are also unable to take up a lipophilic dye, which is usually used to determine proper cilium structure (Hedgecock et al., 1985). daf6 is unique in that the dye cannot enter because the amphid channel is blocked (Albert et al., 1981; Perkins et al., 1986; Perens and Shaham, 2005). daf-6 does not suppress daf-11 (Riddle et al., 1981), indicating that dauer formation in daf-11 mutants does not require contact with the environment further supporting the idea that the Daf-d phenotype of daf-  !  ""!  6 is due to inability to sense the pheromone. daf-6 encodes a patched-related protein expressed in the amphid sheath and glial pocket cells which are required for proper pore formation (Perens and Shaham, 2005). daf-10 is another Daf-d mutant that is also Dyf (Albert et al., 1981; Bell et al., 2006). This is due to inability to form sensory cilia in the mutant. daf-10 encodes a WD repeat protein that is homologous to IFT122. As with daf-10 mutants many other conserved IFT genes cause defects in ciliogenesis and have Dyf and Daf-d phenotypes when mutated (Bell et al., 2006; Vowels and Thomas, 1992; Ailion and Thomas, 2000). Most Dyf mutants, including daf-10 do suppress the Daf-c phenotype of daf-11 indicating that proper cilia structure is required for promotion of the dauer phenotype in the absence of cGMP (Vowels and Thomas, 1992). It has been shown that defects in ciliogenesis lead to expression and secretion of more IIS pathway ligand, which could explain the Daf-d phenotype by excess activation of the IIS pathway (Li et al., 2003). daf-19 mutants are Daf-c and also lack sensory cilia (Perkins et al., 1986; Swoboda et al., 2000; Malone and Thomas, 1994). daf-19 encodes an RFX-type transcription factor that is a major regulator of cilia proteins in C. elegans and other animals. DAF-19 binds DNA at a consensus sequence known as the X-box (Swoboda et al., 2000; Efimenko et al., 2005; Blacque et al., 2005; Chen et al., 2006). This consensus site is found in the promoters of many cilia-related genes. Though it is not quite understood why daf-19 mutants have a strong Daf-c phenotype while other Dyf mutants have a Daf-d phenotype, there are some clues as to why this may be the case. Ablating all the amphid neurons results in a Daf-c phenotype, indicating that daf-19 mutants represent a case closer to non-functional neurons than do  !  "#!  the other Dyf mutants (Bargmann and Horvitz, 1991). One isoform of daf-19 regulates ciliogenesis while another regulates synaptic function (Senti and Swoboda, 2008). Defects in synaptic function often result in weak Daf-c phenotypes, so this may be a contributing cause of the daf-19 Daf-c phenotype. daf-19 mutants may also disrupt the expression of other proteins that have dauer inhibitory properties (e.g. DAF-11, DAF-21, DAF-7, DAF-28) that may not be affected in other Dyf mutants (Murakami et al., 2001; Li et al., 2003). The sensory cilia are structurally required for proper dauer signaling, especially in responding to the environmental signals.  1.2.3 Insulin/insulin-like signaling Perhaps the most extensively studied of all the pathways found in dauer signaling is the IIS pathway. The IIS pathway starts in the sensory neurons (and to some degree other tissues) with expression and secretion of the various insulin-like ligands (Pierce et al., 2001; Li et al., 2003). The most influential (i.e. the one with the strongest mutant phenotype) is daf-28 (Li et al., 2003). daf-28 encodes a insulin receptor (DAF-2) agonist. In daf-28 mutants, there is less activation of daf-2, which results in more dauer formation. Processes and proteins that affect insulin secretion, many of which are conserved with mammals, also influence level of DAF-28 secretion. UNC-31, a homolog of CAPS (Calcium-dependent Activator Protein for Secretion), is required for proper secretion of dense core vesicles, which contain protein hormones including DAF-28 (Avery et al., 1993; Speese et al., 2007; Hammarlund et al., 2008; Lin et al., 2010). unc31 mutants are weak Daf-c mutants in that they enhance most other Daf-c mutants and have a Hid phenotype (High temperature Induced Dauer, abnormal dauer formation at 27  !  "#!  °C) (Ailion and Thomas, 2000; Ohkura et al., 2003; Avery et al., 1993). Another protein that has been shown to affect secretion of DAF-28 is ASNA-1 (Kao et al., 2007). Mutation of asna-1 results in a much-reduced DAF-28 secretion, while over-expression mimics increased insulin secretion. The ASNA-1 human homolog is highly expressed in pancreatic !-cells. Although daf-28 was the only insulin-like protein isolated in forward genetic screens, the C. elegans genome encodes 40 different insulin-like peptides (Malone and Thomas, 1994; Li et al., 2003). Though there are 40 ligands there is only one receptor identified to date, daf-2 (Pierce et al., 2001; Kimura et al., 1997). This indicates that, assuming most of the 40 are expressed and functional, there is a high level of redundancy between agonistic and antagonistic insulin-like proteins. There has been a function associated with a number of them despite this limitation. ins-1 is the one that is most similar to human insulin (Pierce et al., 2001). Knockout of ins-1 doesn’t have a dauer phenotype but over-expression of INS-1 in many tissues, including amphid neurons, ring neurons and intestine, shows a dauer enhancing phenotype in the wild type N2 as well as in the daf-2 (hypomorphic) mutant background. INS-18 over-expression in sensory neurons, intestine and nerve ring also enhances dauer formation. This indicates that INS1 and INS-18 are both antagonists of DAF-2 signaling. No phenotype was observed with over-expression of INS-9, INS-19, INS-22, and INS-31. This could be for many reasons however INS-1 and INS-18 are the only C. elegans insulin-like proteins that have a cpeptide. The daf-2 Insulin/Insulin-like growth factor 1 receptor is a major regulator of dauer formation and longevity in C. elegans (Riddle et al., 1981; Kenyon et al., 1993;  !  "#!  Honda and Honda, 1999; Larsen et al., 1995; Kenyon, 2010; Kimura et al., 1997; Gems et al., 1998). The many alleles of daf-2 have varying severity in their Daf-c phenotype from low dauer formation in hypomorphs (e1365, e1368) to stronger hypomorphs (m41, e1370) to L1 arrest in the strongest alleles (m65, sa223). They also exhibit a muchextended life span. When bound to an agonist the DAF-2 homodimer transautophosphorylates to activate (Kimura et al., 1997). The activated DAF-2 receptor then activates the downstream PI3K (PhosphoInositide 3-Kinase) homolog AGE-1. age-1 is allelic to daf-23 and are Daf-c (Friedman and Johnson, 1988; Malone et al., 1996). daf-18 a Daf-d mutant, homolog of PTEN (phosphoinositide 3-phosphatase), antagonizes AGE1/PI3K (Ogg and Ruvkun, 1998). Activation of AGE-1 results in the activation of the partially redundant AKT-1 and AKT-2 as well as PDK-1 and SGK-1 kinases (Paradis et al., 1999; Paradis and Ruvkun, 1998; Hertweck et al., 2004). AKT-1, AKT-2 and SGK-1 all phosphorylate a forkhead transcription factor DAF-16, the major output of the IIS pathway, to deactivate it by causing sequestration from the nucleus (Ogg et al., 1997; Henderson and Johnson, 2001). This is antagonized by PPTR-1, a PP2A homolog that antagonizes phosphorylation of DAF-16 by AKT-1 (Padmanabhan et al., 2009). Cytoplasmic retention of DAF-16 requires PAR-5 and FTT-2, both of which are 14-3-3 proteins (Berdichevsky et al., 2006; Li et al., 2007). The E3 ubiquitin ligase RLE-1 can bind DAF-16 and attach ubiquitins, which results in proteasome degradation of DAF-16 (Li et al., 2007). Mutations in daf-16 cause a Daf-d phenotype and can suppress any of the Daf-c mutants in the IIS pathway, mentioned above (Riddle et al., 1981; Larsen et al., 1995; Ogg et al., 1997). The DAF-16 transcription factor will be discussed in more detail below.  !  "#!  1.2.4 TGF-! signaling The TGF-! pathway in C. elegans is parallel to the IIS pathway and is downstream of the cGMP pathway (Riddle et al., 1981; Patterson et al., 1997; Thomas et al., 1993; Fielenbach and Antebi, 2008; Hu, 2007). Although genetic analysis puts the TGF-! pathway partially parallel to the cGMP pathway, an allele of daf-11 was isolated in a screen for reduced daf-7 (homolog of TGF-!) transcriptional GFP reporter expression as well as Daf-c phenotypes (Murakami et al., 2001). This indicates a role for TGF-! downstream of the cGMP pathway in that cGMP levels in the ASI neurons, where daf-7 is mainly expressed, control the level of expression and likely secretion of DAF-7. The TGF-! pathway is defined by the Daf-c genes daf-1, daf-4, daf-7, daf-8, and daf-14 as well as the Daf-d genes daf-3 and daf-5 (Thomas et al., 1993; Patterson et al., 1997). The ligand for the TGF-! pathway is DAF-7, which is expressed in the ASI pair amphid neurons (Ren et al., 1996). Its expression is regulated by the amount of pheromone in the environment. It is then secreted from the neurons and detected by the heterodimeric TGF-! receptors DAF-1 (Type I) and DAF-4 (Type II) (Georgi et al., 1990; Estevez et al., 1993; Gunther et al., 2000). When activated the Type II receptor phosphorylates the Type I receptor . This activates the Type I receptor which then phosphorylates an R-Smad (DAF-8 and DAF-14, in this case) (Inoue and Thomas, 2000b; Park et al., 2010). The R-Smads relocate to the nucleus where they either bind or co-regulate genes with other transcription factors such as the Co-Smad DAF-3 or the Sno/Ski homolog DAF-5 (Patterson et al., 1997; da Graca et al., 2004). DAF-3 function appears to be inhibited by the activation of the TGF-! pathway, presumably to allow for  !  "#!  stronger function of the opposing transcription factors DAF-8 and DAF-14 (Inoue and Thomas, 2000b; Park et al., 2010). Other proteins have been identified that participate in the TGF-! signaling pathway outside of the canonical members mentioned above. Three of these, scd-1, scd-2 and scd-3, were identified as suppressors of constitutive dauer (scd) for daf-1, daf-8 and daf-14 (Inoue and Thomas, 2000a). While scd-3 remains unidentified, scd-1 encodes a glutamine-rich protein and scd-2 encodes the homolog of the anaplastic lymphoma kinase (Reiner et al., 2008). Another modifier identified in this pathway is BRA-1, a homolog of BMP-receptor associated molecule (Morita et al., 2001). BRA-1 appears to inhibit TGF-! signaling because bra-1 mutants suppress the Daf-c phenotype of daf-1. bra-1 does not suppress daf-14, which is consistent with its functioning at the level of the receptor. While the phenotypes of most Daf-c and Daf-d genes are quite simple and invariable, this is not the case for daf-3. daf-3 mutants are Daf-d at all temperatures up to 25 °C but at 27 °C they form more dauer larvae than N2, which is the Hid phenotype (Patterson et al., 1997; Ailion and Thomas, 2000). This may be due to an under-defined role in regulating sterol signaling, as daf-3 mutants with ablated XXX neuroendocrine cells are Daf-c (Ohkura et al., 2003). Ablating these two cells, which produce the initial dauer larva related sterol signal (Gerisch and Antebi, 2004), reverses the phenotype of daf-3 mutants.  !  "#!  1.2.5 TOR signaling While the TGF-! and IIS pathway mutants have very discreet dauer phenotypes, either more or less dauer larvae formed, three of the TOR pathway mutants arrest as dauer-like L3 larvae (Long et al., 2002; Jia et al., 2004; Honjoh et al., 2009; Jones et al., 2009). let-363 mutants (homolog of TOR, Target Of Rapamycin) arrest as L3 larvae. LET-363 has been shown to regulate longevity potentially through regulation of protein translation and nutrient metabolism (Kapahi et al., 2004; Teleman et al., 2005; Hansen et al., 2007). TOR forms two complexes, TORC1 with Raptor that regulates Autophagy, translation, and metabolism, and TORC2 which binds Rictor and regulates actin organization and Akt (which regulates dauer formation in C. elegans) (Loewith et al., 2002; Wullschleger et al., 2006). Cosistent with this the mutants in daf-15 (Raptor) arrest as dauer-like L3 larvae and mutants in rict-1 (Rictor) have body morphology, developmental delay, reduced brood, short life span and fat accumulation phenotypes (Jia et al., 2004; Jones et al., 2009). Upstream of TORC1 and TORC2 is the protein Rheb (Ras homology enriched in brain) (Avruch et al., 2006; Honjoh et al., 2009). In C. elegans, reduction in Rheb has been shown to have an L3 arrest phenotype. This is consistent with the data from mammalian studies, which shows that it is a direct upstream activator of LET-363/TOR (Stocker et al., 2003; Saucedo et al., 2003). Upstream of Rheb is Akt, which now gives two points at which the TOR pathway can cross talk with the IIS pathway. The larval arrest phenotype of the TOR pathway mutants is epistatic to the Daf-d phenotype of DAF-16 (Jia et al., 2004), indicating that while the TOR pathway may cross-talk with the IIS pathway, it is genetically downstream.  !  "#!  1.2.6 Steroid hormone pathway The dauer-related steroid hormone pathway in C. elegans is downstream of other dauer pathways and requires exogenous cholesterol (Antebi et al., 2000; Gerisch and Antebi, 2004; Ohkura et al., 2003; Albert et al., 1981; Albert and Riddle, 1988; Jia et al., 2002; Motola et al., 2006). Consistant with this, the two homologs of Niemann-Pick C ncr-1 and ncr-2 show weak Daf-c phenotypes on their own, but have a strong Daf-c phenotype as a double mutant (Li et al., 2004). These proteins carry cholesterol into the cell. Mutation of ncr-1 or ncr-2 results in cholesterol deprivation and can be rescued by increasing the concentration of cholesterol. The arrest phenotype seen after a few generations of cholesterol deprivation is similar to the phenotype of the mutants in the Daf-c gene daf-9 (Gerisch et al., 2001). daf-9 encodes a cytochrome P450 homolog which is similar to steroid hydroxylases (Jia et al., 2002). DAF-9 functions in the last steps of the conversion of cholesterol to the two dauer and heterochronic steroid hormones !4 and !7 dafachronic acids (Motola et al., 2006). DAF-9 adds a carboxy group to the lipophillic side chain found on cholesterol and all the intermediate chemical species. Another modification protein identified in this pathway is the daf-36 gene, which encodes a Rieske-like oxygenase (Rottiers et al., 2006). DAF-36 is predicted to function at the initial stages of !7 dafachronic acid biosynthesis. hsd-1 encodes the homolog to 3"-hydroxysteroid dehydrogenase/!5-!4 isomerases and has also been shown to function the initial stages of !4 dafachronic acid synthesis (Patel et al., 2008). Mutants in hsd-1, and daf-36 form more dauer larvae in the absense of cholesterol, indicating that they produce less of the dauer-inhibiting dafachronic acids.  !  "#!  !4 and !7 dafachronic acids were first identified as the ligands for the DAF-12 (Motola et al., 2006), which is a nuclear hormone receptor homologous to the vitamin D receptor in mammals (Antebi et al., 2000). Certain mutations in the ligand binding domain are Daf-c while all other mutations are Daf-d. DAF-12 is the furthest downstream member of the dauer genetic pathway and Daf-d mutations in daf-12 can suppress mutations in all of the Daf-c genes (Riddle et al., 1981). The two neuroendrocrine cells, XXXL and XXXR are central to the steroid signaling pathway (Gerisch et al., 2001; Jia et al., 2002; Ohkura et al., 2003). All of the genes listed in this section are expressed in these two cells except daf-36 (Gerisch et al., 2001; Jia et al., 2002; Ohkura et al., 2003; Li et al., 2004; Rottiers et al., 2006; Patel et al., 2008). DAF-9 expression, which presumably correlates with dafachronic acid synthesis, is variable in the XXX cells in response to environmental conditions. In a harsh environment, DAF-9 shows strong expression in the XXX cells, which results in low expression of DAF-9 in the hypodermis (Gerisch and Antebi, 2004). Conversely, low expression of DAF-9 in the XXX cells results in high expression in the hypodermis. Both NCR proteins are expressed in the XXX cells as is HSD-1 (homolog of the 3"hydroxysteroid dehydrogenase/!5-!4 isomerases) (Patel et al., 2008). Another gene that shows cholesterol induced dauer formation as well as XXX cell expression is sdf-9 (synthetic dauer formation) (Ohkura et al., 2003; Hu et al., 2006). sdf-9 encodes a phosphatase-dead phosphatase-like protein.  !  "#!  1.2.7 Enhancers of dauer larva formation Many genes have been identified in which mutants were identified in the background of another Daf-c gene. One screen was conducted in the background of unc31, mentioned above. The resulting mutants were named sdf for synthetic dauer formation. Though 14 complementation groups were identified by this screen only two genes were cloned, sdf-9 and sdf-13 (Ohkura et al., 2003; Miyahara et al., 2004). sdf-9 is a cholesterol sensitive dauer enhancer that is only expressed in the XXX cells. Due to these phenotypes it is thought to function in the sterol signaling pathway downstream of DAF-2. sdf-13 encodes a T-box transcription factor and mutants in sdf-13 fail to adapt to prolonged exposure to olfactory stimuli that are detected by the AWC neuron. Another dauer formation enhancer screen was conducted in the akt-1 background (Hu et al., 2006). Mutants identified in this screen were identified as eak genes for enhancers of akt-1. Seven complementation groups resulted from this screen, four of which have been cloned. eak-5 was found to be allelic to sdf-9. eak-4 encodes a novel protein with an N-myristoylation motif and eak-6 encodes a protein tyrosine phosphatase homolog. As with sdf-9, eak-4 and eak-6 are expressed in the XXX cells and likely function in a pathway connected to the steroid signaling pathway. eak-3 has also been cloned and it encodes a novel protein that regulates DAF-16 function in the XXX cells (Zhang et al., 2008). eak-7 encodes a novel protein with an N-myristoylation motif and a TLDc (TBC and LysM Domain Containing) domain (Alam et al., 2010). EAK-7 functions to keep the DAF-16/FOXO transcription factor out of the nucleus where it cannot be active.  !  "#!  1.3 Dauer signaling pathway and aging The idea of a connection between dauer larvae and longevity in C. elegans was postulated early in C. elegans genetic research (Brenner, 1974; Cassada and Russell, 1975; Klass and Hirsh, 1976). Since then it has emerged that many of the genes involved in the dauer developmental decision are also major regulators of longevity (Kenyon et al., 1993; Friedman and Johnson, 1988; Larsen et al., 1995; Kenyon, 2010). Expression profiling was performed on IIS pathway mutants to understand the biological changes associated with longer life (Murphy et al., 2003; McElwee et al., 2003; Jones et al., 2001; Lund et al., 2002; Halaschek-Wiener et al., 2005; Ruzanov et al., 2007). It is hoped that research into the life span of C. elegans will result in a better understanding of human aging.  1.3.1 Life span and Daf mutants Many mutants in the dauer formation pathway have been identified as having a longer life span. The first of these identified was age-1, the PI3K (Friedman and Johnson, 1988). Originally age-1 was not identified as a daf gene. Through later complementation testing it was determined that age-1 was allelic to daf-23 (Malone et al., 1996). This was the first example of a genetic change resulting in an extended life span. The next gene to be identified as a major controller of life span was daf-2 (Kenyon et al., 1993). It was shown that daf-2 mutants live twice as long as wild type and that daf-16 suppresses this effect just as it suppresses the dauer formation phenotype of daf-2. This identified the first pathway involved in the regulation of longevity, the IIS pathway (Friedman and Johnson, 1988; Kenyon et al., 1993; Larsen et al., 1995).  !  ""!  This effect was not limited to the InsR daf-2 or the PI3K age-1. akt-1, akt-2, sgk-1 and pdk-1 all show an extended longevity, dependent on DAF-16 (Paradis et al., 1999; Hertweck et al., 2004). Over-expression of DAF-16 can also extend longevity (Henderson and Johnson, 2001). This indicates that DAF-16 may regulate downstream intercellular processes that regulate longevity. As well as DAF-16, two other transcription factors have been shown to regulate longevity downstream of the IIS pathway, HSF-1 and SKN-1 (Hsu et al., 2003; Tullet et al., 2008). These transcription factors regulated downstream targets that modulate longevity. For DAF-16, this includes genes required for stress-response, anti-microbial peptides, metabolism and chaperones (Jones et al., 2001; Murphy et al., 2003; McElwee et al., 2003; Halaschek-Wiener et al., 2005; Ruzanov et al., 2007). More recently, the TGF-! pathway has been shown to regulate longevity in C. elegans (Shaw et al., 2007; Luo et al., 2009). It was shown that all of the Daf-c mutants from the TGF-! pathway have an extended life span and that the longer life span of daf-7 is suppressed by daf-16. This indicates that the longevity phenotype of the TGF-! pathway signals through DAF-16. The reverse is not true, however, daf-3 does not suppress the long life span of daf-2. The TOR pathway has also been linked to longevity (Jia et al., 2004; Honjoh et al., 2009; Jones et al., 2009; Hansen et al., 2007). The TOR kinase (LET-363) acts as an amino acid and nutrient sensor. It can function to regulate growth, metabolism and autophagy, as well as translation; all of these processes have been linked to longevity. Inhibiting the TOR pathway has been shown to extend longevity in a variety of organisms including C. elegans (Stanfel et al., 2009). Unlike the TGF-! or IIS pathways  !  "#!  this life span enhancement is independent of DAF-16, indicating that it may regulate different processes related to aging (Hansen et al., 2007). The effect of inhibiting the TOR pathway mimics the effect of dietary restriction, as dietary restriction does not extend the life span of TOR mutants. Dietary restriction does extend the life span of daf-2 mutants (Lee et al., 2006). Dietary restriction induced longevity requires the transcription factors SKN-1 and PHA-4 (Panowski et al., 2007; Bishop and Guarente, 2007). PHA-4 is also required for the regulation of autophagy by the TOR pathway in C. elegans (Hansen et al., 2008). There are many other factors that regulate longevity in C. elegans. The ones directly connected to dauer formation and daf mutants are discussed here. Some factors are only discussed briefly. There is a wealth of work on this subject including two reviews (Kenyon, 2010; Stanfel et al., 2009).  1.3.2 DAF-16 target analysis Several groups have deepened the understanding of the transcriptional response to the IIS signaling pathway by assaying transcript levels for the majority of transcripts in C. elegans (Murphy et al., 2003; McElwee et al., 2003; Halaschek-Wiener et al., 2005; Ruzanov et al., 2007; Oh et al., 2006; Furuyama et al., 2000; Lee et al., 2003). These studies have provided insights into the mechanisms regulated by the IIS pathway. The major output of the IIS pathway is the DAF-16/FOXO transcription factor and the majority of the work has been done to analyze the targets of this protein. They take advantage of the activation of DAF-16 in a daf-2 background and often compare the transcripts from daf-2 mutants to those found in daf-2; daf-16 double mutants (Lin et al.,  !  "#!  2001; Henderson and Johnson, 2001). This was accomplished using SAGE, microarrays as well as assaying direct targets by in silico prediction based on conserved DAF-16 binding sites or by chromatin immuno-precipitation (Furuyama et al., 2000; Murphy et al., 2003; McElwee et al., 2003; Oh et al., 2006; Halaschek-Wiener et al., 2005; Ruzanov et al., 2007; Lee et al., 2003). Two microarray studies, completed in 2003, both compared daf-2 mutants to daf2; daf-16 double mutants (Murphy et al., 2003; McElwee et al., 2003). These studies revealed many enriched gene classes in the set of target genes (direct or indirect targets) including anti-microbial genes, metabolism and stress response genes. Many of these genes were also shown to regulate longevity via RNAi experiments. One of the studies concluded that the transcriptional signature of daf-2 mutants is similar to that of dauer larvae. This is intriguing in that the long life span of daf-2 mutants may come from their ‘dauer-like’ metabolism. Three other studies focused on direct targets (Furuyama et al., 2000; Lee et al., 2003; Oh et al., 2006). The first group to identify all the genomic targets looked for the DBE in the upstream regulatory regions of all the annotated genes in C. elegans, though they only did follow-up studies on one of their 159 identified targets (Furuyama et al., 2000). Using comparative genomics another group identified 17 genes that contain the consensus DBE (DAF-16 Binding Element) in C. elegans and Drosophila melanogaster (Lee et al., 2003). Though only four of these 17 genes were shown to have an effect on DAF-16 regulated phenotypes (longevity, fat accumulation, or dauer formation). This assay was utilized RNAi, which results in incomplete knockdown as well as very little knockdown of genes expressed in neurons. This could result in an under-representation  !  "#!  of the true phenotypes associated with these genes. Another study identified direct targets by chromatin immuno-precipitation (Oh et al., 2006). About half of their 103 targets showed change in expression in a daf-16 mutant. 18 of the 33 genes tested by RNAi showed DAF-16 regulated phenotypes (longevity, fat accumulation, or dauer formation). These studies show that the DAF-16 target genes contribute to the phenotypes observed in IIS pathway mutants.  1.3.3 SKN-1 and aging skn-1 encodes a Nrf2-like homolog and is a bZIP transcription factor which lacks the leucine zipper domain (Bowerman et al., 1992). The leucine zipper is used for homodimer or heterodimer formation. The absence of this domain allows SKN-1 to bind DNA as a monomer (Blackwell et al., 1994). It was originally identified as regulating gut and pharyngeal development in the embryo (Bowerman et al., 1992). In adults it plays a role in stress response and longevity, a function that is conserved with its homolog Nrf2 (An and Blackwell, 2003; An et al., 2005; Bishop and Guarente, 2007; Tullet et al., 2008; Oliveira et al., 2009). Many of the identified SKN-1 targets are free radical detoxification proteins that prevent oxidative stress damage. This includes superoxide dismutases, catalases and glutathione s-transferases among others. In addition to functions in stress response, SKN-1 target genes function in other processes including endoderm development. The role for skn-1 in longevity is due to an inducible stress response but also to the regulation of caloric restriction induced longevity (An and Blackwell, 2003; An et al., 2005; Bishop and Guarente, 2007). SKN-1 is required for the life span extension  !  "#!  observed when C. elegans is given a restricted amount of bacteria as opposed to being fed ad libitum. This effect is regulated by SKN-1 in the ASI neurons (Bishop and Guarente, 2007). These neurons play a key role in controlling dauer formation and longevity (Bargmann and Horvitz, 1991; Ren et al., 1996; Alcedo and Kenyon, 2004). SKN-1 is also a downstream target of the IIS pathway (Tullet et al., 2008). Mutation of skn-1 suppressed the long life span of some daf-2 mutants. It was also shown that reducing IIS signaling, through akt-1, akt-2, or sgk-1 causes increased SKN-1 accumulation in the intestine. Indeed, it was also shown that AKT-1, AKT-2 and SGK-1 all phosphorylate SKN-1 to inactivate it. Mutation of an Akt consensus phosphorylation site in SKN-1 was shown to increase function and expression of SKN-1. Also, SKN-1 target genes were up regulated with reduced IIS signaling. This indicates that SKN-1 is regulated by the IIS pathway at the same point, in parallel to DAF-16.  !  "#!  1.4 Thesis objectives The specific goals for the work documented in this thesis are as follows: 1) To identify and characterize the gene encoded by the Daf-c gene daf-25 that falls into the cGMP pathway by epistatic analysis and when mutated causes olfactory phenotypes. 2) To identify and characterize the gene encoded by the dauer enhancer mutant m708 isolated as an enhancer of daf-2. 3) To identify novel SynDaf genes using RNAi with a double mutant enhanced for dauer formation (sdf-9) and RNAi efficacy (eri-1). 4) To characterize zip-5, identified as a target of IIS signaling using SAGE (Serial Analysis of Gene Expression).  !  "#!  1.5 Bibliography Ailion, M., and Thomas, J. H. (2003). Isolation and characterization of high-temperatureinduced Dauer formation mutants in Caenorhabditis elegans. Genetics 165, 12744. Ailion, M., and Thomas, J. H. (2000). 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"#!  CHAPTER 21: Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25  2.1 Introduction The dauer larva of Caenorhabditis elegans is an alternate third larval stage where a stress resistant, non-aging life plan is adopted in harsh environmental conditions (Cassada and Russell, 1975). Dauer larvae disperse and will resume development when conditions improve. The study of dauer formation has elucidated a complex gene network used to control the decision to go into diapause (Hu, 2007). The dauer pathway includes well-recognized members in the canonical TGF-! (Transforming Growth Factor-Beta) and Insulin/Insulin-like signaling (IIS) pathways, as well as proteins affecting olfactory reception, neuron depolarization and peptide hormone secretion. Many mutants isolated as dauer formation defective (Daf-d) or constitutive (Daf-c) have revealed the key signaling components (Hu, 2007). Here we identify DAF-25, a novel member of the olfactory signaling pathway that is associated with cGMP signaling—a signal transduction pathway with established links to cilia (Johnson and Leroux, 2010). We show that the mammalian ortholog, Ankmy2, is expressed in ciliary photoreceptors and interacts with a guanylate cyclase (GC1), as predicted from the C. elegans results. The olfactory signaling cascade has been well characterized in the two C. elegans amphids, organs consisting of a set of twelve bilaterally symmetric pairs of ciliated !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! #!$!%&'()*+!*,!-.)(!/.01-&'!2&&+!0//&1-&3!0+3!)(!)+!1'&((4!(&&5!6&+(&+!784!9)0:0(!;64!  9)(.*1<=>':&?!@84!A*:30?!884!B)30!B4!;C>?&+!DE4!9:0/F>&!GH4!A*:30?!I@4!8&'*>J! AI4!I)33:&!K8!LMN#NO!Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25. PLoS Genetics in press.! !  "#!  sensory neurons (Bargmann, 2006; Perkins et al., 1986). While similar to mammalian olfactory signaling, at least some proteins involved are also homologous to those implicated in mammalian phototransduction (Ward et al., 2008). Chemicals are sensed at the afferent, ciliated ends of sensory neurons where they contact the environment through pores in the cuticle. The cilia are required for chemosensation of chemical attractants and repellants, as well as for dauer entry and exit (Bargmann et al., 1993). For many odorants the specific neurons that detect the odor are known (Bargmann, 2006). For example, the AWA, AWB and AWC neuron pairs sense volatile odorants such as pyrazine, benzaldehyde, trimethyl thiazole and isoamyl alcohol. The ASH pair of ciliated olfactory neurons can detect changes in osmotic pressure. The connection between dauer formation, chemosensory behavior and cilia is well known (Hu, 2007; Inglis et al., 2007). C. elegans hermaphrodites only possess nonmotile (primary) cilia which are found at the dendritic ends of 60 sensory neurons in the head and tail (Perkins et al., 1986; Inglis et al., 2007). Intraflagellar transport (IFT) proteins, normally required for building cilia, are well conserved in C. elegans and several have been discovered in this organism through the identification of sensory mutants (Silverman and Leroux, 2009). Indeed, dauer formation is a sensory behavior dependent on the balanced inputs of dauer pheromone, temperature and food signals (Bargmann, 2006). Proteins in the olfactory component of the dauer pathway include SRBC-64 and SRBC-66 (dauer pheromone receptors), DAF-11, a guanylyl cyclase, G-proteins (gpa-2 and gpa-3), the Hsp90 molecular chaperone DAF-21, the IFT protein DAF-10, and the DAF-19 RFX-type transcription factor (Swoboda et al., 2000; Bell et al., 2006; Birnby et !  "#!  al., 2000; Kim et al., 2009; Zwaal et al., 1997). DAF-19 is strictly required for cilium formation as it regulates the expression of many cilia-related genes through a consensus sequence dubbed ‘x-box’ (Efimenko et al., 2005). daf-11, daf-19 and daf-21 are Daf-c, whereas daf-6 and daf-10 are Daf-d (Riddle et al., 1981). daf-19, daf-6 and daf-10 are all dye-filling defective, indicating that their cilia (if present) are not exposed to the environment (Albert et al., 1981; Swoboda et al., 2000). By contrast, daf-11 and daf-21 mutants show wild-type dye filling (Malone and Thomas, 1994). All five mentioned daf genes are defective in recovery from the dauer diapause, presumably because they cannot detect the bacterial food stimulus (Albert et al., 1981). Dauer recovery defects are present for mutants with broad chemosensory defects caused by abnormal ciliogenesis or signaling, and for many Unc genes, such as unc-31, which encodes a dense core vesicle secretion protein (Albert et al., 1981; Avery et al., 1993; Ailion and Thomas, 2003). Our genetic screen for C. elegans Daf mutants has uncovered a novel ciliary protein, DAF-25, which participates in cGMP-associated signaling by modulating the ciliary localization of a guanylyl cyclase, DAF-11. The mammalian ortholog of DAF-25, Ankmy2, interacts with ciliary photoreceptor guanylyl cyclase 1 (GC1), indicating that the role of the MYND domain protein in cilia function is likely to be conserved and potentially relevant to human retinal disease or other ciliopathies.  2.2 Results 2.2.1 Genetic epistasis analysis places DAF-25 function in the amphid cilia To identify genes potentially implicated in sensory transduction, we uncovered four alleles of daf-25 in various screens for new mutants exhibiting a temperature!  "#!  sensitive Daf-c phenotype. Three alleles (m98, m137, and m362) were isolated from ethyl methanesulfonate (EMS) mutagenesis screens and the fourth, m488, was isolated in a screen for Daf-c mutants with transposon insertions (Brenner, 1974; Kiff et al., 1988). Epistasis tests with the Daf-d mutants daf-12, daf-16, daf-3, daf-6 and daf-10 were used to position daf-25 into the existing genetic pathway. Mutations in the daf-12 nuclear hormone receptor gene suppress most Daf-c mutants (Riddle et al., 1981; Antebi et al., 2000) including daf-25 (0% dauer larva formation, n>200 for daf-25(m362); daf12(m20) compared to 97.5%, n=281 for daf-25(m362) at 25 °C). DAF-16/FOXO is the major downstream effector for Insulin/IGF1 signaling (Ogg et al., 1997) as is DAF-3/CoSmad for the TGF-! pathway (Patterson et al., 1997). Mutations in daf-16 and daf-3 only partially suppress the Daf-c phenotype of daf-25 (37.6% dauer larvae, n=407 for daf25(m362); daf-16(m26) and 60.0%, n=167 for daf-25(m362); daf-3(mgDf90) at #$!%&), indicating that DAF-25 likely functions upstream of both pathways. Importantly, daf-10, which encodes an IFT protein (DAF-10/IFT122) required for ciliogenesis (Bell et al., 2006), suppresses daf-25 (0% dauer larvae, n>200 for daf25(m362); daf-6(e1387) compared to 97.5%, n=281 for daf-25(m362) at 25 °C), suggesting a function for DAF-25 within sensory cilia. daf-6 mutants have closed amphid channels and cannot smell chemoattractants or form dauer larvae even though their cilia are present (Perkins et al., 1986). Interestingly, daf-6 mutations do not suppress the daf25 Daf-c phenotype (97.4% dauer larvae, n=312 for daf-25(m362); daf-6(e1377) at #$! %&', indicating that DAF-25 acts downstream of DAF-6, and that environmental (ciliary)  input is not required for the Daf-c phenotype. DAF-6/PTCHD3 is expressed in the glial (sheath) cell that forms the amphid sensory channel, allowing contact of the sensory cilia !  ""!  to the environment through pores in the cuticle (Perens and Shaham, 2005). 8-bromocGMP rescues the dauer phenotype of daf-25 (0% dauer larva formation for daf25(m362) on 8-bromo-cGMP, n = 72 compared to 32% dauer larva formation on the control, n = 65, both at 20 °C), similar to that previously reported for daf-11 (Birnby et al., 2000) indicating that DAF-25 functions upstream of the cGMP pathway in the cilia. Indeed the Daf-c phenotype of daf-25(m362) is very similar to that of daf-11(m84) at all temperatures tested (Table S1). The epistasis results are also similar to those for daf-11, indicating that both genes function at the same point in the genetic pathway—upstream of cilia formation and cGMP signaling in the cilia, and downstream of environmental input.  2.2.2 daf-25 mutants exhibit chemosensory phenotypes independent of ciliary ultrastructure defects daf-25 mutants are temperature-sensitive Daf-c and defective in dauer recovery. They constitutively form virtually 100% dauer larvae at 25°C, which do not recover upon transfer to 15°C. The Daf-c phenotype is rescued by maternally contributed daf-25 as seen in the progeny of daf-25(m362) heterozygous hermaphrodites which form zero percent dauer larvae at 25 °C (n>200). Moreover, daf-25 animals exhibit defective responses to various chemosensory stimuli as well as a moderate defect in response to osmotic stress (37 of 45 daf-25(m362) adults crossed the sucrose hyperosmotic boundary compared to 1 of 45 for N2, !2-p-value = <0.00001, while 0 of 30 daf-25(m362) and N2 adults crossed a glycerol boundary). Adults are also defective in egg laying. Despite the fact that Daf-c genes in the IIS pathway (like daf-2 and age-1) extend adult lifespan  !  "#!  (Larsen et al., 1995), daf-25 mutants show no significant difference in lifespan from N2 (Figure S1). daf-25 mutants are defective in chemotaxis to at least four volatile odorants (Figure 2.1). Wild-type N2 adults were attracted to the compounds tested, the chemotaxis-defective mutant daf-11 was partially attracted, whereas the two daf-25  Figure 2.1 - daf-25 mutants have chemosensory defects. We assayed the ability of two daf-25 mutants to respond to four attractants. The daf-25 behavior was compared with N2 and with daf-11(m47), which is partially chemotaxis defective. Chemotaxis index scores were calculated as the number of adults at the attractant minus the number at the control, divided by the total number of adults. Neither allele of daf-25 responded to any of the attractants, indicating an olfactory defect in daf-25 mutants that is more severe than that of the daf-11 guanylyl cyclase mutant. Benzaldehyde, trimethyl thiazole and isoamyl alcohol are detected by the AWC neurons, and pyrazine by the AWA neurons. Pyrazine: N2 n=66, daf-25(m98) n=78, daf-25(m362) n=123, daf-11(m47) n=83. Benzaldehyde: N2 n=91, daf-25(m98) n=80, daf-25(m362) n=115, daf-11(m47) n=62. Isoamyl alchohol: N2 n=66, daf-25(m98) n=78, daf-25(m362) n=78, daf-11(m47) n=83. Trimethyl thiazole: N2 n=91, daf-25(m98) n=69, daf-25(m362) n=74, daf-11(m47) n=93. !  "#!  mutants tested were nearly unresponsive (Figure 2.1). DAF-11 and the cGMP pathway are known to regulate responses to the AWC neuron-mediated odors isoamyl alcohol, trimethyl thiazole and benzaldehyde, and our results indicate that DAF-25 is also required in this pathway (Vowels and Thomas, 1994). The AWA-detected scent, pyrazine, is not reported to be detected by the cGMP pathway, suggesting that DAF-25 participates in another signaling pathway in AWA neurons. Interestingly, although it has been shown that the cGMP pathway does not participate in AWA-mediated olfaction, the particular tested allele daf-11(m47) was previously shown to have reduced affinity for pyrazine (Vowels and Thomas, 1994), as we have seen here. To establish if the olfactory phenotypes are associated with ciliary defects, mixedstage populations of daf-25 mutants and N2 were stained with the lipophillic dye, DiI. Mutants with cilia structure anomalies have abrogated dye filling of the olfactory neurons (Starich et al., 1995), whereas daf-25 mutants take up the dye normally at all ages, suggesting that they have structurally intact cilia (Figure S2). To confirm this possibility, we further examined the integrity of ciliary structures by transmission electron microscopy. Ciliary ultrastructures in two daf-25(m362) L2 larvae—including transition zones, middle segments consisting of doublet microtubules, and distal segments composed of singlet microtubules—were indistinguishable from the two N2 controls (Figure S3). We conclude that daf-25 animals have no obvious defects in ciliogenesis or cilia ultrastructure.  !  "#!  2.2.3 Molecular identification of daf-25 To identify the daf-25 genetic locus, we first used three-factor genetic crosses to map the m362 allele to the left arm of Chromosome I. Then, we employed a modified SNP mapping procedure (Wicks et al., 2001), in which we selected for recombinants in the unc-11-daf-25 interval to map daf-25 to the left-most 1 Mbp of Chromosome I. Finally, we used a custom-made high-density array for the left-most ~2.5 Mbp for comparative genomic hybridization (CGH). Two molecular lesions in daf-25(m362) were identified in exon 4 of Y48G1A.3 (Figure 2.2), including a 31 bp deletion and a G>A  Figure 2.2 - daf-25 alleles encode the ortholog of mammalian Ankmy2. We identified the daf-25 gene using three-factor genetic crosses and SNP mapping followed by ArrayCGH. The four alleles of daf-25 include two EMS-induced deletions m98 (996 bp deletion at I:332481-333477) and m362 (31 bp deletion at I:335814-335844 which results in a premature stop 14 codons downstream), a transposon (Tc1) insertion at I:330927 (m488) and an EMS-induced ochre nonsense mutation m137 (at I:336013). DAF-25 is well conserved and has been named Ankmy2 in mammals for its three ankyrin repeats and MYND-type zinc finger domain.  !  "#!  change 72 bp to the right of the deletion. Subsequent sequencing of PCR products from mutant genomic DNA uncovered the lesions in the remaining alleles. The m98 mutant has a 996 bp deletion that removes the first two exons, m137 has an ochre stop in the fourth exon, and m488 has a Tc1 transposon insertion in the third exon (Figure 2.2). Y48G1A.3 encodes the C. elegans ortholog of mammalian Ankmy2 (by reciprocal BLAST), a protein with three ankyrin repeats and a MYND-type zinc finger domain. The C. elegans protein shares throughout its length (388 residues) 52% similarity and 32% identity with mouse Ankmy2 (440 residues). The C. elegans ankyrin repeat domain is 40% identical and the MYND domain is 55% identical to the murine ortholog. Ankmy2 is very well conserved among chordates, with identity percentages compared to human Ankmy2 of 99% for macaque, 93% for cow, 88% for mouse, and 76% for zebrafish (Figure S4). Although the protein is highly conserved, there is no reported functional data for this gene from any organism. The MYND domain is thought to function in protein-protein interactions, although only a small number of MYND domain-containing proteins have been characterized, including the AML1/ETO protein, which binds SMRT/N-CoR through its MYND domain (Liu et al., 2007). To analyze the transcript(s) generated by the daf-25 gene, we employed a PCRbased approach. Using primers for the SL1 transplice sequence or poly-T in combination with gene-specific primers, we were able to amplify only one isoform (Figure S5). This result is consistent with the RNA-Seq and trans-splice data found on Wormbase, which shows a daf-25 transcript sequence identical to that presented in Figure S5, including the 5’ and 3’ UTRs (Hillier et al., 2009; Shin et al., 2008; Rogers et al., 2008). We were  !  "#!  unable to amplify an SL2 trans-spliced product using multiple gene specific primers and an SL2 primer under any conditions tested.  2.2.4 DAF-25 functions in cilia To determine the sub-cellular localization of DAF-25, a GFP-tagged protein was constructed. The daf-25 upstream promoter (approx. 2.0 kb 5’ of the ATG) was fused to the daf-25 cDNA in frame into the pPD95.77 vector (gift from Dr. Andrew Fire) containing GFP (without a nuclear localization signal) and the unc-54 3’UTR. This construct was found to be expressed in many ciliated sensory neurons, including the following pairs of anterior neurons: AFD, ASK, ASI, ASH, ASJ, ASG, ASE, ADF, AWA, AWB, AWC and IL2 (Figure 2.3). It is also expressed in the PQR ciliated neuron and one ventral interneuron. We also show expression of the DAF-25::GFP construct in the 7A ciliated neuron in the male tail though we did not fully examine male expression due to the limited number examined and the mosaic expression associated with extrachromosomal arrays. Most importantly, the fluorescence of the GFP-tagged protein was localized to the cilia of all these cells. The GFP-fusion construct was judged to be functional because it fully rescued the Daf-c phenotype of daf-25(m362) at 25 °C while non-transgenic siblings arrested as dauers (n>200).  !  "#!  Figure 2.3 - daf-25 is expressed in many ciliated cells and encodes a novel ciliary protein. A reporter construct joined the 2.0 kb promoter region 5’ of the AUG for daf-25 to the daf-25 cDNA with the C-terminal GFP coding sequence. Expression is seen in many anterior chemosensory neurons in (A) including AFD, ASK, ASI, ASH, ASJ, ASG, ASE, ADF, AWA, AWB, AWC and IL2. There is a strong DAF-25::GFP signal localized in the sensory cilia (B). Expression of DAF-25::GFP is shown in the PQR neuron (C) and in the male tale neuron 7A (D). To investigate whether the ciliary localization of DAF-25 might depend on the intraflagellar transport (IFT) machinery, the DAF-25::GFP construct was crossed into che-11, which is required for retrograde transport in the cilia. In che-11 mutants, IFTassociated proteins accumulate in the cilia (Blacque et al., 2006). The DAF-25::GFP translational fusion protein accumulated within the cilia and basal body (base of cilia) despite a reduction in total GFP fluorescence (mean DAF-25::GFP fluorescence in che11 (8.7E12) compared to N2 (1.4E13), p<0.00001, n=9 for both), suggesting that the protein is associated with IFT (Figure 2.4). To test for a possible role for DAF-25 in the !  "#!  core IFT complex, GFP translational fusion constructs of two IFT proteins, CHE-2 and CHE-11 [30], were crossed into the daf-25(m362) mutant background and analyzed by time-lapse microscopy. The velocities of IFT transport of CHE-2 and CHE-11, as determined by kymograph analysis, were unchanged in daf-25 compared to that of wild type animals (Figure 2.4). Specifically, transport velocities in the middle segment were ~0.7 !m/s, and in the distal segments ~1.2 !m/s, exactly as reported for all studied IFT proteins (Ou et al., 2007). Collectively, our data show that DAF-25 is not essential for IFT, and is therefore unlikely to be a core component of IFT transport particles— consistent with the findings that the ciliary ultrastructure of the daf-25 mutant is intact (Figure S3). However, its accumulation within cilia in the retrograde IFT mutant does suggest that it is associated with (i.e., transported by) the IFT machinery.  Figure 2.4 - DAF-25 depends on IFT for proper localization within cilia but is not essential for the IFT process. The DAF-25::GFP translational fusion was crossed into che-11(e1810) and assayed for protein accumulation. As seen in (A), DAF-25::GFP localized normally to the cilia in the N2 background, but in the che-11 background DAF25::GFP accumulates in the cilia, indicating that when IFT is disrupted DAF-25 localization is also disrupted. We conclude that DAF-25 requires the IFT complex for proper transport and/or localization within cilia. Translational fusion reporters for CHE2::GFP and CHE-11::GFP were crossed into daf-25(m362). As reported previously (Sengupta et al., 1996) both reporters localize to basal bodies and ciliary axonemes (B), and have normal velocities in both N2 and daf-25 mutants, as measured in the kymographs (C). Slopes in kymographs correlate with IFT complex speeds and were created as described previously (Blacque et al., 2006). In daf-25(m362) mutants there is no change in localization (B) or velocity (C) for either of the two reporters, indicating that DAF-25 is not required for normal rates of IFT transport, and is probably not a core IFT protein. cil = cilia, den = dendrite, TZ/BB = transition zone/basal body, asterisk indicates DAF-25::GFP accumulation, DS = distal segment, and MS = middle segment.  !  "#!  2.2.5 DAF-25 is required for DAF-11 localization to cilia The phenotype of daf-25 is most similar to that of daf-11, and our epistasis results placed daf-25 at the same position in the genetic pathway previously reported for daf-11 (Thomas et al., 1993). To test for possible functional interactions, a strain harboring DAF-11::GFP (gift from Dr. James Thomas), which is known to localize to cilia (Birnby et al., 2000), was crossed with two daf-25 mutants (m98 and m362). In wild-type !  "#!  animals, the DAF-11::GFP protein localized to the sensory cilia of the olfactory neuron pairs ASI, ASJ, ASK, AWB and AWC (Figure 2.5A), all of which express DAF-25::GFP (Figure 2.3). In both daf-25 mutants, the DAF-11::GFP protein was observed only in a region near the base of cilia, rather than along their length (Figure 2.5B). To assess more precisely where the DAF-11::GFP protein is mislocalized, we introduced into the same strain a ciliary (IFT) marker, namely tdTomato-tagged XBX-1 (a gift from Dr. B. Yoder), which localizes at basal bodies and along the ciliary axoneme (Schafer et al., 2003). Visualization of the two fluorescently-tagged proteins in the daf-25 mutant revealed that DAF-11::GFP accumulates at the very distal end of dendrites, with little or no localization to the basal body-ciliary structures (Figure 2.5E). This indicates that DAF-25 is required for the proper localization of DAF-11 to the cilia, providing a likely explanation for the similarities between the daf-11 and daf-25 mutant phenotypes. To test if the DAF-25-DAF-11 functional interaction is specific, GFP-tagged ciliary channel proteins (OSM-9/TRPV4 and TAX-4/CNGA1) and IFT-associated proteins (CHE2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8/TTC8, OSM-5/IFT88 and XBX1/D2LIC) were also crossed into the daf-25(m362) mutant background. All eight reporters showed normal localization to the olfactory cilia in the wild-type N2 and daf25(m362) strains, indicating the possible specificity of DAF-25 for guanylyl cyclases (OSM-9::GFP localization in the daf-25 mutant shown in Figure 2.5C,D; the remaining constructs are presented in Figure S6). The mislocalization of DAF-11::GFP in daf25(m362) was not suppressed by daf-12(sa204) (Figure S7). This indicates that it is the abrogation of DAF-25 rather than entry into dauer that controls the ciliary localization of DAF-11. !  "#!  !  ""!  Figure 2.5 - DAF-25 is required for the localization of a guanylyl cyclase (DAF-11) to cilia. The guanylyl cyclase DAF-11::GFP translational fusion protein was expressed in both N2 and daf-25(m362) genetic backgrounds. In wild type (A), DAF-11::GFP was localized to the ASI, ASJ or ASK sensory cilia, but was limited to the distal end of the dendrites (indicated by arrow) and largely excluded from basal body-ciliary structures in daf-25(m362) cilia (B). Normal ciliary localization was seen for the transient receptor potential channel (TRPV4) OSM-9::GFP reporter gene in both wild type (C) and daf25(m362) (D). Also, no change in localization was seen for TAX-4/CNGA1, CHE2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8/TTC8, OSM-5/IFT88 and XBX1/D2LIC in daf-25 mutants (Figure S6). In (E), DAF-11::GFP and XBX-1::tdTomato are co-expressed in the amphid cilia in daf-25(m362) mutants. XBX-1::tdTomato localizes to the basal body (indicated by arrowhead) and cilia while DAF-11::GFP localizes to the distal end of the dendrite (indicated by arrow). No overlap in protein localization is observed indicating that DAF-11::GFP shows very little, if any localization to the basal body and no expression in the cilia. XBX-1::tdTomato is expressed in all of the amphid cilia while DAF-11::GFP is expressed in a subset. The GFP reporter results suggest a potentially specific function for DAF-25 in cilia. This finding is consistent with the reported regulation of daf-25 by the ciliogenic DAF-19 RFX-type transcription factor (Blacque et al., 2005). Taken together, DAF-25 appears to be an adaptor protein required for the transport or tethering of the guanylyl cyclase DAF-11 within sensory cilia.  2.2.6 Conservation of function for DAF-25/Ankmy2 To ascertain if a functional association between DAF-25/Ankmy2 and guanylyl cyclase is evolutionarily conserved, we used a pull-down experiment to test whether mouse Ankmy2 interacts with the retinal-specific guanylyl cyclase GC1, a mammalian homolog of DAF-11 present within ciliary photoreceptors. We amplified Ankmy2 cDNA from a mouse retinal cDNA preparation (gift from Simon Kaja), and constructed a cDNA clone with the rhodopsin 1D4 epitope to use for co-IP experiments with anti-1D4 monoclonal antibody (Wong et al., 2009). We co-expressed both in HEK293 cells to test !  "#!  for GC1 co-immunoprecipitation with the 1D4 epitope-tagged Ankmy2 (HEK293 cells do not express rhodopsin). Pull-down of Ankmy2 co-precipitated GC1, but not another control protein (retinal membrane protein ABCA4; Figure 2.6). This indicates that the functional interaction between DAF-25/Ankmy2 and guanylyl cyclase observed in ciliated sensory cells may be conserved between mouse and worm.  2.3 Discussion In this study, we have identified in a genetic screen for Dauer formation mutants a novel MYND domain-containing ciliary protein, DAF-25, that is required for the proper localization of a guanylyl cyclase (DAF-11) to sensory cilia. Disruption of DAF-25 does not interfere with intraflagellar transport (IFT) or ciliary ultrastructure, but the protein accumulates in a che-11 retrograde IFT mutant. We therefore propose that DAF-25 is associated with IFT not as a ‘core’ protein but instead as an adaptor for transporting ciliary cargo. In our model, abrogation of DAF-25 would thereby not allow transport of DAF-11, which explains the improper localization of DAF-11 in daf-25 mutants at the very base of cilia and the similarity in phenotype between daf-11 and daf-25 mutants. The amino acid sequence and domain structure similarity between DAF-25 and Ankmy2 suggests an important function for the latter mammalian protein that may be similar to DAF-25 in C. elegans. We attempted to co-immunoprecipitate DAF-25 and DAF-11 in C. elegans but were unable to satisfactorily remove a sufficient amount of background proteins to avoid confounding any identified interaction (data not shown). We also showed that the retinal guanylyl cyclase GC1 binds to Ankmy2, and we propose  !  "#!  Figure 2.6 - Ankmy2 and GC1 can be co-precipitated in HEK293 cells. In (A), detergent-solubilized extracts of HEK293 cells co-expressing Ankmy2-1D4 and either GC1 of ABCA4 were immununoprecipitated on a Rho 1D4-Sepharose matrix and the bound protein was analyzed on Western blots labeled with Rho 1D4 for detection of Ankmy2 and antibodies to GC1 or ABCA4 to detect co-precipitating proteins. Precipitation of 1D4-tagged Ankmy2 with 1D4 antibody also pulls down GC1 (retinal guanylyl cyclase), but not ABCA4 (retinal expressed ATP-binding Cassette, sub-family A, member 4). This indicates that GC1 forms a protein complex with Ankmy2, implying conservation of the functional interaction between DAF-11 and DAF-25. Lane 1 indicates the input proteins (whole cell lysate) and lane 2 indicates elution from immunoaffinity matrix. In (B) detergent-solubilized extracts of HEK293 cells expressing only Ankmy21D4 or GC1 were immunoprecipitated on a Rho 1D4 immunoaffinity matrix and analyzed on Western blots labeled with an anti-GC1 antibody or Rho 1D4 antibody. Lane 1: Input; lane 2: bound protein. The presence of Ankmy2-1D4 but not GC1 in the bound fractions indicates that GC1 does not nonspecifically interact with the Rho 1D4 immunoaffinity matrix. In (C), HEK293 cells co-expressing Ankmy2-1D4 and GC1 were co-immunoprecipitated on a Rho 1D4 immunoaffinity matrix in the absence or the presence of excess competing 1D4 peptide. Both Ankmy2-1D4 and GC1 bound in the absence of peptide. In the presence of the 1D4 peptide less than 10% of the Ankmy2 bound.  !  "#!  that the functional relationship between DAF-25 and DAF-11 is conserved between Ankmy2 and GC1 in ciliated photoreceptor cells. Indeed, Ankmy2 may be required for the transport of not only GC1 but perhaps other cilia-targeted guanylyl cyclases as well as other cilia-targeted proteins in mammals. Further studies will be required to experimentally confirm whether Ankmy2 is required for transport of GC1 to the rod outer segment, and to test if Ankmy2 lesions result in retinal disease or a ciliopathy syndrome that includes retinopathies. Mutations in ciliogenesis and cilia related genes cause human disease phenotypes including Bardet-Biedl syndrome, retinopathies, obesity, situs inversus and polycystic kidney disease, among others (Sharma et al., 2008; Lancaster and Gleeson, 2009). Interestingly, GC1 and the nuclear hormone receptor Nr2e3 shown to regulate Ankmy2 expression in mouse retina both harbor mutations in patients with retinal disease (Kitiratschky et al., 2008; Haider et al., 2009). While this research was being conducted we became aware of another group that cloned and characterized chb-3 (Y48G1A.3/daf-25/Ankmy2) as a suppressor of the che-2 body size phenotype (Fujiwara et al., 2002). Fujiwara et al., (in press) describe the cloning of chb-3/daf-25 and its essential role in GCY-12 cilia localization. They show that DAF-25 is required in a subset of sensory neurons to rescue the phenotypes they assayed (dauer formation and body size) using a tax-4 promoter. This indicates that DAF25 function is required in the neurons where cGMP signaling takes place (TAX-4 is a subunit of cGMP-gated calcium channel). They also show expression of DAF-25 in the ASJ neurons (one pair of neurons where DAF-11 is expressed) is required for rescue of the dauer phenotype, also indicating a cell autonomous role for DAF-25. It is interesting that screens for the Daf-c and Chb (che-2 body size suppressor) phenotypes both resulted !  "#!  in the identification of daf-25/chb-3 and separately identified its apparent ciliary cargos daf-11 and gcy-12, guanylyl cyclases that specifically work in dauer formation and body size, respectively. This indicates that DAF-25/CHB-3/Ankmy2 may interact with ciliatargeted guanylyl cyclases in a general manner and that much of the phenotype of daf25/chb-3 mutants reflects a global defect in cGMP signaling, potentially along with other unidentified cargo proteins. In conclusion, our findings uncover a novel ciliary protein that plays an important role in modulating the localization/function of cGMP signaling components, which are known to play a critical role in the function of ciliary photoreceptors (Wensel, 2008). DAF-25/Ankmy2 may also play a role in the ciliary targeting of other as of yet identified proteins. As such, Ankmy2 could participate in phototransduction and be associated with retinopathies, and more generally, could be implicated in other ciliary diseases (ciliopathies).  2.4 Methods 2.4.1 Mapping, epistasis and phenotyping daf-25 daf-25 mutations were created by treatment of N2 with 0.25 M EMS, or by mut-2 transposon mobility, and selection for constitutive dauer formation as previously described (Kiff et al., 1988). For 3-factor mapping, fog-1(e2121) unc-11(e47) was crossed with daf-25(m362) and daf-25(m362) unc-35(e259) was crossed with dpy-5(e61). Scoring the genotypes of the F2 progeny required the phenotyping of F3 progeny (due to the maternal effect of the daf-25 dauer phenotype). Pooled SNP mapping was completed as previously described (Wicks et al., 2001) with some changes. In the Po generation, !  "#!  CB4856 males were crossed to daf-25;unc-11 double mutant hermaphrodites. The F1 males were crossed with CB4856 hermaphrodites. F2 hermaphrodites were selected by absence of Unc progeny. F3 hermaphrodites were placed one to a plate and were selected into wild type or mutant pools based on absence or presence of dauers in the F4. Wild type and mutant pools of F3 hermaphrodites were subject to SNP analysis as previously described (Wicks et al., 2001). ArrayCGH was done as previously described (Maydan et al., 2009) for the leftmost 2.4 Mbp of Chromosome I with 50 base probes spaced every four base pairs. Epistasis analysis was performed by crossing daf-25(m362) into daf-12(m20), daf-16(m26), daf-3(mgDf90), daf-10(e1387) and daf-6(e1377). Once the double mutants were isolated, the dauer phenotype was assayed to determine if daf-25 was suppressed fully (no constitutive dauer larvae formed at 25 °C), partially (fewer dauer larvae than daf-25(m362) control) or no suppression. Treatment with cGMP was performed as previously described (Birnby et al., 2000) with 5 mM 8-bromo-cGMP (Sigma). Neuronal dye-filling was assayed by incubating a mixed-stage population of each genotype in Vibrant DiI (Molecular Probes) 1000-fold diluted in M9 buffer for one hour followed by washing in M9 and one hour destaining on plates. Chemotaxis assays were performed with synchronized day-1 adults as previously described with the volatile attractants trimethyl-thiazole, pyrazine, benzaldehye and isoamyl alcohol (Saeki et al., 2001). The DAF-25::GFP construct was created by inserting the 2.0 kb promoter region 5’ of the AUG followed by daf-25 cDNA the into the pPD95.77 vector (gift from Dr. Andrew Fire). After microinjection into N2 adults (Evans, 2006) with 10 $%&'(!of pRF4 (contains rol-6(su1006)), and 90 ug/ml of DAF-25::GFP plasmid (described above), !  "#!  transgenics lines were established based on the roller phenotype. The extra-chromosomal array mEX179(pdaf-25::DAF-25::GFP, rol-6(su1006)) was crossed into daf-25(m362) and rescue of the Daf-c phenotype was detected by normal non-dauer development in the F3 progeny grown at 25°C. GFP fluorescence was visualized on a Zeiss Axioskop with a Qimaging Retiga 2000R camera.  2.4.2 Intraflagellar transport and ciliary protein localization analyses To measure the integrity of IFT within the daf-25(m362) mutant, kymograph analyses were performed using GFP-tagged CHE-11 and CHE-2 IFT markers. Timelapse movies were obtained for the different strains, including N2, and kymographs were generated from the resulting stacked tiff images using Metamorph software (Universal Imaging, West Chester, PA). Rates of fluorescent IFT particle motility along middle and distal segments were measured as described previously (Blacque et al., 2006; Snow et al., 2004). To assess how disrupting IFT affects the ciliary localization of DAF-25::GFP, mEX179 was crossed into che-11 mutants and visualized by microscopy essentially as described (Blacque et al., 2006). Fluorescence intensity was measured by analyzing images in ImageJ by highlighting the entire head region for each animal, then measuring pixel density minus the pixel density for an equal sized adjacent region. The localization of several GFP–tagged proteins in daf-25(m362) animals, namely DAF-11, OSM-9, TAX-4, CHE-2, CHE-11, CHE-13, BBS-8, OSM-5 and XBX-1, were ascertained by crossing the reporter into the mutant, followed by visualization using standard microscopy. Co-localization was carried out by injecting the osm-5p::XBX-1::tdTomato  !  "#!  into daf-25(m362);daf-12(sa204) and crossing it into TJ9386 which carries the DAF11::GFP reporter (Birnby et al., 2000).  2.4.3 Electron microscopy Staged N2 and daf-25 L2 larvae were produced by harvesting eggs from gravid adults by alkaline hypochlorite treatment, followed by overnight hatching in M9 buffer, and subsequent incubation of hatched L1 larvae on seeded NGM plates for 26 hours at 16$C. Worms were then washed directly into a primary fixative of 2.5% glutaraldehyde in 0.1 M Sorensen phosphate buffer. To facilitate rapid ingress of fixative, worms were cut in half using a razor blade under a dissecting microscope, transferred to 1.5 ml Ependorf tubes and fixed for one hour at room temperature. Samples were then centrifuged at 3,000 rpm for two minutes, the supernatant removed and the pellet washed for ten minutes in 0.1M Sorensen phosphate buffer. The worms were then post-fixed in 1% osmium tetroxide in 0.1 M Sorensen phosphate buffer for one hour at room temperature. Following washing in Sorensen phosphate buffer, specimens were processed for electron microscopy by standard methods. Briefly, they were dehydrated in ascending grades of alcohol to 100%, infiltrated with Epon and placed in aluminum planchetes orientated in a longitudinal aspect and polymerized at 60$C for 24 hours. Using a Leica UC6 ultramicrotome individual worms were sectioned in cross section from anterior tip, at 1µm until the area of interest was located as judged by examining the sections stained with toluidine blue by light microscopy. Thereafter, serial ultra-thin sections of 80nm were taken for electron microscopical examination. These !  "#!  were picked up onto 100 mesh copper grids and stained with uranyl acetate and lead citrate. Using a Tecnai Twin (FEI) electron microscope, sections were examined to locate, in the first instance, the most distal (anterior) region of the cilia, then to the more proximal regions of the ciliary apparatus. At each strategic point, distal segment, middle segment and transition zone/fiber regions were tilted using the Compustage of the Tecnai to ensure that the axonemal microtubules were imaged in an exact geometrical normalcy to the imaging system. All images were recorded, at an accelerating voltage (120kV) and objective aperture of 10µm, using a MegaView 3 digital recording system.  2.4.4 Co-expression and co-immunoprecipitation of Ankmy2 and Guanylate Cyclase 1 (GC1) Mouse ankmy2 cDNA, amplified from retinal RNA, was engineered to contain a sequence encoding a 9 amino acid 1D4 C-terminal epitope as previously described (Wong et al., 2009). Ankmy2-1D4 and either human GC1 or the retinal ABC transporter ABCA4 as a control were co-expressed in HEK 293 cell. HEK 293 cell extracts were solubilized in 18mM CHAPS in TBS (20mM Tris, 150mM NaCl, 1mM EDTA, 1mM MgCl2 and Complete inhibitor). 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Mutations affecting the chemosensory neurons of Caenorhabditis elegans. Genetics 139, 171-188. Swoboda, P., Adler, H. T., and Thomas, J. H. (2000). The RFX-type transcription factor DAF-19 regulates sensory neuron cilium formation in C. elegans. Molecular cell 5, 411-21. Thomas, J. H., Birnby, D. A., and Vowels, J. J. (1993). Evidence for parallel processing of sensory information controlling dauer formation in Caenorhabditis elegans. Genetics 134, 1105-17. Vowels, J. J., and Thomas, J. H. (1994). Multiple chemosensory defects in daf-11 and daf-21 mutants of Caenorhabditis elegans. Genetics 138, 303-316. Ward, A., Liu, J., Feng, Z., and Xu, X. Z. S. (2008). Light-sensitive neurons and channels mediate phototaxis in C. elegans. Nat. Neurosci 11, 916-922. Wensel, T. G. (2008). Signal transducing membrane complexes of photoreceptor outer segments. Vision Res 48, 2052-2061. Wicks, S. R., Yeh, R. T., Gish, W. R., Waterston, R. H., and Plasterk, R. H. (2001). Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map. Nat. Genet 28, 160-164. Wong, J. P., Reboul, E., Molday, R. S., and Kast, J. (2009). A Carboxy-Terminal Affinity !  "#!  Tag for the Purification and Mass Spectrometric Characterization of Integral Membrane Proteins. Journal of Proteome Research 8, 2388-2396. Zwaal, R. R., Mendel, J. E., Sternberg, P. W., and Plasterk, R. (1997). Two Neuronal G Proteins are Involved in Chemosensation of the Caenorhabditis elegans DauerInducing Pheromone. Genetics 145, 715-727.  !  "#!  CHAPTER 31: Caenorhabditis elegans SDF-9 enhances insulin/insulin-like signaling through interaction with DAF-2  3.1 Introduction In an environment favorable for reproduction, C. elegans develops directly to the adult through four larval stages (L1-L4). Under conditions of overcrowding, limited food or high temperature, larvae arrest development at the second molt to form dauer larvae (Cassada and Russell 1975). Dauer larvae can remain in diapause for months, essentially not aging, until conditions improve (Klass and Hirsh 1976). The signaling pathways that control the developmental switch have been revealed by studying two broad classes of mutants, those that block dauer formation (Daf-d, dauer formation defective) and those that result in constitutive dauer or dauer-like arrest (Daf-c), even when conditions are favorable (reviewed by Riddle and Albert 1997). The daf genes encode elements of pathways conserved in humans, including Insulin/IGF-1 (IIS), TGF-! (Transforming Growth Factor), TOR (Target Of Rapamycin) and guanylate cyclase / G protein mediated pathways (reviewed by Hafen 2004; Levy and Hill 2006). In addition to the Daf-c phenotype, mutants with reduced IIS signaling also display an Age (adult life span extension) phenotype (Kenyon et al. 1993; Larsen et al. 1995). Higher growth temperatures favor dauer formation, as observed in wild-type strains (C. elegans N2 and wild-type C. briggsae) exposed to exogenous dauer  1  A version of this chapter has been published, see: Jensen VL, Albert PS and Riddle DL (2007) Caenorhabditis elegans SDF-9 enhances insulin/insulin-like signaling through interaction with DAF-2. Genetics 177; 661-666. 71  pheromone (Golden and Riddle 1984a; Jeong et al. 2005; Butcher et al. 2007), and in hypomorphic Daf-c mutants, which convey increased pheromone sensitivity (Golden and Riddle 1984b). The concentration of dauer pheromone indicates population density. Among the Daf-c mutants, null mutants of TGF-! pathway genes convey a temperaturesensitive (ts) phenotype, whereas severe daf-2 or age-1 mutants (IIS pathway) arrest at the dauer stage non-conditionally (Larsen et al. 1995; Ren et al. 1996). The IIS pathway is essential for larval maturation beyond the dauer stage, whereas the TGF-! pathway is essential only at higher temperatures so long as the IIS pathway is intact. Most Daf-c alleles are temperature sensitive, not as a result of a thermolabile protein product but because temperature is an input into dauer formation (Golden and Riddle 1984a). One truly temperature-sensitive allele has been described, daf-2(m41), which is wild-type at 15°, but severe at 25° (Gems et al. 1998). This is unlike other daf-2 alleles, which have partially penetrant Daf-c phenotypes at 15º that correlate with their severity at 20º, indicating they are hypomorphic. Forward mutagenesis screens have been useful, not only for understanding dauer biology, but because the cloning of Daf-c and Daf-d genes has identified novel members of these conserved pathways (Georgi et al. 1990; Ren et al. 1996; Patterson et al. 1997; Ogg et al. 1997; Paradis and Ruvkun 1998; da Graca et al. 2004). There are likely to be more genes involved that modulate or coordinate these pathways (Tewari et al. 2004). There is also value in creating new alleles for genes already known to be involved in the dauer pathways. Transposon insertion mutants allow isolation of knockout mutations or in situ knockin alterations to the gene (Plasterk and Groenen 1992; Barrett et al. 2004). In the mut-2 (mutator) genetic background, mobilized transposons of the Tc family insert  72  into or near genes preferentially at consensus sites (Collins et al. 1987). We used mut-2 in a screen to identify novel Daf-c mutants and to obtain transposon insertion alleles for genes already known (Caldicott 1995).  3.2 Results and methods 3.2.1 Isolation and mapping of m708 One non-conditional Daf-c mutant from the mut-2 screen failed to complement daf-2(e1370). Upon subsequent backcrossing it was determined to contain not only a ts allele of daf-2, m637, but also an independently segregating daf-2 enhancer, m708. The m708 homozygous single mutant exhibited only a weak Egl (egg laying) phenotype, and no evidence of Daf-c, Hid (Daf-c at 27°), Dyf (neuronal dye-filling) or Age phenotypes previously associated with Daf mutants (Malone and Thomas 1994; Larsen et al. 1995; Ailion and Thomas 2000). Three-factor mapping placed m708 on Chromosome V to the right of unc-51. Polymerase Chain Reaction (PCR) amplification of six candidate genes using genespecific primers revealed that one gene, sdf-9 (synthetic dauer formation), had a 1.2 kb transposon insertion in exon 4. Based on dauer formation on NGM agar plates without cholesterol (Ohkura et al. 2003), m708 failed to complement sdf-9(ut163). The insert is contained between C515 and T516 of the coding sequence, and is nine bases to the left of the lesion in ut163 and nine bases to the right of the lesion in the ut169 and ut174 alleles (Ohkura et al. 2003). The m708 open reading frame ends with an amber stop 24 codons into the insert. No sdf-9 mRNAs were detected in either of two sdf-9(m708) RNA  73  preparations, but we cannot eliminate the possibility that an altered gene product may be produced.  3.2.2 Cemar1 transposition Unexpectedly, sdf-9(m708) did not contain a transposon normally mobilized by mut-2 (i.e., Tc1 or Tc5). Instead, it contained Cemar1 (C. elegans Mariner 1), found in 66 copies in the haploid genome of the N2 strain, but not previously reported to be mobile (Witherspoon and Robertson 2003). Cemar1 was originally identified by the repeat identifying program RECON (Bao and Eddy 2002), and is currently listed by WormBase as Ce000178 (release WS172, 2007, http://www.wormbase.org/). The sequence of the inserted element differed from the consensus by an 11 base pair deletion and a T > C mutation. The only copy of Cemar1 with the corresponding sequence is located on Chromosome V, 10 Mbp to the left of sdf-9 in an intron of the gene D1054.5, indicating that the transposon inserted in sdf-9(m708) originated from this copy. The parental mut-2 strain used for the mutant screen contained no transposon in sdf-9, whereas the original isolate of sdf-9(m708) still contained the parent transposon in D1054.5 as well as the insert in sdf-9(m708). Most copies of Cemar1 encode a functional transposase (Witherspoon and Robertson 2003), the expression of which has been observed in at least two expression studies (Kim et al., 2001, Murphy et al., 2003). We conclude that Cemar1 was mobilized in the mut-2 background, and other uncharacterized mutants isolated in this background may bear insertions of Cemar1 rather than members of the Tc family.  74  TABLE 3.1 - Interaction between sdf-9 and daf-2 15° 20° Genotype Daf-c N Daf-c daf-2(m41) 0 129 10 ± 5 sdf-9(m708) 0 300 0 m41; m708 0 72 5±6 sdf-9(ut163) 0 258 0 m41; ut163 0 340 4±2  N 365 370 133 667 339  22° Daf-c 33 ± 3 0 30 ± 5 0 31 ± 6  23° N 393 280 332 321 395  Daf-c 100 100 -  N >500a >500a -  25° Daf-c 100 0 100 17 ± 2 100  N 131 140 96 110 126  daf-2(e1368) e1368; m708  -  -  1±1 60 ± 17  195 424  6±1 89 ± 6  388 340  8±3 100  250 220  -  -  daf-2(e1365) e1365; m708  -  -  1 ± .3 14 ± 4  381 280  4±2 53 ± 15  544 131  16 ± 8 100  1277 1120  -  -  daf-2(e1370) e1370; m708 e1370; ut163  0 92 ± 3 0  683 628 766  3 ± .4 100 50 ± 4  1131 >500a 959  -  -  -  -  -  -  daf-2(e979) 10b 30 e979; m708 100b 30 Values are percent dauer larvae ± standard error, including dauer and dauer-like larvae counted on the first day of adulthood for nondauer siblings. Populations were synchronized by hatching alkaline hypochlorite treated embryos in M9 buffer at room temperature for 24 hours, then transferring them to NGM plates with E. coli OP50. N indicates population size. Alleles are listed in order of severity except m41, which is listed first because of its unique phenotype. Strains were genotyped as follows: PCR for sdf-9(m708), sequencing for daf-2(m41) and sdf-9(ut163) and by phenotype for all other daf-2 alleles. a Based on visual inspection of multiple samples (>500 animals) with no larvae growing past dauer. b Includes genotyped progeny from one daf-2(e979)/daf-2(e979); sdf-9(m708)/+ including 12 dauers, 35 non-recovering dauers (allowed to recover at 15° for three days after cold shock at 4° overnight) and 53 adults (!2 p-value < 0.001).  75 75  3.2.3 Epistatic analysis of sdf-9 and daf-c mutants Since sdf-9(m708) enhanced daf-2(m637), double mutants were created with other representative alleles of daf-2 to determine if the enhancement is allele specific. As was the case with daf-2(m637), the daf-2(e1370); sdf-9(m708) double mutant showed a much stronger Daf-c phenotype compared to daf-2(e1370) (Table 3.1). Double mutants between sdf-9(m708) and the strong ts daf-2 alleles, e979 (Table 3.1) and m637 (data not shown), were non-conditional, forming only non-recovering dauer larvae at 15º. We also tested daf-2(e1365) and daf-2(e1368), two alleles with very weak Daf-c phenotypes, and daf-2(m41), a unique ts mutant exhibiting no Daf.c or Age phenotype at 15°, but stronger Daf-c phenotype at 22.5° than other daf-2 alleles that are Age at 15° (GEMS et al. 1998). At 20°, 22° and 23° m708 strongly enhanced both e1365 and e1368. However, m708 did not enhance m41 at any temperature at which enhancement might be detected (Table 3.1). To investigate possible sdf-9 allele specificity in the interaction with daf-2, we made sdf-9(ut163) double mutants with daf-2(m41) and daf-2(e1370). As was the case with m708, ut163 enhanced daf-2(e1370) dauer formation but not daf-2(m41) (Table 3.1). The ut163 allele appears to be ts because it has a very weak phenotype at 15° (no enhancement of daf-2(e1370)), a moderate phenotype at 20° (weaker enhancement than sdf-9(m708)) and a stronger Daf-c phenotype than m708 at 25° (Table 3.1). Ohkura et al. (2003) reported that the ut163 Daf-c phenotype was the second strongest among the tested sdf-9 alleles at 25° but the weakest allele at 20° on NGM plates lacking cholesterol, also indicating that the ut163 protein may be thermolabile.  76  Since sdf-9 enhanced all of the daf-2 alleles tested except for m41, it could be judged to fall into a parallel dauer formation pathway. sdf-9(m708) also enhanced daf7(e1372) and daf-8(m85), which encode components of the TGF-! pathway (Table 3.2). In fact, double mutants between sdf-9(m708) and the type I and type II TGF-! receptors, daf-1(m40) and daf-4(e1364) respectively, constitutively formed dauer larvae that could not recover at 15º (data not shown). Furthermore, the Daf-c phenotype of daf-11, encoding a transmembrane guanylate cyclase (Birnby et al. 2000), was enhanced by sdf9. daf-11(m47) sdf-9(m708) exhibited strong dauer formation at 15º (Table 3.2), with many dauer larvae unable to resume development. Taken together it appears that SDF-9 may work in parallel to the TGF-! and guanylate cyclase pathways. TABLE 3.2 - Effect of sdf-9 on mutants in other dauer pathways  daf-7(e1372) e1372; m708  15° Daf-ca N 4 ± 2 465 64 ± 3 177  daf-8(m85) m85; m708  1±0 63 ± 6  481 175b  daf-11(m47) 4 ± 2 330 m47 m708 85 ± 6 106 a Percent constitutive dauer formation ± standard error, with populations scored as in Table 3.1. Genotype was confirmed by phenotype for daf-7(e1372), daf-8(m85) and daf11(m47), and by PCR for sdf-9(m708). b Approximately one quarter of the animals in this sample were multivulva, small, embryonic lethal, grew slowly or had other morphological defects. These were not included in the counts. sdf-9 not only enhanced the phenotypes of all tested Daf-c mutants except for daf2(m41), it also proved to be hypersensitive to dauer pheromone, as are the Daf-c mutants (Golden and Riddle 1984b). sdf-9(m708) formed far more dauer larvae than N2 at all  77  temperatures and concentrations of pheromone tested (Figure 3.1). We then subjected sdf-9(m708); daf-d double mutants from the IIS (daf-16) and TGF-! (daf-3) pathways to dauer pheromone at 2.5 µl per plate to see which pathway mediates the hypersensitivity. daf-16(m26); sdf-9(m708) failed to form dauer larvae in response to pheromone (N = 114), but sdf-9(m708); daf-3(e1376) formed only slightly fewer dauer larvae (76 ± 6%, N = 119) than sdf-9(m708) alone (100%, N = 124). Neither the daf-3(e1376) nor the daf16(m26) single mutant formed any dauer larvae (N = 116 and 102, respectively). This suggests that SDF-9 modulates the IIS pathway, since daf-16(+) is required for sdf-9 to manifest its effect, whereas daf-3(+) is not. daf-16 suppression of sdf-9 hypersensitivity to dauer pheromone is dominant. We crossed daf-16(m26)/+; sdf-9(m708)/+ males to sdf-9(m708); dpy-3(e27) double mutants and exposed the progeny to 2.5 "l of dauer pheromone. If daf-16 were a recessive suppressor we would expect 50% dauer larvae among the hermaphrodite progeny, but we observed only 27 ± 7% (N = 64) for the line used in the pheromone assay, and 21 ± 6% (N = 63) for another isolate of the same genotype. This is compared to 100% dauer larvae in the control sdf-9(m708) strain (N = 120) and 0% for N2 (N = 114). Since 50% reduction in daf-16(+) gene dosage suppresses dauer formation, high levels of DAF-16 activity must be required for expression of the sdf-9 mutant phenotype. Enhancement of daf-2 by sdf-9(m708) was also semi-dominant. As shown in Table 3.1, 100% of the progeny of daf-2(e1368); sdf-9(m708) formed dauer larvae compared to 8% dauer larvae for daf-2(e1368) at 23°. If sdf-9(m708) were recessive, the expected number of dauer larvae segregated from daf-2(e1368); sdf-9(m708)/ +  78  79  FIGURE 3.1 - Response of sdf-9(m708) to dauer pheromone. The pheromone extract and plates were made as previously described (Golden and Riddle, 1984b) and used in the amounts given. (A and B) Each data point ± standard error represents results from 2 or 3 plates, each started with approximately 40 eggs laid in situ by three gravid adults, which were subsequently removed. Plates were scored for dauer formation on the first day of adulthood. At 25.5°, sdf-9(m708) showed increased sensitivity to pheromone for repeated experiments even when compared to unc-31(e928), previously reported to be sensitive to pheromone at 25.4° (Ailion and Thomas, 2000). (C) Using the same method, hypersensitivity to pheromone was also observed at 20°. heterozygotes would be 31% (equals 25% sdf-9(-/-) + 8% X 75% sdf-9(+/- and +/+) = 31%), but we observed 41 ± 3% (N = 624). We conclude that sdf-9(m708) is a semidominant enhancer of daf-2 (p-value from !2= 4.5 X 10-4). A 50% reduction in sdf-9(+) gene dosage enhances the phenotype of weak daf-2 mutants. SDF-9 and DAF-16 are both points of fine tuning for insulin-like signaling. To test whether sdf-9 mutants may be long lived, as are the Daf-c mutants in the IIS pathway (Kenyon et al. 1993; Larsen et al. 1995), we compared the life spans of sdf9(m708) with N2, and daf-2(e1370); sdf-9(m708) with daf-2(e1370). In agreement with previous results for sdf-9 (Ohkura et al. 2003; Hu et al. 2006), we saw no increase in the life span of sdf-9(m708) relative to N2. Similarly, sdf-9(m708) had no effect on the life span of daf-2 (data not shown), despite enhancing its Daf-c phenotype. SDF-9 may function to enhance IIS primarily during larval development, and not during adulthood. By contrast, treatment of wild-type adults with daf-2 RNAi is sufficient to increase longevity (Dillin et al. 2002). Slow maturation of Daf-c mutants to the adult at intermediate temperatures results from entry into an L2d-like state with a delayed second molt (Swanson and Riddle 1981). ut163 fully suppressed the slow growth of daf-2(m41) at 20°, whereas m708 had no effect (Table 3.3). At 22° both alleles partially suppressed the slow growth phenotype.  80  Suppression of the L2d delay would suggest a gain of SDF-9 function at the intermediate temperatures, but this is not supported by the dauer formation data (Table 3.1), which show no obvious suppression of the Daf-c phenotype at 20° or 22°. Instead, suppression of the slow growth phenotype suggests that SDF-9 may interact with other pathways required for growth. For example, sterol deprivation has already been shown to enhance the weak Daf-c phenotype of sdf-9 (Ohkura et al. 2003) TABLE 3.3 - Maturation time Genotype N2 daf-2(m41) sdf-9(m708) sdf-9(ut163) m41; m708 m41; ut163  20° 64 104 64 64 104 64  22° 56 96 56 56 88 80  a  Populations of 50 to 120 animals per plate (eight to ten plates per genotype) were synchronized as in Table 3.1 prior to transfer to NGM agar plates with E. coli at 20° and 22°. They were observed every 8 hours until all eight to ten of the plates for each sample had eggs present. Within each strain there was no variation observed among plates. 3.3 Discussion We isolated an allele of sdf-9 as an enhancer of daf-2. This gene was previously detected as an enhancer of akt-1 (Hu et al. 2006) and unc-31 (Ohkura et al. 2003). We found that sdf-9 enhanced all Daf-c mutants tested, except daf-2(m41). We propose that the allele specificity of interaction with daf-2 and the requirement for daf-16(+), not daf3(+), for SDF-9 function demonstrate genetically that SDF-9 modulates the IIS pathway, at least in larvae. These data support previous interpretations of SDF-9 function (Ohkura et al. 2003; Hu et al. 2006), which were based on its identity as a tyrosine phosphataselike protein lacking the necessary catalytic cysteine residue and membrane-bound subcellular localization. These authors suggested that SDF-9 might act along with EAK81  6 (another likely inactive tyrosine phosphatase that enhances the akt-1 mutant phenotype,) to bind the DAF-2 activating phosphotyrosine, but they did not test for genetic or molecular interaction with daf-2. Tyrosine kinase receptors like DAF-2 function via ligand binding, dimerization, activation by trans-autophosphorylation, phosphorylation of target proteins, and deactivation by a tyrosine phosphatase (reviewed by Romano 2003). It is possible that SDF-9 enhances IIS signaling by protecting phosphorylated DAF-2 from inactivation by a tyrosine phosphatase, or it may act as an adaptor protein to enhance binding of a DAF-2 target to the DAF-2 kinase. Hypomorphic daf-2 alleles would be sensitive to loss of SDF-9 as long as DAF-2 is able to trans-autophosphorylate. The daf-2(m41) mutation is a G to A substitution that changes a glycine to glutamic acid at position 383 (Yu and Larsen 2001). The glycine is part of a Caenorhabditis conserved GP turn motif adjacent to a conserved cysteine in the cysteinerich region in the extracellular domain. This structural change may disrupt a disulfide bond formed by the conserved cysteine and could make the overall structure of the daf2(m41) gene product unstable, or unable to dimerize at higher temperatures. Mutations in a similar domain of the EGFR protein cause inability to dimerize, preventing transautophosphorylation (Macdonald et al. 2006). Lack of trans-autophosphorylation would render SDF-9 unable to modify m41 activity, so loss of SDF-9 function would not affect the m41 phenotype. Alternatively, thermolability of the daf-2(m41) protein may prevent binding of SDF-9 to DAF-2, also resulting in no enhancement of daf-2(m41) by sdf9(m708). We propose that the hypomorphic daf-2 alleles that are enhanced by sdf9(m708) and sdf-9(ut163) all trans-autophosphorylate at some level.  82  Formally, SDF-9 could bind to a phosphorylated target of DAF-2, rather than DAF-2 itself. However, one would not expect daf-2 allele specificity in that case, since the enhancement would only be affected by the phosphorylation state of the particular DAF-2 target. This explanation seems far less likely than our posited lack of m41 protein dimerization. STYX family proteins have tyrosine phosphatase domains that are phosphatase inactive (Wishart and Dixon, 1998). They have been shown to bind proteins with phosphorylated serine, threonine or tyrosine to act as adaptor proteins, or to protect the phosphorylated tyrosine. Co-incubation of the mammalian STYX protein Sbf1 with SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9 (suppressor of variegation), was shown to stabilize the phosphorylated state of SUV39H1, whereas engineering Sbf1 to restore catalytic phosphatase activity eliminated such stabilization (Firestein et al. 2000). In C. elegans, the STYX protein IDA-1 enhances daf-2(e1370), apparently by functioning in several neurons likely to regulate insulin secretion (Cai et al. 2004). Based on the genetic evidence provided by the daf-2 allele specificity of interactions with sdf-9 and the requirement for daf-16(+) to exhibit the sdf-9 pheromone sensitivity, we propose that SDF-9 functions in the IIS pathway, and binds to DAF-2 at one or more of the activating phospho-tyrosines to enhance DAF-2 signaling during larval development. 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J Mol Biol 314, 1017-1028.  87  CHAPTER 41: RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation  4.1 Introduction The C. elegans dauer larva is a facultative diapause and dispersal stage that develops in response to adverse environmental stimuli such as high temperature, high population density or limited food (Cassada and Russell, 1975). Mutations in genes affecting the signal transduction pathways controlling the developmental switch may result either in constitutive dauer formation in favorable environments (dauerconstitutive, or Daf-c) or a lack of dauer formation in adverse environments (dauerdefective, or Daf-d) (Riddle et al., 1981). Though there are nearly 30 identified dauer formation (daf) genes in C. elegans, there may be many more genes that have minor effects on the known pathways that are not detectable as single mutants (Tewari et al., 2004; Hu et al., 2006; Liu et al., 2004). The major pathways involved in dauer formation are the transforming growth factor ! (TGF-!), insulin/insulin-like (IIS) and guanylyl cyclase pathways (Jensen et al., 2006). Transcriptional targets of the DAF-3/Smad (Patterson et al., 1997), DAF-16/FOXO (Ogg et al., 1997) and DAF-12 (Antebi et al., 2000) transcription factors are the effectors for parallel processes that execute the dauer/non-dauer switch.  1  A version of this chapter has been submitted. Jensen VL, Simonsen KT, Lee Y, Park D and Riddle DL (2010) RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation. 88  Some of the genes involved in dauer formation function within neurons, and affect neurosensory perception or neuropeptide secretion (Thomas et al., 1993; Ailion and Thomas, 2003; Speese et al., 2007; Ailion et al., 1999; Alcedo and Kenyon, 2004). The dauer pheromone and the competing food signal both require proper sensory perception to elicit a response (Golden and Riddle, 1984). Genes shown to be involved in dauer formation include a guanylyl cyclase, G-proteins and genes required for proper amphid cilia formation (Thomas et al., 1993; Bell et al., 2006; Swoboda et al., 2000). Neural tissue in C. elegans has been previously shown to be refractory to gene expression knockdown by RNAi (Kennedy et al., 2004). This effect can be reduced with mutants that affect the RNAi process including eri-1, a gene that encodes a siRNAase (Kennedy et al., 2004). This mutant shows a weak Daf-c phenotype when treated with RNAi targeted for the strong Daf-c genes daf-2 and daf-19. Here we use a strain that contains eri-1 as a double mutant with the synthetic dauer formation (SynDaf) mutant sdf-9 (Hu et al., 2006; Ohkura et al., 2003; Jensen et al., 2007). The genetic data suggest that it interacts directly with the DAF-2 insulin receptor to stabilize its phosphorylated state, thereby increasing insulin signaling (Jensen et al., 2007). Although sdf-9(m708) has little or no Daf-c phenotype as a single mutant, it strongly enhances most Daf-c mutants, and results in a synthetic Daf-c phenotype with other genes (Hu et al., 2006; Ohkura et al., 2003; Jensen et al., 2007). The eri-1; sdf-9 double mutant proved itself useful for assaying enhanced dauer formation resulting from gene knockdown via RNAi. It is known that the long-lived mutant daf-2 has increased resistance to pathogenic bacteria (Garsin et al., 2003) as well as other stresses (Honda and Honda, 1999). Increased pathogen resistance has been shown to be dependent on the DAF-16/FOXO 89  transcription factor (Garsin et al., 2003) and many of the DAF-16 transcriptional target genes are predicted to function in innate immunity (McElwee et al., 2003; Murphy et al., 2003). Here we describe an RNAi screen of candidate SynDaf genes (by their identity as DAF-16 transcriptional targets) that identified eight genes associated with innate immunity. This suggests that C. elegans uses dauer formation and subsequent dispersal as a defensive response to pathogens in the environment.  4.2 Results 4.2.1 RNAi screen for enhanced dauer formation As proof of concept for the use of eri-1(mg366); sdf-9(m708) as a sensitized genetic background to detect SynDaf mutations, we tested the effect of akt-1 RNAi on this strain. AKT-1 is involved in transmitting the signal from the DAF-2 receptor to the DAF-16/FOXO transcription factor (Paradis and Ruvkun, 1998). An akt-1 knockout has no Daf-c phenotype as a single mutant, but forms 82% dauer larvae as a double mutant with sdf-9 (Hu et al., 2006). The akt-1 RNAi treatment resulted in a median constitutive dauer formation of 44% compared to 6% for the control RNAi. For our screen, we chose genes that were repressed four-fold by DAF-16 activity (in a daf-2 background) from two microarray analyses (McElwee et al., 2003; Murphy et al., 2003), as well as those identified to be direct targets by chromatin immuneprecipitation (Oh et al., 2006). From the RNAi library [23] we obtained clones corresponding to 513 identified target genes. Sixty-nine of these genes (13%) were obtained from two of our three sources. None were found in all three. Since DAF-16 is a major regulator of dauer formation, we hypothesized that many of its target genes may 90  have small effects on dauer formation, detectable only in a sensitized genetic background.  4.2.2 21 SynDaf genes For the primary screen a qualitative assessment of dauer formation was completed for each target gene. 131 of the 513 RNAi clones were judged by visual inspection to result in increased dauer formation (compared to the control), and these were kept for further assessment (for complete target list see Table S1). These included clones that appeared to have only slightly higher dauer formation. In subsequent quantitative screens we required a target gene RNAi treatment to reproduce higher dauer formation significantly (p<0.05) in three consecutive independent trials. In the three retests, we counted each population (dauer and non-dauer larvae) and compared it to the control, if a clone failed to repeat once it was deemed to be negative. Thirty-one genes remained after a first quantitative pass, twenty-three after a second and twenty-one remained after a third and final re-test. Weighted averages for fold increase in dauer formation are given in Table 4.1 (actual counts included in Table S2).  91  Table 4.1 - Set of 21 SynDaf genes Gene  Predicted Functiona  Previously Identified SynDaf Gene srh-100 Predicted olfactory G-protein coupled receptor New SynDaf Genes C53A3.2 p-Nitrophenyl phosphatase (Synthetic small brood size with daf-18) skr-8 skp1 protein (Regulated by DAF-12) dct-5 zinc finger transcription factor cyp-35A3 Cytochrome P450 CYP2 subfamily C24G6.6 Flavin-containing amine oxidase lase-1 Aminoacylase ACY1 unc-84 Transmembrane protein with a SUN domain ccb-1 Beta subunit of dihydropyridine sensitive L-type calcium channel dct-14 DNAk, heat shock protein E02C12.8 CHK kinase like, like SRC kinase F59B1.2 Gene F44D12.8 SRRM1 (serine arginine repeat nuclear matrix protein) Innate Immunity Related New SynDaf Genes F35E12.9 CUB domain c F35E12.10 CUB domain ZK896.5c CUB domain c dct-17 CUB domain and inorganic phosphatase c clc-1 Claudin c lys-1 Lysozyme c cpr-1 Cysteine proteinase, cathepsin L c F52E1.5 Homology to chondroitin proteoglycan  Fold N Dauer Increaseb 4  417  9  842  6 4 6 5 10 6  420 780 289 329 467 430  3  631  5 8 8 8  509 927 708 560  7 4 8 5  276 645 436 769  6 7 17 9  378 565 433 842  a  Predicted functions are based on previous research and Wormbase annotations.  b  Fold increase is weighted over three independent replicates compared to control.  c  Upregulated upon infection (Troemel et al., 2006; Shapira et al., 2006).  Whereas 69 of the 513 candidate target genes were found in two of the three 92  sources, (McElwee et al., 2003; Murphy et al., 2003; Oh et al., 2006), eight of the 21 positives were among these 69. The probability that this was random had a p-value (!2 test) of 0.001. Hence, genes from multiple sources were enriched among the 21 positives (Table 4.1). Nevertheless, most of the positives originated from only one of the three sources. Each of the three source studies (McElwee et al., 2003; Murphy et al., 2003; Oh et al., 2006) identified gene classes that were enriched in each of their own data sets. The most enriched protein domain in both the 21 positives we report (Table 4.1) and the 513 target genes is the CUB (or CUB-like) domain (C1r/C1s, Uegf, Bmp1) (Bork and Beckmann, 1993). It has been suggested that CUB-domain proteins function in innate immunity due to the organization of their genes in large clusters, the similarity of CUB domains to immunoglobulins and their localization at the cell surface (Thomas, 2006; Shivers et al., 2008). In addition, a CUB domain protein has been identified in a recent RNAi screen for sensitivity to Pseudomonas auruginosa PA14 infection and arsenic stress (Nandakumar and Tan, 2008).  4.2.3 Genes known to affect dauer formation or insulin secretion Several genes identified in our screen function in pathways that have already been associated with dauer formation. This includes one gene that has already been identified as SynDaf, srh-100 (Oh et al., 2006; Lee et al., 2003). SRH-100 is a predicted olfactory G-protein coupled receptor (GPCR) (Robertson, 2000). Detection of this gene shows that our screen can replicate previous results.  93  A previously unreported SynDaf gene, ccb-1, encodes the !-subunit of the L-type calcium channel, a protein involved in insulin secretion in mammals (Davalli et al., 1996). Calcium signaling in C. elegans has been shown to affect dauer formation and insulin secretion (Speese et al., 2007). It is likely that loss of ccb-1 results in lower insulin output, which has been previously shown in other insulin secretion mutants to result in a SynDaf phenotype (Speese et al., 2007).  4.2.4 Genes with unknown function Most of the 21 SynDaf genes we identified have predicted protein domains but no assigned functions (Table 4.1). Five have been shown to interact with daf genes. C53A3.2 encodes a HAD-superfamily hydrolase and was shown to have a synthetic small brood-size phenotype with daf-18/PTEN (Suzuki and Han, 2006). skr-8, a Skp1 homolog that is part of the proteasomal E3 ubiquitin ligase complex, has been shown to be regulated by DAF-12 (Shostak et al., 2004) as well as DAF-16. Three genes (ZK896.5, F35E12.9 and dct-5) are differentially regulated in TGF-! mutants during dauer entry as measured by microarray analysis (Liu et al., 2004). Three of the 21 genes have been previously shown to suppress the tumorous gonad phenotype of gld-1 mutants in an RNAi screen of DAF-16 targets (Pinkston-Gosse and Kenyon, 2007). dct-5 (DAF-16-controlled tumor suppressor) encodes a zinc finger transcription factor (Pinkston-Gosse and Kenyon, 2007), dct-14 encodes a heat shock protein possibly involved in germ cell apoptosis, and dct-17 encodes a protein with CUB and inorganic phosphatase domains. This overlap between the gld-1-tumor-suppressor genes and the SynDaf positives in this study suggest that these overlapping genes could 94  be involved in the IIS pathway. Finally, three genes, F44D12.8, C24G6.6, and F59B1.2, were SynDaf under our conditions but they have no previously identified involvement in any biological process. F44D12.8 encodes a serine arginine repeat nuclear matrix protein (SRRM), which may function in alternative splicing or mRNA stability (Rogers et al., 2008). C24G6.6 encodes a flavin-containing amine oxidase and may function in neurotransmission. F59B1.2 encodes a protein with no known or predicted domains.  4.2.5 Innate immunity genes The most notable trend within our list of 21 SynDaf genes is that eight genes have a connection to innate immunity (Table 4.1). Four genes encode proteins that contain CUB domains and are members of large clusters of paralogs. Several genes in these clusters are induced upon infection (Troemel et al., 2006; Shapira et al., 2006; Thomas, 2006), so we include these in our list of immunity genes that are SynDaf. A total of seven of the eight innate immunity related genes found in our screen, including three CUB domain proteins, dct-17, clc-1, cpr-1 and lys-1 are reported to be induced upon infection (Troemel et al., 2006; Shapira et al., 2006). To determine whether the innate immunity-related genes were required for pathogen resistance, we tested all eight immunity genes using RNAi in the rrf-3 RNAi hypersensitive background (Simmer et al., 2002), and challenged them with Staphylococcus aureus. Under these conditions, two of the eight, lys-1 and clc-1, had significantly reduced survival on S. aureus (Figure 4.1). Sensitivity to pathogenic bacteria has not been previously reported for either of these two genes, but LYS-1 over95  expression has been shown to confer resistance to Serratia marcescens (Mallo et al., 2002). It is predicted that clc-1, which encodes a claudin-like protein, plays a role in epithelial cohesion (Asano et al., 2003). It is possible that the epithelial layers in C. elegans become more permeable to S. aureus as a result of clc-1 RNAi.  Figure 4.1 - Survival of RNAi treated adults on S. aureus. clc-1 and lys-1 RNAi treatment increased pathogen sensitivity compared to the RNAi control (GFP). One of two independent tests is shown. The TD50 (time required for 50% of the nematodes to die) for lys-1 was 4.7 days (p<0.0001) and for clc-1 was 5.4 days (p=0.001) compared to 6.8 days for the GFP RNAi control. p-values were calculated using the log-rank test.  4.2.6 Dauer formation on pathogenic bacteria To determine if pathogenesis affects dauer formation, we challenged C. elegans with different pathogenic bacteria. We selected the strong pathogen Pseudomonas  96  aeruginosa PA14 (Tan et al., 1999), and three weaker pathogens that previously have been tested with C. elegans, S. aureus, Agrobacterium tumefaciens and S. marcescens (Mallo et al., 2002; Couillault and Ewbank, 2002; Sifri et al., 2003). The strains we used reduced survival (compared to the standard laboratory food E. coli OP50) similarly to the previous reports (data not shown). We also used Bacillus subtilis, because it had been previously shown to increase the survival of C. elegans compared to E. coli OP50 (Garsin et al., 2003). We first challenged the daf-8(m85ts) Daf-c mutant with all the bacterial strains at an intermediate temperature (22.5°C), except for PA14 which we tested at 15°C, a permissive temperature for daf-8(m85). The percent dauer formation seen for daf-8 increased on all three pathogens tested compared to OP50, but was reduced on B. subtilis (Table 4.2). Similarly, sdf-9(m708) formed ~20% dauer larvae on OP50 (at 26°C) and 2% on B. subtilis, but formed more than twice as many dauer larvae on A. tumefaciens or S. marcescens and three times as many on S. aureus (Table 4.3). Table 4.2 - Percent dauer formation of daf-8(m85) on pathogenic bacteria at 22 °C.  a  Bacteria E. coli OP50 B. subtilis A. tumfaciens S. marcescens  daf-8 65.3 12.1 84.1 79.4  N 101 107 195 102  E. coli OP50a P. aeruginosa PA14a  0 70.6  86 68  p-value 6.3E-31 3.7E-8 2.8E-3  5.9E-9  These tests were carried out at 15 °C  97  Table 4.3 - Percent dauer formation of sdf-9(m708) on pathogenic bacteria at 26 °C. Bacteria E. coli OP50 B. subtilis A. tumfaciens S. marcescens S. aureus  sdf-9 19.6 2.6 49.4 42.2 60.0  N 97 76 79 90 40  p-value 2.0E-4 2.6E-11 6.3E-08 1.2E-10  We tested N2 for its response to pathogens at 27°C (a condition that induces ~510% dauer larvae on OP50) to ensure the effect we observed was not unique to Daf-c mutants (Ailion and Thomas, 2003). The same trend seen with the two weak Daf-c mutants was repeated in N2 with A. tumefaciens and S. marcescens significantly enhancing dauer formation (Table 4.4). We conclude that part of the C. elegans response to a pathogenic environment is to enter the dauer stage at greater frequency. Table 4.4 - Percent dauer formation of N2 and daf-22(m130) on pathogenic bacteria at 27 °C. Bacteria E. coli OP50 B. subtilis A. tumfaciens S. marcescens a  N2 11.7 0.78 27.6 30.6  N 231 129 98 111  p-valuea 1.1E-4 1.0E-6 5.2E-10  daf-22 3.9 0 0 0  N 246 131 133 95  p-valuea 0.021 0.020 0.049  p-values given are relative to OP50 sample for each genotype.  We performed epistasis analysis to determine which part of the dauer signaling pathway affects pathogenesis. We surmised that olfactory sensation might be involved because C. elegans is able to discriminate between bacteria (Zhang et al., 2005). To test this we used the daf-8(m85); daf-6(e1377) double mutant that can form dauer larvae constitutively (due to the daf-8 mutation), but is defective in chemosensory behavior due to daf-6 with improper formation of the sensory channel, preventing the olfactory 98  neurons from contacting the environment (Perens and Shaham, 2005; Albert et al., 1981). While the daf-8 single mutant (which has normal olfactory behavior) responded to pathogenic bacteria by forming a higher percentage of dauer larvae (Table 4.2), the daf8;daf-6 double mutant formed fewer dauer larvae on the pathogenic bacteria (Table 4.5). This indicates that olfactory sensation is required for the increase in dauer formation on pathogenic bacteria. Table 4.5 - Percent dauer formation of daf-8(m85) ; daf-6(e1377) on pathogenic bacteria at 22 °C. Bacteria E. coli OP50 B. subtilis A. tumfaciens S. marcescens  daf-8 ; daf-6 64.5 41.1 35.7 27.6  N 96 73 140 105  p-value 2.7E-5 9.2E-13 2.4E-15  Our initial observation of increased infection causing higher dauer formation involved RNAi tests using the same bacterial strain (HT115) for control and sample. Hence, the dauer stimulus must not originate from the bacteria, but instead from the worms themselves. To test if the dauer pheromone served as an olfactory cue, we used the daf-22(m130) mutant that is unable to produce the pheromone (Butcher et al., 2009; Golden and Riddle, 1985). It has been reported that the expression of daf-22 increases upon infection with PA14 (Troemel et al., 2006). Interestingly, daf-22 was required for the increase in dauer formation. While a daf-8(e1393) unc-13(e51) strain formed more dauer larvae on pathogenic bacteria, a daf-8(e1393) unc-13(e51); daf-22(m130) mutant did not (Table 4.6). In these tests, the unc-13 mutation (which does not affect dauer formation) served to prevent the strain from avoiding the pathogen.  99  Table 4.6 - Percent dauer formation of daf-8(e1393) unc-13(e51) and daf-8(e1393) unc-13(e51) ; daf-22(m130) on pathogenic bacteria at 20 °C. Bacteria  daf-8 unc-13  E. coli OP50 B. subtilis A. tumfaciens S. marcescens  39.2 34.4 61.9 54.5  N  p-value  daf-8 unc-13 ; daf-22 245 40.5 93 0.35 33.7 160 4.1E-09 39.2 101 0.0017 35.4  N  p-value  116 86 0.20 79 0.82 96 0.31  It was previously reported that daf-22 mutants are able to form dauer larvae at a frequency similar to N2 at 27°C (Ailion and Thomas, 2000). We compared daf-22 dauer formation on pathogens at 27°C with that of N2. Whereas N2 formed more dauer larvae on the pathogenic bacteria, the daf-22 mutant did not, forming only a few dauer larvae on the laboratory food OP50 and none on the pathogens tested (Table 4.4). Finally, we used the pdaf-7::GFP reporter gene that exhibits decreased expression with increased pheromone concentrations (Ren et al., 1996). Indeed, GFP expression in L2 larvae decreased markedly after exposure to PA14 (Figure S1). Taken together this indicates that increased dauer pheromone production is a mechanism for increased dauer formation in response to bacterial pathogenesis.  4.3 Discussion Mutations in sdf-9 have been independently isolated three times as enhancers of unc-31, akt-1 and daf-2 mutants (Hu et al., 2006; Ohkura et al., 2003; Jensen et al., 2007). Because sdf-9 enhances the phenotype of most Daf-c mutants tested, we utilized it as a sensitized background for identifying new SynDaf genes. Of 20 previously unreported SynDaf genes, three have been shown and five are predicted to play roles in 100  innate immunity. Five genes have been previously linked to insulin or TGF-! signaling. For example, skr-8 is regulated by DAF-12 (Shostak et al., 2004). It is possible that some of our selected 513 target genes may not be SynDaf with sdf-9, similar to akt-2 or the Eak genes (Hu et al., 2006), but still have a SynDaf phenotype with other mutants. Acknowledging this limitation, our screen nevertheless allowed for the detection of a new set of SynDaf genes and identification of a novel input into the dauer developmental decision. It is not surprising to see an enrichment of target genes identified in two of the three sources in our positive gene set. There is not a strong consensus among the three gene sets we used (McElwee et al., 2003; Murphy et al., 2003; Oh et al., 2006), with about 13% (69/513) overlap. Eight of the 21 positives were present in two of the sources.  4.3.1 CCB-1 and possible feedback regulation of insulin signaling Since ccb-1 was detected in our screen, we conclude that its activity normally inhibits dauer formation. It encodes the !-subunit of the L-type calcium channel, which may modulate the sensitivity of the channel (Davalli et al., 1996). This gene is thought to be a direct target of DAF-16 because it was identified using DAF-16 ChIP, and its promoter contains a DAF-16 binding site (Oh et al., 2006). It is possible that DAF-16 regulates the expression of ccb-1 to modulate calcium signaling, which has been linked to insulin secretion in mammals and worms (Speese et al., 2007; Davalli et al., 1996). The interaction between DAF-16 and ccb-1 may be part of a feedback mechanism to reduce insulin secretion during the dauer development. The IIS pathway acts to inhibit DAF-16, but once DAF-16 activity reaches a critical threshold, it could antagonize insulin 101  secretion to stabilize the dauer developmental decision.  4.3.2 Germ line and dct genes Three dct genes were found in our screen. These are putative DAF-16 targets that are gld-1 (Germ Line Defective) tumor suppressors (Pinkston-Gosse and Kenyon, 2007). When dct expression is reduced the endomitotic tumors that grow within the germ lines of gld-1 mutants are reduced. Germ line proliferation is actively suppressed in dauer larvae (Narbonne and Roy, 2006), so it is reasonable that the mechanisms governing cell proliferation in adults and dauer larvae may overlap. However, it is not clear why reduction of dct activity would trigger constitutive dauer formation in our screen. It is as though inhibition of mitotic progression (e.g., in response to starvation) feeds back to reduce TGF-! and/or insulin signaling and favor dauer arrest, but the point of feedback regulation is not known. Other genes that regulate both germ line proliferation and dauer formation have already been identified, including AKT-1 and DAF-18/PTEN (Narbonne and Roy, 2006; Suzuki and Han, 2006). In our positive gene set, C53A3.2 and skr-8 have been previously shown to have a synthetic small brood size phenotype with daf-18, an indication of poor germ line proliferation (Suzuki and Han, 2006; Sonnichsen et al., 2005). Although it is well known that dauer formation arrests germ line proliferation, these results suggest that the converse may also be true.  102  4.3.3 Immunity related genes Eight of the 21 positives have been previously implicated in innate immunity, four of which contain the CUB (or CUB-like) domain. The CUB domain consists of a !barrel with similarity to immunoglobulins, and is predicted to be extracellular (Troemel et al., 2006). Three of the four CUB domain proteins found in our screen are induced by infection, as are the four remaining innate immunity genes (Troemel et al., 2006; Shapira et al., 2006). The lys-1 lysozyme is an antimicrobial peptidoglycan Nacetylmuramoylhydrolase that has been shown to protect against infection in C. elegans (Mallo et al., 2002). The cpr-1, F52E1.5 and clc-1 genes are also predicted to be protective genes because they are induced upon infection (Troemel et al., 2006; Shapira et al., 2006; Wong et al., 2007). We propose that under the conditions of the RNAi screen, targeting of these innate immunity genes increases the animal’s susceptibility to, or perception of, infection by the E. coli food (Garsin et al., 2003; Darby, 2005; Garigan et al., 2002). As a response to this increased sensitivity to infection, the developing larvae may be predisposed to dauer dispersal. This leads to the hypothesis that it is the process of pathogenesis that stimulates increased dauer formation. Indeed, we have shown that pathogenic bacteria enhance dauer formation, and this requires the dauer pheromone. Two genes, lys-1 and clc-1, were required for normal resistance to S. aureus. The remaining six genes may not affect pathogenesis by S. aureus under our conditions for various reasons, including redundancy among gene families or pathogen-specific interactions. Over-expression of lys-1 had been previously shown to increase survival on 103  S. marcescens (Mallo et al., 2002). LYS-1 is a putative lysozyme, an antimicrobial protein, so we expected that loss of lys-1 might make the worm sensitive to infection in spite of possible redundancy with lys-2. Reduction in survival has not been previously shown for lys-1, but the conditions and pathogens used were different (Mallo et al., 2002). clc-1 encodes a claudin-like protein, and its expression has been seen to be induced upon infection (Shapira et al., 2006; Ren et al., 2009). Claudins are predicted to function in epithelial cohesion, indicating that loss of clc-1 function may cause the epithelial layer to loosen. In C. elegans, clc-1 RNAi was reported to increase the permeability of the pharynx to a high molecular mass dye, TRITC-dextran (Asano et al., 2003). Thus, increased CLC-1 in response to infection could strengthen the epithelial layers to resist pathogenesis. Indeed, we have shown that survival of C. elegans is significantly reduced when treated with clc-1 RNAi followed by exposure to S. aureus from the L4 stage.  4.3.4 Mechanism for pathogenic input into dauer formation DAF-2 and DAF-16 have been previously linked to innate immunity. daf-2 mutants are resistant to infection (Garsin et al., 2003). DAF-16 is required for the immunity phenotype of daf-2, just as it is for the longevity and dauer formation phenotypes (Garsin et al., 2003; Larsen et al., 1995; Kenyon et al., 1993). We have shown that the production of dauer pheromone is required for pathogen induced dauer formation with the requirement of daf-22 for the dauer induction. Olfactory sensation is also required for the increase in dauer formation, probably through the sensation of dauer 104  pheromone. Worms infected with PA14 increase expression of daf-22, a gene that encodes a pheromone biosynthetic enzyme (Troemel et al., 2006; Butcher et al., 2009), and by reducing expression of daf-7, an indicator of higher pheromone levels in the environment (Ren et al., 1996). Taken together, the data indicate that when C. elegans encounters a pathogenic environment it increases pheromone production to elicit dauer formation. At higher concentrations purified components of the dauer pheromone were found to be a chemo-repellent (Srinivasan et al., 2008) suggesting that increased dauer pheromone could deter other worms from entering the toxic environment. Pheromone deposited by 100 worms over 60 minutes decreases the response to a chemo-attractant (Matsuura et al., 2005). We rule out starvation as the cause of increased dauer formation on pathogenic bacteria. It is well known that food limitation increases dauer formation (Cassada and Russell, 1975; Hu, 2007) and that C. elegans can display avoidance to pathogenic bacteria on plates (Zhang et al., 2005; Pradel et al., 2007). The AWB ciliated chemosensory neurons are required for this avoidance (Zhang et al., 2005). Chemosensory function is required for the pathogen induced dauer formation because no increase is observed in a chemosensory mutant, daf-6 (Table 4.5). However, in the unc13 background we show increased dauer formation on pathogenic bacteria, which is suppressed by the daf-22 mutation (Table 4.6). daf-22 single mutants also show no increase in dauer formation on pathogenic bacteria (Table 4.4) despite normal chemosensory behavior (Matsuura et al., 2005). This indicates that decreased nutrition is not part of the mechanism of pathogen induced dauer formation. Animals may use cues to recognize infection in other individuals (Hamilton and 105  Zuk, 1982). The original observations included changes in feather brightness or songs of songbirds affecting mate selection. A bird could select for those with genetic resistance to a pathogen by avoidance of potential mates that are infected (Hamilton and Zuk, 1982). Bullfrog tadpoles receive a chemical cue from infected tadpoles, and they spend less time in the presence of those tadpoles to mitigate the risk of infection (Kiesecker et al., 1999). We suggest that the dauer pheromone can work in a similar way, as it is used for avoidance and sexual attraction as well as dauer formation (Srinivasan et al., 2008). The benefit of dauer formation in a pathogenic environment could accrue from three dauer traits. First, dauer larvae do not feed, which should convey resistance to enteric infection. Second, the dauer stage is used for dispersal, permitting flight from local concentrations of pathogenic bacteria. Third, dauer larvae have a stronger cuticle (Cassada and Russell, 1975), which could also defend against attachment or entry of pathogenic bacteria (Darby, 2005). We have identified 21 SynDaf genes, each of which provides insight into dauer formation. Some genes fall into pathways and processes that have already been associated with dauer formation, whereas others suggest a new input into dauer formation, pathogenesis. Indeed, we show that pathogenic bacteria do enhance dauer formation possibly through increased pheromone production. We have explored the connection between dauer formation and suppression of germ line proliferation as well as innate immunity. Our screen is defined by the 513 putative DAF-16 target genes we used. A genome-wide screen should detect additional environmental inputs for dauer formation that do not require DAF-16.  106  4.4 Methods 4.4.1 Gene target selection and RNAi screen DAF-16 target genes were selected from two microarray studies, 336 from one (McElwee et al., 2003) and 250 from another (Murphy et al., 2003). The targets chosen were at least four-fold down-regulated in a daf-2 background (McElwee et al., 2003; Murphy et al., 2003). An additional 87 targets were selected from a DAF-16 ChIP study (Oh et al., 2006). Only the target genes that were in the Arhinger RNAi library were kept (Kamath et al., 2003). Bacterial cultures in the library that did not grow after three attempts were also not included, leaving a total of 513 target genes (full target list in Table S1). The eri-1(mg366); sdf-9(m708) strain was constructed by crossing sdf-9 males with eri-1 hermaphrodites. The double mutants were selected in the F2 generation by PCR tests for the deletion in eri-1 and the transposon insert in sdf-9 (Kennedy et al., 2004; Jensen et al., 2007). The screen was performed by first spotting (in duplicate) 50 !l of each RNAi clone (thawed from an overnight liquid culture frozen at -80 °C in 30% glycerol) onto 10 ml NGM agar plates containing 100 mg/ml ampicillin and 1 mM IPTG, followed by overnight incubation at room temperature. The clones containing sequences specific for GFP and akt-1 were used for the negative and positive controls, respectively. These controls were run with each test. eri-1; sdf-9 was exposed to RNAi for two generations. The two initial 60 mm plates per clone were inoculated with 2-3 L2 or L3 larvae and incubated at 20°C. On the seventh day of incubation, five F1 gravid adults were transferred to fresh RNAi plates (two per clone), made as above, and allowed to lay eggs for approximately 3 hours at 107  room temperature. The plates were then transferred to 25.5°C, where the transferred adults become sterile (Kennedy et al., 2004), until the 10th day when the populations were scored for percentage dauer larvae. For the initial screen the two plates per clone were checked visually for an increase in constitutive dauer formation by comparison with the negative (GFP RNAi) and positive (akt-1) controls. For subsequent re-screening the dauer larvae (identified visually in the Zeiss Stemi SV 11 stereomicroscope at 660x magnification by the presence of dauer alae and radial constriction of the body) and nondauer larvae were counted. RNAi clones were only re-tested if they produced a significant difference (p < 0.05, !2 test). To be kept as a positive, a clone had to show a significant difference in three independent trials with the smallest sample size >30 and largest being >100 (actual counts included in Table S2). Positive clones were confirmed by PCR using a T7 transcriptional start site primer followed by digestion by two different restriction enzymes.  4.4.2 Classification and comparison of positives Function was inferred from previous work, including the “Gene Summary” page on Wormbase (Rogers et al., 2008), as stated for biological process enrichment assessment. Assessing GO term enrichment was completed by using the Wormbase BioMart function (WormMart) to retrieve identifiers for all genes (Rogers et al., 2008; Smedley et al., 2009). These identities were put into DAVID to identify enriched GO terms (Dennis et al., 2003). The number of genes overlapping in the target and positive gene sets were compared by a !2 test and the p-value reported.  108  4.4.3 Dauer formation, daf-7 expression, and adult survival on pathogenic bacteria Bacterial strains used were P. aeruginosa PA14, A. tumefaciens GV3101, S. marcescens ATCC 8100, S. aureus SH1000, E. coli HT115, and E. coli OP50. Fresh overnight cultures of each bacterial strain were spread on each plate to cover approximately half of the plate surface. Bacteria were not spread to the edges in order to minimize the number of dauer larvae crawling off the plate. To assay percent dauer formation, hypochlorite-purified eggs (Brenner, 1974) were spotted on to two 60 mm plates for each bacterial strain. Dauer and non-dauer larvae were then counted as the first non-dauers reached egg-laying age. Bacterial strains were compared for nematode pathogenesis as previously described (Garsin et al., 2003) by daily assay of the percent survival on each bacterial strain. PA14 plates were made as described above for the pdaf-7::GFP expression analysis. L2 larvae were placed on PA14 or OP50 control plates and were assayed for GFP expression after 4 hours. GFP worms were imaged on a Zeiss Axioskop with a Qimaging Retiga 2000R camera. For the survival assays with the eight pathogen-related RNAi treatments, rrf3(pk1426) was treated with each of the RNAi expressing bacteria for two generations to maximize silencing. F2 larvae were synchronized by hypochlorite treatment followed by hatching overnight in M9 buffer. The synchronized L1’s were then put on a 50-50 mixture of RNAi bacteria for the pathogen related gene and for cdc-25.1 to sterilize them for the survival assay. L4 larvae were transferred from the RNAi plates to NG plates seeded with S. aureus and scored daily for survival. GraphPad Prism software was used  109  to calculate significance using the log-rank test and to calculate the TD50 for each strain for each of two replicates. Figure 4.1 was created in Microsoft Excel.  110  4.5 Bibliography Ailion, M., Inoue, T., Weaver, C. I., Holdcraft, R. W., and Thomas, J. H. (1999). Neurosecretory control of aging in Caenorhabditis elegans. 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(2006). p38 MAPK regulates expression of immune response genes and contributes to longevity in C. elegans. PLoS genetics 2, e183. Wong, D., Bazopoulou, D., Pujol, N., Tavernarakis, N., and Ewbank, J. J. (2007). Genome-wide investigation reveals pathogen-specific and shared signatures in the response of Caenorhabditis elegans to infection. Genome biology 8, R194. Zhang, Y., Lu, H., and Bargmann, C. I. (2005). Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 438, 179-184.  117  CHAPTER 51: ZIP-5 acts downstream of the insulin signaling pathway to regulate longevity dependent on skn-1  5.1 Introduction Many genes have been shown to be involved in control of C. elegans life span. Several signaling pathways have been implicated in this process including the Insulin/Insulin-like Signaling (IIS) and TGF-! pathways (Larsen et al., 1995; Murphy et al., 2003; McElwee et al., 2003; Shaw et al., 2007). Other genes linked to aging in C. elegans include some affecting mitochondrial function (Rea and Johnson, 2003; ArtalSanz and Tavernarakis, 2009) and germline signaling (Berman and Kenyon, 2006). The intestinal GATA transcription factors have also been linked to aging (Budovskaya et al., 2008). Another longevity associated transcription factor is SKN-1, a bZIP-like (basic leucine ZIPper) protein that also regulates stress response as well as endoderm differentiation (An et al., 2005; Tullet et al., 2008; Oliveira et al., 2009). The major downstream effecter of the IIS pathway is the DAF-16/FOXO transcription factor (Ogg et al., 1997; Jensen et al., 2006; Murphy et al., 2003; McElwee et al., 2003; Kenyon et al., 1993; Larsen et al., 1995; Oh et al., 2006; Lee et al., 2003). DAF-16 is required for the long life of the IIS pathway mutants such as daf-2 and age-1 (Friedman and Johnson, 1988; Kenyon et al., 1993). Several studies have focused on the targets of DAF-16 to try to identify targets involved in the control of longevity (Murphy et al., 2003; McElwee et al., 2003; Lee et al., 2003; Halaschek-Wiener et al., 2005; Oh et !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! 1 A version of this chapter is under preparation for submission. Jensen VL, Baillie DL & Riddle DL (2010) ZIP-5 acts downstream of the insulin signaling pathway to regulate longevity dependent on skn-1. !  ""#!  al., 2006; Ruzanov et al., 2007). We focused our attention on just one of these target genes, C34D1.5, a bZIP transcription factor we have named zip-5.  5.2 Results and discussion We first identified zip-5 as C34D1.5, a candidate longevity gene from SAGE tag analysis comparing daf-2 mutants to daf-2;daf-16 double mutants (Halaschek-Wiener et al., 2005). zip-5 encodes a bZIP transcription factor. We received a deletion strain zip5(tm2308) obtained in a UV-Trimethylpsoralen mutagenesis screen (Gengyo-Ando and Mitani, 2000), and back-crossed it six times to the wild-type N2. We tested the longevity phenotype of zip-5(tm2308) compared to N2 at 25 °C. There was a significant increase in survival for the zip-5 mutants (Table 5.1). We then compared the double mutant daf-2(e1370); zip-5(tm2308) to daf-2(e1370) alone. There was no significant enhancement of the daf-2 extended longevity, indicating that zip-5 is downstream of daf-2. Table 5.1 - Life span analysis at 25 °C.  !  Genotype  Mean Life Span (Days)  p-Value  N  N2  11.0  zip-5  15.1  daf-2(e1370)  40.1  daf-2(e1370); zip-5(tm2038)  43.8  zip-5(tm2038); Control RNAi  16.7  zip-5(tm2038); skn-1 RNAi  13.3  1.1E-8  42  N2; skn-1 RNAi  12.6  0.27  51  73 1.3E-5  70 48  0.13  47 68  ""#!  !  "#$!  Figure 5.1 - zip-5 expression analysis. In (A), the expression of the translational fusion protein ZIP-5::GFP is shown for the integrated transgenic. In the L2 (shown) expression is seen in the ASI neurons in the head, the posterior and anterior gut as well as the PQR neuron in the tail. In (B), elt-2 RNAi greatly reduces expression of the ZIP-5::GFP reporter in the gut while the ASI expression is not changed in L2 larvae. In (C) Shown four representative worms for the control RNAi vs. daf-16 RNAi for the ASI neurons, which show increased expression when daf-16 is silenced. In the posterior gut cells, a smaller number of synchronized worms express GFP in the gut in the control RNAi (50%, n=54) compared to daf-16 RNAi (75%, n=61), p=5.8E-5 (!2). In (D) the promoter truncation analysis results are shown. In the 986 bp promoter, expression is similar to the translational fusion. The 747 bp promoter, with four less IRE elements, reduces the expression. The 473 bp promoter, with no IRE elements has a greatly reduced GFP expression level. While the 412 bp promoter, no DBE has an expression level similar or higher than the 986 bp promoter. The 212 bp promoter has no expression and is not shown, it removes a consensus GATA site. Taken together, the GATA and IRE sites enhance GFP expression while the DBE is required to inhibit expression. We created a translational fusion reporter construct using the genomic region of zip-5 including 3 kb 5’ of the start translation site as well as all the exons and introns connected to a C-terminal GFP. This fusion protein is expressed in the ASI neurons and PQR neuron in every life stage, as well as in the gut during the L1 and L2 stages (Figure 5.1). Because the 5’ upstream region from the isoform tested (the other isoforms being identified after this work was completed) included binding sites for DAF-16 and ELT-2 (GATA), we assayed the reporter expression after feeding daf-16 and elt-2 RNAi. daf-16 RNAi increased expression of the transgene, whereas elt-2 RNAi decreased expression (Figure 5.1). This result further indicates that zip-5 is downstream of the IIS. We used serial promoter truncations of the 5’ regulatory region to further assay transcriptional control of zip-5 (Figure 5.1). Expression of GFP in the ASI neurons and the gut is regulated by the 986 bp upstream of the translational start site. A 753 bp region showed the same expression but weaker, this truncation deletes a cluster of insulin response elements (IRE) which appear to be required for enhancing expression of zip-5.  !  "#"!  A 479 bp promoter removes another IRE and shows weaker expression. A 418 bp promoter restores expression to the same level as the 986 bp promoter and removes a DAF-16 binding element (DBE) (Furuyama et al., 2000), indicating that DAF-16 represses expression of zip-5. This is consistent with the SAGE data and the RNAi experiment. A 208 bp promoter, which removes the strongest consensus GATA binding site, removes expression of ZIP-5::GFP completely, agreeing with the elt-2 RNAi experiment above. The DNA binding domain of ZIP-5 is predicted to bind the same DNA binding site as SKN-1 and F45H11.6, which encodes the ortholog of mammalian c-Maf (Kim and Struhl, 1995). Since bZIP proteins dimerize based on the amino acid sequence of the leucine zipper domain, binding partners can be predicted (Vinson et al., 1993). The best ZIP-5 dimerization partner is predicted to be F45H11.6. This is interesting as the best reciprocal BLAST homolog of ZIP-5 is BACH1 when using the bZIP domain. BACH1 and the ortholog of SKN-1 (Nrf2) both bind c-Maf, bind to the same site and have the opposite effect on transcriptional target genes (Igarashi and Sun, 2006). We showed that SKN-1 is required for the life span extension seen in zip-5 mutants (Table 5.1), indicating that the same relationship is conserved in C. elegans. We show that zip-5 regulates life span downstream of the IIS pathway through four lines of evidence. The DBE is required to inhibit expression of zip-5 while the IRE elements, which DAF-16 may also bind (Nasrin et al., 2000), seems to be required to enhance expression. Perhaps the IRE element can recruit DAF-16 away from the DBE to reduce the inhibition of zip-5 expression by DAF-16. The life span extension seen in zip5 mutants requires SKN-1 a transcription factor linked to longevity and regulation of  !  "##!  detoxification pathways (Oliveira et al., 2009). ZIP-5 may act in promoters that contain multiple SKN-1 binding sites, which is common to many SKN-1 targets (An and Blackwell, 2003). This may be a conserved interaction because the mammalian homologs are involved in stress response and senescence (Goven et al., 2008). Further research will elucidate what role ZIP-5 plays in longevity.  5.3 Experimental procedures 5.3.1 Life span analysis Survival for each test was determined as described previously (Larsen et al., 1995). Starting with approximately 100 eggs for each strain listed, worms that crawled off the plate or died from internal hatching were removed from the assay. In the longevity assays that included RNAi worms were grown on RNAi bacteria from the RNAi library (Kamath et al., 2003) for one generation prior to synchronization by timed egg-lay. The p-values given were calculated using the student’s t-test. Shown for each life span are the values for one replicate of two or three.  5.3.2 Translational ZIP-5::GFP reporter and promoter truncation analysis The translational ZIP-5::GFP reporter was created by cloning the genomic region including the exons, introns and 3.0 kb upstream regulatory region into the pPD95.77 GFP expression vector (Gift from Dr. Andrew Fire). RNAi was administered to this strain by feeding E. coli HT115 carrying a daf-16 RNAi clone (Gift from Dr. Di Chen) or the elt-2 RNAi clone from the RNAi library (Kamath et al., 2003). The promoter truncation analysis was performed as previously described (Mah et al., 2010). Specific  !  "#$!  promoter truncation sizes were chosen to include or exclude the IRE, DBE and GATA binding elements within the zip-5 promoter. Four to five independent transgenic strains were created for each promoter size.  !  "#$!  5.4 Bibliography An, J. H., and Blackwell, T. K. (2003). SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. Genes & Development 17, 1882-1893. An, J. H., Vranas, K., Lucke, M., Inoue, H., Hisamoto, N., Matsumoto, K., and Blackwell, T. K. (2005). Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3. Proc. Natl. Acad. Sci. U.S.A 102, 16275-16280. Artal-Sanz, M., and Tavernarakis, N. (2009). Prohibitin couples diapause signalling to mitochondrial metabolism during ageing in C. elegans. Nature 461, 793-797. Berman, J. R., and Kenyon, C. (2006). Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell 124, 1055-1068. Budovskaya, Y., Wu, K., Southworth, L., Jiang, M., Tedesco, P., Johnson, T., and Kim, S. (2008). An elt-3/elt-5/elt-6 GATA Transcription Circuit Guides Aging in C. elegans. Cell 134, 291-303. Friedman, D. B., and Johnson, T. E. (1988). A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility. Genetics 118, 75-86. Furuyama, T., Nakazawa, T., Nakano, I., and Mori, N. (2000). Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues. Biochem. J 349, 629-634. Gengyo-Ando, K., and Mitani, S. (2000). Characterization of mutations induced by ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans. Biochem. Biophys. Res. Commun 269, 64-69. Goven, D., Boutten, A., Leçon-Malas, V., Marchal-Sommé, J., Amara, N., Crestani, B., Fournier, M., Lesèche, G., Soler, P., Boczkowski, J., et al. (2008). Altered Nrf2/Keap1-Bach1 equilibrium in pulmonary emphysema. Thorax 63, 916-924. Halaschek-Wiener, J., Khattra, J. S., McKay, S., Pouzyrev, A., Stott, J. M., Yang, G. S., Holt, R. A., Jones, S. J. M., Marra, M. A., Brooks-Wilson, A. R., et al. (2005). Analysis of long-lived C. elegans daf-2 mutants using serial analysis of gene expression. Genome Res 15, 603-615. Igarashi, K., and Sun, J. (2006). The heme-Bach1 pathway in the regulation of oxidative stress response and erythroid differentiation. Antioxid. Redox Signal 8, 107-118.  !  "#$!  Jensen, V. L., Gallo, M., and Riddle, D. L. (2006). Targets of DAF-16 involved in Caenorhabditis elegans adult longevity and dauer formation. Experimental gerontology 41, 922-7. Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231-7. Kenyon, C., Chang, J., Gensch, E., Rudner, A., and Tabtiang, R. (1993). A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464. Kim, J., and Struhl, K. (1995). Determinants of half-site spacing preferences that distinguish AP-1 and ATF/CREB bZIP domains. Nucleic Acids Res 23, 25312537. Larsen, P. L., Albert, P. S., and Riddle, D. L. (1995). Genes that regulate both development and longevity in Caenorhabditis elegans. Genetics 139, 1567-83. Lee, S. S., Kennedy, S., Tolonen, A. C., and Ruvkun, G. (2003). DAF-16 target genes that control C. elegans life-span and metabolism. Science (New York, N.Y 300, 644-7. Mah, A., Tu, D., Johnsen, R., Chu, J., Chen, N., and Baillie, D. (2010). Characterization of the octamer, a cis-regulatory element that modulates excretory cell geneexpression in Caenorhabditis elegans. BMC Molecular Biology 11, 19. McElwee, J., Bubb, K., and Thomas, J. H. (2003). Transcriptional outputs of the Caenorhabditis elegans forkhead protein DAF-16. Aging cell 2, 111-21. Murphy, C. T., McCarroll, S. A., Bargmann, C. I., Fraser, A., Kamath, R. S., Ahringer, J., Li, H., and Kenyon, C. (2003). Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277-83. Nasrin, N., Ogg, S., Cahill, C. M., Biggs, W., Nui, S., Dore, J., Calvo, D., Shi, Y., Ruvkun, G., and Alexander-Bridges, M. C. (2000). DAF-16 recruits the CREBbinding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells. Proceedings of the National Academy of Sciences of the United States of America 97, 10412 -10417. Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A., and Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389, 994-999. Oh, S. W., Mukhopadhyay, A., Dixit, B. L., Raha, T., Green, M. R., and Tissenbaum, H. A. (2006). Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation. Nature Genetics 38,  !  "#$!  251-7. Oliveira, R. P., Porter Abate, J., Dilks, K., Landis, J., Ashraf, J., Murphy, C. T., and Blackwell, T. K. (2009). Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell 8, 524-541. Rea, S., and Johnson, T. E. (2003). A metabolic model for life span determination in Caenorhabditis elegans. Dev. Cell 5, 197-203. Ruzanov, P., Riddle, D. L., Marra, M. A., McKay, S. J., and Jones, S. M. (2007). Genes that may modulate longevity in C. elegans in both dauer larvae and long-lived daf-2 adults. Exp. Gerontol 42, 825-839. Shaw, W. M., Luo, S., Landis, J., Ashraf, J., and Murphy, C. T. (2007). The C. elegans TGF-beta Dauer pathway regulates longevity via insulin signaling. Curr. Biol 17, 1635-1645. Tullet, J. M. A., Hertweck, M., An, J. H., Baker, J., Hwang, J. Y., Liu, S., Oliveira, R. P., Baumeister, R., and Blackwell, T. K. (2008). Direct inhibition of the longevitypromoting factor SKN-1 by insulin-like signaling in C. elegans. Cell 132, 10251038. Vinson, C. R., Hai, T., and Boyd, S. M. (1993). Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design. Genes Dev 7, 1047-1058.  !  "#$!  CHAPTER 6: General discussion  6.1 DAF-25/Ankmy2 as a novel cilia protein The cloning and characterization of DAF-25/Ankmy2 described in this thesis sheds some light on the function of this conserved but previously uncharacterized protein. As shown in this thesis, DAF-25 is required for the proper cilia localization of DAF-11. A co-immunoprecipitation between the mammalian orthologs hints that this interaction may be direct and conserved. DAF-25 has two protein binding domains a MYND-type zinc finger and an Ankyrin repeat domain. Few MYND domain proteins have been functionally characterized but for those that have been examined the MYND domain is protein binding (Liu et al., 2007). It is not known which domain of DAF-25 binds DAF11. The MYND or Ankyrin repeats may bind the guanylyl cyclase. It could also be shown which guanylyl cyclase sequences are required for this interaction. The other domains of DAF-25 it could bind an IFT protein or another IFT cargo protein allowing for transport into the cilia. This would not be a novel mechanism as it has been shown for retinal membrane receptor proteins that one is required to transport the other indicating an order to assemble IFT cargo complexes (Karan et al., 2008). Another future direction for DAF-25/Ankmy2 research would be to sequence patients that carry Mendelian inherited disorders, which are known to be ciliopathies (Sharma et al., 2008). This would include LCA (Leber’s Congenital Amaurosis) and RP (Retinitis Pigmentosa) that result from mutations in GUCY2D (a homolog of DAF-11) and Nr2e3, which regulates Ankmy2 in mice (Kitiratschky et al., 2008; Haider et al., 2009). Another possible direction would be to determine the range of proteins that require  !  "#$!  DAF-25 for proper localization. This could be determined by yeast-2-hybrid analysis or co-IP followed by HLPC-MS/MS (Li et al., 2004; Moresco et al., 2010). There are some hints that the spectrum of interacting proteins may be more than guanylyl cyclases. In this thesis it is shown that daf-25 mutants have phenotypes that are not associated with the cGMP pathway, including Osm and inability to chemotax to pyrazine (Komatsu et al., 1996; Colbert et al., 1997).  6.2 SDF-9 interacts with DAF-2 SDF-9 is a phosphatase-like protein that lacks catalytic activity due to the mutation of the catalytic cysteine (Ohkura et al., 2003; Hu et al., 2006; Jensen et al., 2007). While not a lot of these proteins are well characterized this thesis represents the third time sdf-9 has been cloned based on the Daf phenotype. Proteins similar to SDF-9 that have been characterized have two functions. They bind to a phosphorylated protein at the phosphate to either protect the phosphate from removal by phosphatases or to act as an adaptor to bring another protein closer to the phosphorylated substrate (Wishart and Dixon, 1998). The genetic interactions reported to date for sdf-9 as well as those reported in this thesis show that SDF-9 may bind to DAF-2. This would cause further activation of the DAF-2 receptor, by keeping the activating phosphate protected from phosphatases. It would also enhance IIS signaling if SDF-9 were an adaptor protein to enhance interaction of DAF-2 with one or more of its downstream phosphorylation targets. Further refining the genetic interactions for SDF-9 have enabled us to better pinpoint the site of action for SDF-9.  !  "#$!  Further study of SDF-9 should include assaying for direct interaction with DAF-2. Co-IP with a DAF-2 peptide may have to be completed in vitro; co-IP from worms could be troublesome due to the size of DAF-2. Interacting proteins potentially could be identified through a yeast-2-hybrid screen or co-IP HPLC-MS/MS (Li et al., 2004; Moresco et al., 2010). This should be done in a background where DAF-2 is hyperactivated so that the interaction with SDF-9 is favored. Also, further characterization of the role of SDF-9 with the other EAK proteins may elucidate a novel-signaling pathway, possibly regulated by IIS signaling or as a novel downstream pathway from the DAF-2 receptor (Hu et al., 2006; Alam et al., 2010; Zhang et al., 2008). Because sdf-9 mutants do not exhibit a Daf-c phenotype, it makes for an optimal genetic background for a genetic screen. Combined with an RNAi enhancing background, sdf-9 proved useful for an RNAi screen for novel Daf or SynDaf genes (Kennedy et al., 2004; this thesis). The molecular lesion that was identified in sdf-9(m708) was a translocation of a Cemar1 transposon. This was first identified translocation event for this transposon in C. elegans. Cemar1 transposons encode what appears to be a functional transposase (Witherspoon and Robertson, 2003). However, the copy that hopped into sdf-9 has a frameshift (11 bp deletion) in the transposase unique among the copies of Cemar1. Because functional Cemar1 transposase is present and it is the largest copy number transposon in C. elegans it was presumed to be functional and active (Witherspoon and Robertson, 2003). Indeed this was shown with sdf-9(m708). Since identification of this allele of sdf-9 another suspected transposition event has been identified (Vergara et al., 2009).  !  "#$!  6.3 Novel SynDaf genes 21 SynDaf genes were identified in this thesis. Though one of these has been previously shown to have a SynDaf phenotype none of these has been confirmed by mutation of the corresponding gene (Oh et al., 2006). There is also inferred function for many of these proteins including a role in pathogen defense. For example, ccb-1 encodes the homolog of the regulatory ! subunit of the T-type calcium channel (Mathews et al., 2003). Though calcium signaling has been implicated in dauer formation in a conserved role of regulating insulin secretion, this has not been confirmed for CCB-1 (Speese et al., 2007; Komatsu et al., 1996). Despite the CUB domain being implicated in innate immunity only one example has been shown for the requirement of a CUB domain protein for proper defense against a pathogen (Shapira et al., 2006). Full characterization of each of these proteins may be taken up individually. The role identified in this thesis for each of these in dauer formation may stimulate interest in studying them further.  6.4 Pathogenesis as a novel input into the dauer larva signaling In this thesis bacterial pathogenesis was identified as a novel input into the dauer larva signaling pathway. Enhanced dauer larva formation in response to pathogenesis would be a favorable evolutionary trait. Firstly, dauer larvae do not eat, which would reduce individual uptake of the pathogenic bacteria. Dauer larvae have a thicker cuticle with most orifices plugged, which would also reduce ability to invade the host (Cassada and Russell, 1975). Dauer larvae are also a dispersal stage that could escape from the pathogenic environment.  !  "#"!  The requirement of the dauer pheromone for this increased dauer formation is intriguing. This indicates that upon infection there may be an increased production of pheromone or an increased sensitivity to the dauer pheromone. Using exogenous pheromone in the daf-22 mutant background, which doesn’t produce its own pheromone, could identify the distinction between the two possibilities (Golden and Riddle, 1985; Butcher et al., 2009). The pheromone is also a chemo-repellant to adult hermaphrodites (Srinivasan et al., 2008). This suggests that the pheromone may also demarcate a pathogenic environment to allow individual adults to sense this by pheromone concentration. This would be a population level control of behavior and development by secretion of a pheromone.  6.5 ZIP-5 as a SKN-1 opposing longevity inhibitor The link between SKN-1 and longevity has been well established and extensively examined (An et al., 2005; Tullet et al., 2008; Oliveira et al., 2009). The IIS pathway’s role in longevity has also been the subject of a large body of work (Kenyon et al., 1993; Kenyon, 2010). It has also been shown that the IIS pathway regulates SKN-1. This thesis shows that ZIP-5 function inhibits longevity and that ZIP-5 is a downstream target of the IIS pathway. It is also shown that the extension in life span exhibited by zip-5 mutants requires SKN-1. SKN-1 has been shown to regulate the increase in longevity induced by dietary restriction as well as endoderm development (Bishop and Guarente, 2007; Bowerman et al., 1992). Regulation of dietary restriction induced longevity requires the ASI pair of neurons. Because ZIP-5 is also expressed in these two neurons, it may also play a role in  !  "#$!  dietary restriction induced longevity. SKN-1 also plays a role in the signaling cascade that controls endoderm differentiation. ZIP-5 is highly expressed at the comma stage of embryogenesis when there are eight gut cells. It is also not determined if zip-5 transcripts are packaged in the embryo as are skn-1 (Bowerman et al., 1993). It would be of great interest to determine if the opposing roles SKN-1 and ZIP-5 play are found not only in adult life span, but also in dietary restriction as well as development.  !  "##!  References Alam, H., Williams, T. W., Dumas, K. J., Guo, C., Yoshina, S., Mitani, S., and Hu, P. J. (2010). EAK-7 controls development and life span by regulating nuclear DAF16/FoxO activity. Cell Metab 12, 30-41. An, J. H., Vranas, K., Lucke, M., Inoue, H., Hisamoto, N., Matsumoto, K., and Blackwell, T. K. (2005). Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3. Proc. Natl. Acad. Sci. U.S.A 102, 16275-16280. Bishop, N. A., and Guarente, L. (2007). Two neurons mediate diet-restriction-induced longevity in C. elegans. Nature 447, 545-549. Bowerman, B., Draper, B., Mello, C., and Priess, J. (1993). The maternal gene skn-1 encodes a protein that is distributed unequally in early C. elegans embryos. Cell 74, 443-452. Bowerman, B., Eaton, B. A., and Priess, J. R. (1992). skn-1, a maternally expressed gene required to specify the fate of ventral blastomeres in the early C. elegans embryo. Cell 68, 1061-1075. Butcher, R. A., Ragains, J. R., Li, W., Ruvkun, G., Clardy, J., and Mak, H. Y. (2009). 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(2008). A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115-1118. Tullet, J. M. A., Hertweck, M., An, J. H., Baker, J., Hwang, J. Y., Liu, S., Oliveira, R. P., Baumeister, R., and Blackwell, T. K. (2008). Direct inhibition of the longevitypromoting factor SKN-1 by insulin-like signaling in C. elegans. Cell 132, 10251038. Vergara, I., Mah, A., Huang, J., Tarailo-Graovac, M., Johnsen, R., Baillie, D., and Chen, N. (2009). Polymorphic segmental duplication in the nematode Caenorhabditis elegans. BMC Genomics 10, 329. Wishart, M. J., and Dixon, J. E. (1998). Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem. Sci 23, 301306. Witherspoon, D. J., and Robertson, H. M. (2003). Neutral Evolution of Ten Types of mariner Transposons in the Genomes of Caenorhabditis elegans and Caenorhabditis briggsae. Journal of Molecular Evolution 56, 751-769.  !  "#$!  Zhang, Y., Xu, J., Puscau, C., Kim, Y., Wang, X., Alam, H., and Hu, P. J. (2008). Caenorhabditis elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity. Dev. Biol 315, 290-302.  !  "#$!  APPENDIX A: Supplementary information for Chapter 2 Table S1 - Dauer Formation of daf-25(m362) compared to daf-11(m84). Genotype daf-25(m362) daf-11(m84)  !  % dauer 25 °C 99.3% 99.1%  N 286 343  % dauer 20 °C 15.1% 15.6%  N 126 77  % dauer 15 °C 0% 0%  N >200 >200  "#$!  Figure S1 - Lifespan phenotype of daf-25. Lifespan of daf-25(m362) does not significantly differ from wild type N2. Mean lifespan was 12.3 for daf-25 (n=96) compared to 13.2 for N2 (n=88) while the maximum lifespan was 20 days for both (p = 0.08, t-test). Shown is one replicate of two. Survival was assayed at 25 °C.  !  "#$!  Figure S2 - Dye filling of daf-25 mutants. Dye filling assay showing daf-25(m362) and daf-25(m98) compared to the wild type N2. Worms were incubated for 1 hour in 0.1% DiI in M9 buffer. No difference was detected between the two daf-25 alleles m98 and the wild type N2.  !  "#$!  !  "#"!  Figure S3 - Cilium ultrastructure is normal in daf-25 mutants. Shown are TEM serial cross sections of an amphid channel from N2 and daf-25(m362) L2-staged worms. In the six pairs of images, low magnification images (B, D, F, H, J, L) are presented on the left and one axoneme from the left image is shown in high magnification on the right (C, E, G, I, K, M). (A) Schematic of an amphid pore and channel from wild-type adult N2 worms. 10 ciliary axonemes (only three shown in longitudinal section) extend from the distal dendrite tips (den) into the lumen of the amphid pore, which is created by channel cilia invaginating surrounding support cells (sheath, socket). Channel have a ~1 µm long transition zone (tz) at the ciliary base, consisting of a constricted ring of 9 outer doublet microtubules (MTs), connected to the ciliary membrane via Y-link connections. This is followed by a 'middle segment' of ~4 µm, consisting of a ring of 9 outer doublet MTs, along with a varying number of inner singlet MTs. At the middle segment tip, the Btubule of each doublet MT terminates, with the A-tubule extending to form the characteristic singlet MT structure of the 'distal segment'. (B-E) Distal segment region of amphid cilia showing that N2 (B, C) and daf-25 (C-E) worms both possess 10 MT-singlet containing axonemes. (F-I) 4 µm (N2) or 5 µm (daf-25) proximal to B-E (through middle segments). Both N2 and daf-25 animals possess axonemes of similar number and MT ultrastructure (e.g., doublet MTs). Interestingly, 9 outer doublet MTs are not always observed in N2 and daf-25 worms (F, H), indicating that L2-staged worms lack a full complement of MTs (currently under investigation in Blacque lab). (J-M) 6 µm proximal to B-E (through transition zones and distal dendrites). Transition zones appear identical in N2 and daf-25 worms, with Y-links (arrow) and the internal apical ring (arrowhead) clearly visible and intact. Scale bars; 200 nm. !  "#$!  !  "#$!  Figure S4 - Alignment of DAF-25 with Ankmy2. C. elegans (Ce) DAF-25 was aligned with Homo sapiens (Hs), Bos taurus (Bt), Mus musculus (Mm), and Danio rerio (Dr). The red bar indicates the ankyrin repeat domain and the blue bar indicates the zinc finger MYND domain. White font on black background indicates conservation in all five species, white font on grey indicates four, and black font on grey indicates three. Ankmy2 is very well conserved among chordates, with identity percentages compared to human Ankmy2 of 93% for cow, 88% for mouse, and 76% for zebrafish while DAF-25 shares 32% identity.  !  "##!  !  "#$!  Figure S5 - The daf-25 transcript including allele and UTR information. Displayed is the sequence of the daf-25 transcript including the molecular lesions in the four daf-25 alleles. A line over the sequence indicates the extent of the deletion. A line under the amino acid sequence indicates the two protein domains including the ankyrin repeat domain in the first half of the sequence and the zinc-finger MYND domain near the Cterminus of the sequence.  !  "#$!  !  "#$!  Figure S6 - Many cilia targeted proteins localize normally in daf-25(m362). Shown are the localization patterns of the translational fusion constructs BBS-8::GFP, CHE2::GFP, CHE-11::GFP, CHE-13::GFP, OSM-5::GFP and XBX-1::GFP. All six of these GFP-tagged proteins localize normally to the cilia in both N2 and daf-25(m362) mutants[comma] indicating that DAF-25 is unlikely to be a core IFT complex component. For each genotype and transgenic construct the left panels are the anterior or amphid cilia and the right panels are the posterior or phasmid cilia. Arrowheads denote basal body regions whereas brackets show the ciliary axonemes.  !  "#$!  !  "#$!  Figure S7 - DAF-11::GFP localization in N2, daf-25(m362), and daf-25(m362); daf12(sa204). Despite suppressing the dauer phenotype of daf-25, daf-12 does not suppress the cilia mislocalization of DAF-11::GFP in daf-25(m362). This indicates that entry into the dauer stage does not cause the mislocalization of DAF-11::GFP.  !  !  "#$!  Appendix B: Supplementary information for Chapter 4  Figure S1 - Reduction in daf-7 expression on PA14. In (A), the native GFP expression on the standard laboratory food E. coli OP50 from a daf-7 promoter driving expression of GFP. The expression of daf-7 is much reduced after are exposure to the strong pathogen PA14, as seen in (B). Images were taken with a 100x objective and 10x ocular lenses, eight hours after L2 larvae were transferred to either OP50 or PA14 from OP50 plates.  !  "#"!  Table S1 - Full list of target RNAi clones B0024.4 B0024.6 B0213.15 B0281.3 B0281.5 B0365.6 B0491.5 B0496.4 B0496.7 B0511.8 B0513.5 B0554.6 C01A2.2 C01A2.3 C01B7.1 C01B7.3 C01F1.2 C03C10.2 C03C10.5 C03E10.6 C03G6.15 C04F5.1 C04F6.1 C05A9.1 C05B5.1 C05B5.2 C05C10.4 C05D12.2 C05D2.7 C05D2.8 C05D9.8 C05E11.5 C05G5.6 C06B3.3 C06E1.6 C07A9.4 C07B5.5 C07E3.9 C07G3.9 C07H6.5 C08B11.4 C08B6.10 C08F11.11 C08F11.12 C08F11.8 C08F8.5 C08H9.5 C09D4.5 C09F12.1 F57F4.3  !  C09G1.1 C09G4.5 C10C5.4 C10C5.5 C10G8.4 C12C8.2 C12D12.1 C12D5.9 C14A6.1 C14E2.2 C14F5.1 C15C6.3 C15H9.7 C15H9.9 C16A3.10 C16C10.5 C16C4.4 C16H3.2 C17B7.1 C17D12.2 C17D12.6 C17H12.6 C17H12.8 C18E3.9 C18E9.5 C18H7.4 C18H7.6 C18H9.6 C24G6.6 C25A8.4 C25D7.5 C25E10.1 C25H3.9 C27A7.1 C28F5.3 C29A12.3 C29F3.7 C29F7.3 C31A11.5 C32D5.5 C32E8.11 C32H11.1 C32H11.10 C32H11.12 C32H11.13 C32H11.2 C32H11.4 C32H11.9 C34H4.1 K12H4.7  C34H4.2 C35E7.1 C35E7.5 C36A4.9 C37A2.1 C39D10.7 C39E9.1 C41H7.7 C42C1.7 C43D7.4 C43D7.5 C44B12.1 C44B7.5 C44F1.2 C46A5.5 C46E10.3 C46E10.4 C46E10.7 C47B2.6 C47B2.8 C47D12.6 C48B4.1 C48E7.2 C49C3.9 C50E3.12 C52D10.7 C52D10.9 C52E4.1 C53A3.2 C53B7.2 C54D1.2 C54G4.6 C56E6.1 D1025.4 D1054.10 D1054.11 D2013.7 D2023.7 D2045.6 D2085.3 E02C12.6 E02C12.7 E02C12.8 E03H4.11 EGAP2.3 F01D4.2 F01D5.3 F01D5.5 F01G10.3 T12D8.1  F02A9.3 F02G3.1 F07F6.1 F07F6.5 F08A8.2 F08C6.2 F08D12.2 F08F1.4 F08F3.4 F08G5.6 F08H9.8 F08H9.9 F09B9.1 F09E10.1 F09F3.9 F09G8.8 F10A3.9 F10D11.1 F10D7.5 F10G2.3 F11A5.10 F11C1.3 F12B6.2 F13A7.11 F13A7.9 F13C5.4 F13H6.3 F14B4.3 F14B6.3 F14B8.3 F14H3.12 F15E11.1 F15E11.12 F15E11.7 F15E11.9 F16H6.1 F17E5.1 F17E9.11 F18A1.7 F18E2.1 F18E9.5 F19C6.4 F19C7.1 F19C7.2 F19C7.4 F19C7.6 F19F10.8 F20C5.2 F20H11.5 Y119D3_451.B  F21D5.3 F21F8.7 F22D6.3 F22H10.3 F23A7.4 F23B2.12 F23F12.3 F23H11.7 F26E4.4 F27B3.5 F28B4.3 F28C1.3 F28D1.4 F28D1.5 F28G4.1 F29G9.1 F31C3.6 F31F7.1 F32A5.3 F32H5.1 F34D10.4 F35C5.9 F35E12.10 F35E12.5 F35E12.7 F35E12.8 F35E12.9 F35E2.2 F35E8.11 F35F10.10 F36F2.1 F36G9.12 F36H5.8 F37F2.3 F38A1.10 F38A1.9 F40A3.7 F40F12.2 F40F12.3 F40F4.1 F40F4.2 F40F4.4 F41A4.1 F41E7.4 F42G10.1 F42G4.5 F44D12.8 F44E7.2 F44G4.1 Y71H10B.1  F45C12.7 F45D3.2 F46B6.8 F46E10.1 F46F3.3 F46G10.3 F46G10.4 F47C10.2 F47G4.3 F48E3.4 F49C12.14 F49C12.7 F49E12.1 F49E12.2 F49E12.9 F49F1.1 F49F1.5 F52D2.6 F52E1.1 F52E1.5 F53A9.8 F53C11.1 F53C3.2 F53F4.8 F53G12.5 F53G2.6 F53H4.2 F54B11.3 F54D10.8 F54D5.4 F54E2.1 F54F11.2 F54F2.2 F54F3.1 F54F7.2 F55B11.1 F55G11.2 F55G11.5 F55G11.8 F56A4.G F56A4.j F56A4.K F56B6.1 F56C9.7 F56D5.5 F56F3.2 F56G4.2 F56G4.3 F57C2.4  "#$!  F57F4.4 F57F5.1 F57G4.1 F58A3.1 F58A4.3 F58A4.7 F58B3.9 F58B4.5 F58G1.4 F59A3.3 F59B1.2 F59C6.4 F59C6.5 F59D8.A F59D8.B F59D8.C F59D8.d F59D8.E F59D8.F H04D03.1 H12C20.2 H13N06.6 H19N07.1 H20E11.1 K01A2.5 K01C8.5 K01C8.6 K01G5.3 K02B12.2 K02H11.2 K04E7.2 K04H4.6 K05F1.10 K06A9.1 K06B9.4 K06G5.1 K07C6.5 K08C7.6 K08D8.5 K08D8.6 K08E5.3 K09D9.1 K09D9.2 K09F5.2 K09H11.7 K10D11.1 K11B4.1 K11D9.2  !  M01F1.7 M01H9.3 M02F4.3 M02F4.7 M03C11.5 M03F4.7 M04D8.1 M163.3 M28.8 M60.1 M7.2 R03G5.5 R03G8.3 R03G8.6 R05F9.10 R05F9.12 R07B1.10 R08E3.1 R09B5.3 R09H10.5 R09H3.3 R102.4 R107.8 R10H10.3 R13H4.3 R193.D R53.4 T01D3.6 T03D3.1 T03E6.7 T03F6.1 T05A10.1 T05A8.5 T05D4.2 T05E7.1 T06G6.11 T07F10.1 T07H3.3 T08A9.8 T08G5.10 T09E11.11 T09E11.2 T10B11.3 T10C6.8 T10G3.3 T11F9.12 T11F9.3 T12B5.10  T13B5.3 T13F2.8 T15B7.9 T16A9.1 T16G1.4 T16G1.6 T16G12.1 T16G12.4 T16G12.7 T16H12.1 T16H12.8 T17A3.7 T19C9.8 T19D12.4 T20B3.12 T21C12.2 T21G5.2 T22G5.2 T24B8.3 T24B8.5 T25B6.2 T25C12.3 T25E12.6 T27A10.4 T28F2.5 VW02B12L.1 W01A11.4 W01B11.3 W02B12.1 W02D9.6 W02D9.7 W02H3.1 W03G1.7 W04A4.2 W04E12.6 W04G3.6 W05F2.2 W06B11.3 W07G4.2 W07G4.3 W08D2.3 W09C3.7 Y106G6H.10 Y106G6H.9 Y116A8A.2 Y116A8C.17 Y116A8C.35 Y116F11A.H  Y119D3_457.C Y14H12B.2 Y17G7B.8 Y17G9A.E Y19D10A.j Y19D10A.K Y22F5A.4 Y37D8A.12 Y38H6C.1 Y38H6C.3 Y38H6C.5 Y39C12A.A Y39G10A_243.C Y41D4B_7946.B Y43C5B.2 Y43F4A.3 Y43F8B.9 Y43F8C.16 Y43F8C.2 Y45F10A.2 Y45F10C.2 Y45F10C.4 Y46C8_100.B Y46C8_103.a Y46C8_95.B Y46H3B.B Y46H3C_14.c Y47H10A.F Y47H9C.1 Y48A6B.6 Y48A6B.7 Y48A6C.1 Y49E10.1 Y49E10.8 Y51A2D.13 Y51A2D.4 Y53F4C.J Y55F3A_747.a Y55F3A_748.b Y56A3A.15 Y57A10C.7 Y57G11B.5 Y59E1B.2 Y62H9A.3 Y62H9A.4 Y62H9A.5 Y62H9A.6 Y67D8A_380.C  Y75B8A.4 ZC155.6 ZC266.2 ZC302.2 ZC416.6 ZC443.5 ZK105.F ZK1127.10 ZK1127.3 ZK1193.2 ZK1193.4 ZK1251.2 ZK1290.6 ZK1320.1 ZK177.8 ZK218.6 Zk218.8 ZK228.3 ZK228.4 ZK484.2 ZK546.13 ZK546.17 ZK6.10 ZK6.7 ZK688.6 ZK757.1 ZK757.2 ZK813.2 ZK896.2 ZK896.5 ZK896.7 ZK896.8 ZK899.8  "#$!  Table S2 - Dauer and Non-dauer worm counts for positive genes. Count 1 Gene  Non-dauer  Dauer larva  Total  Percent Dauer  !2 p-value  GFP  71  9  80  11.3%  akt-1  88  58  146  39.7%  1.3E-27  srh-100  42  28  70  40.0%  2.7E-14  104  61  165  37.0%  1.4E-25  F44D12.8  73  17  90  18.9%  2.2E-02  ZK896.5  53  19  72  26.4%  4.8E-05  skr-8  80  29  109  26.6%  3.9E-07  dct-5  16  10  26  38.5%  1.1E-05  F52E1.5  43  11  54  20.4%  3.4E-02  cpr-1  37  24  61  39.3%  3.8E-12  Total  Percent Dauer  !2 p-value  lase-1  Count 2 Gene  Dauer larva  GFP  150  11  161  6.8%  akt-1  14  32  46  69.6%  8.3E-64  srh-100  35  50  85  58.8%  1.7E-80  lase-1  9  26  35  74.3%  2.4E-56  F44D12.8  2  6  8  75.0%  2.1E-14  cyp-35A3  8  34  42  81.0%  8.1E-81  C24G6.6  14  33  47  70.2%  1.8E-66  dct-17 F35E12.9 F35E12.10 dct-14 clc-1 unc-84 ccb-1 F59B1.2 C53A3.2 E02C12.8 lys-1 Count 3 Gene GFP akt-1  2 49 46 91 14 36 72 71 69 75 39  19 31 40 49 18 9 30 64 25 32 15  21 80 86 140 32 45 102 135 94 107 54  90.5% 38.8% 46.5% 35.0% 56.3% 20.0% 29.4% 35.4% 47.4% 26.6% 29.9%  4.0E-52 1.1E-29 3.5E-48 7.7E-40 1.6E-28 4.6E-04 1.6E-19 1.2E-28 6.5E-78 3.1E-14 3.1E-21  Total 109 417  Percent Dauer 1.8% 71.9%  !2 p-value  2 300  Non-dauer 107 117  Dauer larva  0  ZK896.5  29  38  67  56.7%  1.2E-245  skr-8  56  22  78  28.2%  1.9E-67  cyp-35A3  119  16  135  11.9%  4.2E-18  C24G6.6  23  9  32  28.1%  1.5E-28  259  39  298  13.1%  1.8E-47  93  13  106  12.3%  1.2E-15  dct-17 F35E12.9  !  Non-dauer  "#$!  F35E12.10  290  29  319  9.1%  4.6E-22  dct-5  428  41  469  8.7%  7.5E-29  F59B1.2  135  53  188  28.2%  1.1E-159  C53A3.2  244  77  321  24.0%  3.3E-192  F52E1.5  78  8  86  9.3%  2.5E-07  E02C12.8  275  121  396  30.6%  0  lys-1  121  30  151  19.9%  3.1E-61  cpr-1 Count 4 Gene GFP akt-1  71  27  98  27.6%  3.1E-80  Non-dauer 633 213  Dauer larva 37 28  Total 670 241  Percent Dauer 5.5% 11.6%  !2 p-value  srh-100  224  38  262  14.5%  2.0E-10  51  216  267  80.9%  0  F44D12.8  142  225  367  61.3%  0  ZK896.5  228  69  297  23.2%  1.0E-40  skr-8  173  60  233  25.8%  1.2E-41  cyp-35A3  98  14  112  12.5%  1.2E-03  C24G6.6  205  45  250  18.0%  5.8E-18  dct-17  305  50  355  14.1%  1.6E-12  F35E12.9  51  39  90  43.3%  1.4E-55  F35E12.10  215  25  240  10.4%  9.0E-04  dct-14  108  49  157  31.2%  4.3E-45  clc-1  38  42  80  52.5%  1.4E-75  unc-84  147  114  261  43.7%  2.1E-160  ccb-1  223  44  267  16.5%  4.6E-15  Total 452 342  Percent Dauer 1.3% 14.9%  !2 p-value  6 51  lase-1  Count 5 Gene GFP akt-1  Non-dauer 446 291  F59B1.2  356  29  385  7.5%  2.0E-26  C53A3.2  390  37  427  8.7%  4.6E-40  F52E1.5  595  50  645  7.8%  4.1E-46  E02C12.8  399  25  424  5.9%  2.0E-16  lys-1  333  27  360  7.5%  1.4E-24  cpr-1 Count 6 Gene GFP akt-1  196  78  274  28.5%  0  Non-dauer 209 227  Dauer larva 18 53  Total 227 280  Percent Dauer 7.9% 18.9%  !2 p-value  dct-14  141  71  212  33.5%  3.6E-43  clc-1  177  89  266  33.5%  1.4E-53  99  25  124  20.2%  4.6E-07  unc-84  !  3.4E-05  Dauer larva  8.4E-107  9.7E-12  "##!  !  ccb-1  188  74  262  28.2%  4.5E-34  dct-5 Count 7 Gene GFP akt-1  150  22  172  12.8%  1.8E-02  Non-dauer 217 18  Dauer larva 10 71  Total 227 89  Percent Dauer 4.4% 79.8%  !2 p-value  F44D12.8  82  21  103  20.4%  2.7E-15  dct-17  83  33  116  28.4%  1.7E-36  dct-5  117  22  139  15.8%  5.3E-11  4.6E-263  "#$!  APPENDIX C1: Targets of DAF-16 involved in Caenorhabditis elegans adult longevity and dauer formation  !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! "!%!&'()*+,!+-!./*)!0/12.'(!/1)!3'',!2435*)/'67!)''8!9',)',!:;7!<155+!=!1,6!>*665'!?;! @ABBCD!E1(F'.)!+-!?%GH"C!*,&+5&'6!*,!!"#$%&'"()*+*,-#.#/"$,!1645.!5+,F'&*.I!1,6! 614'(!-+(J1.*+,K!012#&*3#$+".-4#&%$+%.%/5!L"MNAAHNA$K! !  "#$!  !  "#$!  !  "#$!  !  "#$!  !  "#"!  !  "#$!  

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