David S Peal et al. (2003) International Worm Meeting "Negative Regulation of FGF Signaling By Heparan Sulfate in Caenorhabditis elegans"

David S Peal, Laura L Kiessling, Beth Stadtmueller, Judith Kimble

[International Worm Meeting, 2003]

Heparan sulfate (HS) proteoglycans are proteins that display sulfated carbohydrate polymers. HS proteoglycans are implicated in numerous biological processes, including signal transduction and modulation of proteolytic cascades. The 3-O sulfate group found on the N-acetylglucosamine moiety of HS is particularly intriguing. The only known role of this group is to modulate the coagulation cascade in mammals, yet multiple genes encoding 3-O sulfotransferases and their spliced isoforms are expressed in complex and tissue-specific patterns during development. These observations imply that 3-O sulfation may have other functions. To investigate the role of 3-O sulfation, we have isolated a null mutant of the 3-O sulfotransferase (3OST) gene in C. elegans. This mutant is clear (clr) at a low penetrance (35%). This previously described phenotype is caused by upregulation of the FGF signaling pathway(1). Our results suggest that the 3-O sulfate group of HS is involved in negative regulation of FGF signaling. We are testing this hypothesis through genetic and biochemical experiments. (1) Kokel, M, Borland, CZ, DeLong, L, Horvitz, HR, Stern, MJ. Genes Dev 1998; 12: 1425-37.

Simon, Matt A et al. (2011) International Worm Meeting "The DAF-2 somatic longevity pathway interfaces with PRG-1-mediated germ cell immortality."

Simon, Matt A, Miska, Eric, Ahmed, Shawn

[International Worm Meeting, 2011]

Mutations in the C. elegans Piwi Argonaute orthologue prg-1 have previously been reported to result in reduced levels of thousands of germline-specific piRNAs as well as temperature-sensitive sterility (1, 2, 3). We find that outcrossing of prg-1 mutations results in homozygous strains with normal levels of fertility, but that propagation of these strains for 15 to 40 generations results in sterility. prg-1 mutants have previously been reported to display increased levels of Tc3 transposition. However, stereotypical germ cell development phenotypes occur at sterility, suggesting that progressive sterility is not induced by random Tc3-mediated mutations but rather by another form of heritable stress. Repression of daf-2 is well known to result in stress resistance and increased somatic lifespan in C. elegans (4). Independent daf-2 mutations were able to suppress the progressive sterility phenotype of prg-1. Thus, reduced DAF-2-mediated insulin/IGF1 signaling can repress post-mitotic aging in somatic cells as well as proliferative aging in the germ line. These observations defy a prediction of the antagonistic pleiotropy theory of aging (5), which suggests that prolonged lifespan might result in compromised fertility. References: 1. Batista PJ, Ruby JG, Claycomb JM, Chiang R, Fahlgren N, Kasschau KD, Chaves DA, Gu W, Vasale JJ, Duan S, Conte Jr. D, Luo S, Schroth GP, Carrington JC, Bartel DP, Mello CC. Molecular Cell (2008) 2. Das PP, Bagijn MP, Goldstein LD, Woolford JR, Lehrbach NJ, Sapetschnig A, Buhecha HR, Gilchrist MJ, Howe KL, Stark R, Matthews N, Berezikov E, Ketting RF, Tavare S, Miska EA. Molecular Cell (2008) 3. Wang G, Reinke V. Current Biology (2008) 4. Kenyon C, Chang J, Gensch E, Rudner A. Nature (1993) 5. Willims GC. Evolution (1957).

Adler CE et al. (2000) West Coast Worm Meeting "In vivo imaging of HSN outgrowth"

Bargmann CI, Adler CE

[West Coast Worm Meeting, 2000]

Axons in developing organisms must migrate and extend long distances in order to synapse onto their proper targets. Little is known about how extracellular guidance cues elicit the changes in cytoskeletal dynamics that drive axon outgrowth. In order to understand the factors that control the development of the HSN motorneuron, we are undertaking an observational approach. We are utilizing two-photon microscopy to analyze the trajectory of HSN as it grows ventrally in late L2/early L3 stage worms. Using worms expressing GFP under the unc-86 promoter, we are able to visualize HSN during the course of its outgrowth. Preliminary imaging indicates that HSN initially extends filopodia in all directions from its cell body, but that only those filopodia facing the ventral side persist. Eventually these filopodia develop into a large lamellopod that extends ventrally and stalls at the ventral muscle barrier. A few processes protrude through the body wall muscle attachment sites, as seen in VD and DD motorneuron growth through the dorsal muscle attachment sites.1 Although multiple processes extend toward the ventral nerve cord, only one is stabilized and develops into the axon. We plan to characterize HSN outgrowth over the course of its development. With this information, we can analyze the HSN axon in mutant worms known to have defects in ventral growth. In particular, we will examine worms containing mutations in the extracellular guidance cues to which HSN responds as well as in cytoplasmic proteins thought to participate in the regulation of cytoskeletal dynamics. In addition, we plan to label the actin and microtubule cytoskeletons and visualize their dynamics during HSN outgrowth. This approach will allow us to elucidate the roles of particular proteins in the process of HSN outgrowth. 1. Knobel KM, Jorgensen EM, Bastiani MJ. Development. 1999 Oct;126(20):4489-98.

Carolyn Adler et al. (2001) International C. elegans Meeting "In Vivo Imaging of HSN Growth Cones"

Carolyn Adler, Cori Bargmann

[International C. elegans Meeting, 2001]

A growth cone at the leading edge of a developing neuron responds to environmental cues to spatially direct growth by regulating the underlying cytoskeleton. We are studying the development of the HSN motorneuron in C. elegans using time-lapse confocal microscopy, as pioneered by Knobel et al. for VD motorneurons. This approach allows us to combine in vivo imaging with genetics so that we can begin to understand the extracellular and intracellular factors that control axon guidance in worms. Using an unc-86 transgene to drive expression of soluble GFP, we have developed a detailed description of growth cone dynamics in HSN during its development. The cell body of HSN is located in a lateral position slightly posterior to the vulva and has a single ventral process that fasciculates onto the ventral nerve cord and grows anteriorly to the nerve ring. In wild-type worms, HSN begins to extend filopodia and lamellipodia ventrally in the late L2 stage. Ventral growth of the lamellopod continues until it is impeded by the muscle attachment sites between the ventral body wall muscles and the hypodermis. After stalling for approximately 2-4 hours, HSN sends multiple processes through these attachment sites toward the ventral nerve cord. These processes also extend filopodia in all directions. Eventually only one of these processes is stabilized and becomes the axon, while the others retract. The stalling/outgrowth/retraction sequence is similar to what Knobel et al. have described for the VD motorneurons. By early L4, ventral growth is complete and the growth cone has begun its anterior trajectory. HSN ventral guidance is controlled by attraction to a ventral source of UNC-6/netrin and by repulsion from a dorsal source of SLIT. In unc-6 and unc-40 null mutants, HSN lacks the ventral component of axon outgrowth and instead grows anteriorly along the lateral hypodermis. We are currently asking when this defect arises. We are also generating other markers to visualize the cytoskeleton and the leading edge of growth cones. ________________ Knobel KM, Jorgensen EM, Bastiani MJ. (1999 ) Development . 126:4489-98.

Akay, Alper et al. (2015) International Worm Meeting "Proteomic analysis of the nuclear RNAi pathway identifies EMB-4/AQR in transposable element regulation."

Larance, Mark, Bradshaw, Charles, Miska, Eric A., Di Domenico, Tomas, Akay, Alper, Lamond, Angus I., Allen, George, Medhi, Ragini

[International Worm Meeting, 2015]

Endogenous small RNA pathways can induce transcriptional gene silencing in C. elegans. piRNAs bound to the PRG-1 PIWI protein recognise their target by sequence complementarity and lead to the recruitment of RNA-dependent RNA polymerases (RdRPs). RdRPs, in turn, generate secondary siRNAs that are antisense to the target RNA and are 22 nt long with a 5' G (22G-RNAs). Other endogenous small RNA pathways such as the 26G-RNA pathway use similar amplification steps leading to 22G-RNA synthesis. In the C. elegans germline, the amplified 22G-RNAs are loaded into the argonaute protein HRDE-1 which localises to the nucleus initiating the nuclear RNAi pathway. The nuclear RNAi pathway leads to the transcriptional silencing of target genes and requires multiple chromatin factors. Silenced genes accumulate the repressive chromatin mark H3K9me3 and, remarkably, silencing of genes can last for several generations (1-6). The majority of the data linking small RNA mediated transcriptional and heritable silencing is based on evidence from genetic experiments. In order to better understand the mechanism of transcriptional and heritable silencing, it is essential to understand how key proteins in this silencing pathway function. We identified the intron binding protein EMB-4/AQR as an interaction partner of HRDE-1 in our immunoprecipitation experiments coupled to SILAC (7) based quantitative proteomics. Similar to hrde-1, emb-4/AQR is required for piRNA-mediated transgene silencing and the recently described 22G-RNA spreading (8) is inhibited in emb-4/AQR mutant animals. RNA-seq experiments revealed that emb-4/AQR is required for the repression of a subset of piRNA targets including transposable elements. In addition, some transposable elements which are de-silenced in emb-4/AQR mutants are not targeted by the piRNA pathway, indicating a more global role for emb-4/AQR in transposable element regulation. We are currently elucidating the molecular mechanism by which emb-4/AQR communicates with the nuclear RNAi machinery. 1. Ashe A et al. Cell. 150(1):88. (2012) 2. Buckley BA et al. Nature. 489(7416):447. (2012) 3. Luteijn MJ et al. EMBO J. 31(16):3422. (2012) 4. Shirayama M et al. Cell. 150(1):65. (2012) 5. Rechavi O et al. Cell. 158(2):277. (2014) 6. Han T et al. Proc Natl Acad Sci USA. 106(44):18674. (2009) 7. Larance M et al. Nat Meth. 8(10):849. (2011) 8. Sapetschnig A et al., PLoS Genet. 11(3):e1005078. (2015).

Harfe BD et al. (1998) East Coast Worm Meeting "Characterization of NK-2 Class Homeobox Genes in C. elegans"

Harfe BD, Fire A

[East Coast Worm Meeting, 1998]

Homeobox-containing genes have been identified in a large number of eukaryotic organisms, where they are thought to be involved in a wide range of developmental processes. In C. elegans, the imminent completion of the genome sequencing project will offer us the unique opportunity to identify and characterize the entire NK-2 class homeobox gene family in a multi-cellular organism. Presently, we have identified four NK-2 homeobox genes in C. elegans: ceh-22, ceh-24, ceh-27, and ceh-28 (>85% of the genome has now been sequenced). The molecular and genetic characterization of two of these genes, ceh-22 and ceh-24, has been described previously (Okkema and Fire, 1994; Harfe and Fire, 1998). The characterization of the remaining two NK-2 homeobox genes, ceh-27 and ceh-28 are discussed below. 1. ceh-28 - ceh-28 appears to be transcribed at extremely low levels; we were unable to obtain ceh-28 cDNAs from three different libraries. In addition, no EST hits have been reported for this gene. As a first step in understanding the role ofceh-28 in development, we created transgenic animals carrying a ceh-28::gfp transcriptional fusion. Strong GFP expression in transgenic animals was reproducibly observed in a single pharyngeal neuron, M4. 2. ceh-27 - We obtained a putative full length ceh-27 cDNA from an embryonic library. This cDNA was used to produce dsRNA for RNA inhibition (RNAi; Fire et al., 1998) and as a probe for RNA in situ hybridization analysis. ceh-27 RNAi injections resulted in 100% F1 embryonic lethality. Anaylis of ceh-27 RNAi animals using 4-D time-lapse imaging revealed defects in the ventral hyperdermis. ceh-27 RNAi animals appeared wild-type until they began moving inside the egg shell. The onset of embryonic muscle contractions appeared to cause the embryo to burst at a breach in the ventral hyperdermis. Localization of ceh-27 RNA by in situ hybridization and examination of transgenic animals containing ceh-27::gfp and lacZ expression constructs demonstrated that ceh-27 was expressed in a very dynamic pattern during development. Expression was first seen in four (possibly MS-derived) cells in ~100 cell embryos. Later, numerous cells in the anterior head region were ceh-27 positive. Presently, we are attempting to better characterize the ceh-27 expression pattern and identify the cause of the ceh-27 loss of function hyperdermal defect. Meeting fees and transportation costs for B.D.H were generously provided by MJ Research Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. (1998) Nature 391, 806-811 Harfe, B.D. and Fire, A. (1998) Development 125, 421-429. Okkema, P.G. and Fire, A. (1994) Development 120, 2175-2186.

Martha Kirouac et al. (2001) International C. elegans Meeting "Identification of Cis -Regulatory Response Elements in Vulva Cell Fate Markers"

Martha Kirouac, Paul Sternberg

[International C. elegans Meeting, 2001]

After induction of primary and secondary fates by the anchor cell and lin-12 , and undergoing two rounds of cell division the final vulval cell types are specified A, B1, B2, C, D, E and F. A number of transcription factors have been identified that play a role in vulva development. However we know little about how these transcription factors interact with each other, about their interactions with the inductive pathway, or about how they are regulated. The identification of cis -regulatory regions that confer cell specificity and respond to the ras pathway would be very helpful in determining such relationships. Three such target genes are egl-17 , cdh-3 and zmp-1 . These genes offer the opportunity to find response regions for multiple vulva cell types: vulE, vulF, vulC, vulD, and vulA as well as the anchor cell. In addition, egl-17 is an early cell fate marker for the response to the inductive signal; the isolation of a cis – regulatory element that drives this early expression and identification of genes that regulate this expression would be informative. We are analyzing these regions using a PCR based strategy to generate defined deletions to test the necessity and the sufficiency of fragments to confer regulation on the naïve promoter pes-10 . egl-17::gfp comes on in P6.p, P6.p daughters and granddaughters; goes off in early L4; staying off in mid-L4; on in vulC and vulD cells in mid L4 [Burdine RD, Branda CS, and Stern MJ, Development Mar;125(6):1083,1998]. This GFP marker strain contains 3.8 kb of cis- regulatory region. Since this gene is the earliest known maker in response to the inductive signal, it makes a good candidate for a immediate response element to the ras pathway. We have found a 0.2 kb region that is necessary and sufficient for vulC and vulD cell specificity and a 1.5 kb region that confers early expression in P6.p daughters. zmp-1::gfp is expressed during late L4 in vulE and vulA cells and in the L3 animals it is also expressed in the anchor cell [J. Butler & J. Krug, personal communication]. This GFP marker also contains 3.8 kb of upstream regulatory sequence. We have defined a 0.35 kb region that is necessary and sufficient vulE, vulA and anchor cell specificity. cdh-3::gfp is expressed during L3 in vulE and vulF cells and in L4 animals it is expressed in the vulC and vulD cells. cdh-3 is also expressed in the anchor cell starting in the late L2 stage. The gfp marker strain contains 6.0 kb of upstream regulatory sequence. We have found a 0.6 kb region that confers specificity to the anchor cell element and 0.5 kb region that drives expression in vulE and vul F cells.

Anne Hart (2001) International C. elegans Meeting "Using C elegans to teach genetics: SNP mapping in three weeks!"

Anne Hart

[International C. elegans Meeting, 2001]

C. elegans has been used to the teach fundamentals of genetic mapping for the last three years in the Neurobiology summer course at the Marine Biological Laboratories (MBL). The nine week course is primarily aimed at graduate students and postdoctoral fellows. The molecular section of the course I help teach lasts three weeks including lectures in the morning and lab work in the afternoons and evenings. Two students work with each of the six faculty members in the molecular section of the course. The two students assigned to me spend about 120 hours each working in the lab spread out over a three week period. Consistently, the students have had no experience in C. elegans and, often, little or no prior experience in molecular biology. Given the short duration the molecular section, and the desire to accomplish something of substance, the project has to be chosen with care. The short life cycle of C. elegans makes it an ideal organism to teach the fundamentals of genetic mapping in a limited amount of time. The project chosen last year was recombinant mapping of an interesting mutation, rt70 , using single nucleotide polymorphisms (SNPs) from the Hawaiian strain, CB4856. rt70 causes ASH neuron degeneration and is described in an abstract/poster by Emily Bates, et al. Prior to the start of the course last year, a lon-2(e678) rt70 egl-15(n484) chromosome was generated. All mutations are recessive. Heterozygous lon-2 rt70 egl-15 / CB4856 animals were generated and picked one per plate. These heterozygous animals were transported to MBL along with hundreds of seeded plates. The students practiced picking C. elegans , then singled animals carrying recombinant chromosomes/ lon-2 rt70 egl-15 for the first few days (putative recombinant lines). We attempted to generate homozygous recombinant lines by picking single 9 animals in the next generation and were successful about 80%of the time; the remaining 20% were repicked for analysis at a later date. The homozygous recombinant lines were 1) frozen in a 96 well format using Michael Koelle's protocol and 2) allowed to starve and lysed for PCR analysis using previously defined SNPs identified by Stephen Wicks. To facilitate analysis, only previously characterized SNPs creating a restriction site polymorphism were utilized. Screening more than 100 lines by PCR was not a problem and we were able to refine the rt70 map position. The biggest difficulty we had was scoring egl-15(e484) . In retrospect, I should have chosen marker mutations with very obvious phenotypes so they could be scored unambiguously and easily. PCR reactions were optimized while we were waiting for the recombinant strains to grow. The MBL students also had time to learn how to generate transgenic C. elegans by microinjection. PCR machines for the course were generously loaned by MJ Research, Taq was kindly provided by Fisher and NEB generously donated restriction enzymes. Thanks to all who donated their time, reagents and help- especially MBL students Michael Long and Ryohei Yasuda for their unflagging enthusiasm!