Taylor, Remington et al. (2021) International Worm Meeting "Snip-SNP mapping of a trembler C. elegans mutant strain with undergraduate students"

Pham, Kelvin, Henderson, Jacklyn, Soebianto, Sarita, Mathai, Kristina, Taylor, Remington, Nguyen, Gina, LaForce, Michael, Simmons, Dr. Alexandra

[International Worm Meeting, 2021]

Caenorhabditis elegans is a valued model organism that is easy to maintain in the lab. We obtained a mutant strain of C. elegans that exhibits constant involuntary muscle spasms, a phenotype that has not been described before in the literature (MCW230, curtesy of Dr Wang, BCM, Houston, TX). The 'trembler' mutant strain has a nre-1(hd20) lin-15b(hd126) genetic background. We use snip-SNP mapping to seek the causal mutant gene responsible for the trembling phenotype in the MCW230 strain. MCW230 worms were backcrossed with wild-type N2 worms to eliminate the double mutant background (nre-1, lin-15b). For snip-SNP gross mapping, we crossed our mutant with CB4856 worms. DNA was extracted from 43 F2 individuals resulting from the MCW230 x CB4856 cross. The F2 samples were studied to identify the presence of specific single nucleotide polymorphisms at 18 genomic locations (3 per chromosome) and assess how the alleles segregate relative to the trembler phenotype. We completed genotyping at all genomic locations and our data supports the idea that the trembler phenotype seems to be linked to one of the markers on chromosome III. Our work is an example of how research using C. elegans can be done in the undergraduate environment with basic laboratory equipment.

Crittenden SL et al. (1997) International C. elegans Meeting "TEMPERATURE-SENSITIVE REPRESSION OF TRANSGENES CARRYING A fem-3(gf) MUTANT 3'UTR"

Crittenden SL, Kimble JE

[International C. elegans Meeting, 1997]

We are developing a method to control gene expression in a temperature dependent manner. Our strategy is to regulate reporter expression with a well-defined promoter at the 5' end and a mutant fem-3 3'UTR at the 3' end. The fem-3(gf) mutations map to the fem-3 3'UTR and release fem-3 from post-transcriptional repression at 25 C, but not at 15 C (1,2). We have tested expression of a lag-2 promoter::GFP construct that carries either a strong or a weak fem-3(gf) 3'UTR. The lag-2 promoter drives expression in the distal tip cells of adults (3-5). We find that transgenes express GFP at much lower levels at 15 C than at 25 C when carrying a fem-3(gf) 3'UTR. We are currently trying to optimize repression at 15 C using the lag-2 promoter and GFP as a reporter. In addition, we are testing whether temperature sensitive repression can be achieved in other tissues, and we are starting to explore the biological activity of various proteins expressed in the distal tip cell. 1. Ahringer, J., and Kimble, J. (1991). Nature 349, 346-348. 2. Barton, M. K., Schedl, T. B., and Kimble, J. (1987). Genetics 115, 107-119. 3. Fitzgerald, K., and Greenwald, I. (1995). Development 121, 4275-4282. 4. Gao, D. and J. Kimble, midwest worm meeting 1996. 5. Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924.

Gao DL et al. (1996) Midwest Worm Meeting "Analysis of the Distal Tip Cell (DTC)/Germline Interaction in C. elegans Gonad"

Kimble JE, Gao DL

[Midwest Worm Meeting, 1996]

Analysis of the individual components of a signal transduction pathway is greatly facilitated by the separation of the signal and receptor into well-defined cells or tissues. The cells participating in an inductive interaction are clearly defined as signaling or receiving members of the interaction. We have focused on the induction of the germ line by the somatic distal tip cell (DTC). The DTC resides at the distal end of the germ line and induces mitotic divisions in the germ line throughout postembryonic development. We have previously shown that lag-2 is expressed in the signaling DTC cell and that a LAG-2::b-Gal fusion protein is taken up into the receiving germline tissue (1). Others in the Kimble lab have shown that glp-1 is expressed in the receiving tissue the germ line (2). This is consistent with the idea that LAG-2 is a signaling ligand for the receptor GLP-1. Therefore, this GLP-1 mediated inductive interaction is both well-defined and accessible to manipulation. We have built a battery of lag-2::reporter constructs with myc, b-gal, and GFP epitope tags inserted either in the extracellular or intracellular domain or fused to the promoter of the lag-2 gene. By utilizing these reporter constructs and anti-GLP-1 antibodies, we have systematically investigated the distribution of LAG-2 at different developmental stages of the animal and in different mutant backgrounds. In addition, we have explored the ability of our tagged proteins to signal GLP-1 and induce germline mitosis. In the course of these experiments, we have discovered a novel dominant-negative LAG-2 created by insertion into the extracellular domain, suggesting that LAG-2 may normally function as oligomers or dimers. (1) Henderson, Gao, Lambie & Kimble(1994). Development 120, 2913-2924. (2) Crittenden,Troemel,Evans & Kimble(1994). Development 120, 2901-2911.

Henderson ST et al. (1997) International C. elegans Meeting "REGULATION OF CELL FATE BY LAG-2 AND OTHER DSL PROTEINS"

Crittenden SL, Kimble JE, Henderson ST, Gao DL

[International C. elegans Meeting, 1997]

DSL (for DELTA, SERRATE, LAG-2) ligands act with LNG (for LIN-12, NOTCH, GLP-1) receptors to regulate cell fates in many organisms. To understand how DSL proteins shape metazoan development, we have investigated control by LAG-2 as well as two new DSL proteins. LAG-2, a predicted transmembrane protein, acts in many cell fate decisions (1,2,3). A systematic study of LAG-2 functional domains lead to four new conclusions. First, the non-conserved N-terminal region is critical for LAG-2 activity. Second, EGF-like repeats are not essential. Third, membrane association is required for rescue, but not signaling. Curiously, fusion to GFP can confer rescuing activity upon secreted LAG-2 forms, suggesting that GFP fusion proteins, when secreted, may stick in the membrane or aggregate outside the cell. Fourth, the intracellular domain normally lowers activity: LAG-2 mutants lacking this domain are hyperactive. In addition, a secreted LAG-2 placed under control of the ges-1 intestinal promoter can induce germline tumors, but membrane bound LAG-2 under the same control cannot. Therefore, the secreted form appears to retain activity across two basement membranes. How does LAG-2 control germline mitosis? One idea is that the length of distal tip cell processes may determine the extent of LAG-2 signaling (4). However, we have not been able to confirm this idea. We propose that the LAG-2 DTC signal is not limited to areas of cell-cell contact. Instead, we suggest that the signal is propagated in the germline by downstream factors. The sequencing project has identified two other genes that have the potential to encode DSL proteins, one on cosmid F15B9 and another on F58B3. We have constructed reporter constructs and begun antisense injections of both. Results on expression patterns of these DSL homologs and their antisense phenotypes will be reported. (1) Lambie, E. J., and Kimble, J. (1991). Development 112, 231-240. (2) Henderson, S. T., Gao, D., Lambie, E. J., and Kimble, J. (1994). Development 120, 2913-2924. (3) Tax, F. E., Yeargers, J. J., and Thomas, J. H. (1994). Nature 368, 150-154. (4) Fitzgerald, K., and Greenwald, I. (1995). Development 121, 275-4282.

Wilson Berry L et al. (1997) International C. elegans Meeting "MORE ABOUT TEGS (TUMOROUS ENHANCERS OF GLP-1(OZ112OZ120))"

Wilson Berry L, Schedl TB, Read R

[International C. elegans Meeting, 1997]

Normal hermaphrodite germline development requires coordination of molecules controlling proliferation, entry into meiosis, progression through meiotic prophase, and differentiation of sperm and oocytes. We have devised a mutational scheme to identify molecules involved in two of these processes, the control of germ cell proliferation and entry into the meiotic pathway. We reasoned that a reduction in products normally involved in either promoting entry into meiosis or inhibiting proliferation should result in excess proliferation. However, this phenotype may not be observed in a wild-type background, since proliferation in the distal germline requires a localized source of ligand [1,2,3]. To circumvent this problem, we conducted the mutational analysis in a glp-1(oz112oz120) genetic background where ligand-independent excess proliferation is observed in 0.05% of gonad arms [4]. From 9000 haploid genomes, we isolated 12 enhancers, designated as teg (tumorous enhancers of glp-1(oz112oz120)) alleles, that cause a glp-1(oz112oz120) dependent tumorous phenotype. In a glp-1(+) background, none of the Teg mutants display an obvious germline phenotype. The teg loci have been mapped to individual chromosomes on the basis of their enhancer phenotypes with detailed three-factor mapping continuing. In addition, our screen may yield loss-of-function mutations in negative regulators of the GLP-1 signalling pathway [5, 6, 7]. Potential negative regulators have been identified in previous screens for suppressors and enhancers of glp-1 (sog, suppressor of glp-1) and lin-12 (sel, suppressor and enhancer of lin-12) [5, 6, 7]. To determine if any of the sog or sel genes enhance the tumorous germline phenotype of glp-1(oz112oz120), we are currently constructing strains mutant for glp-1(oz112oz120) and selected sog or sel mutations. Several of the sog genes, specifically sog-1, 3, 5, and 10 are capable of enhancing the tumorous phenotype of glp-1(oz112oz120). However, at least three of the Teg mutants appear to identify genes distinct from known sog or sel genes. The possibility that these teg genes can suppress glp-1(lf) phenotypes or enhance lin-12(gf) mutants is being investigated. [1] Crittenden et al., 1994; [2] Tax et al., 1994; [3] Henderson et al., 1994; [4] Berry et al., 1997; [5] Maine and Kimble, 1993; [6] Sundaram and Greenwald, 1993; [7] Tax et al., in preparation. This work was supported by NIH Grant HD25614.

Marie McGovern et al. (2008) Development & Evolution Meeting "A Mechanism for Tumor Initiation in the C. elegans germ line"

John Maciejowski, E. Jane Hubbard, Marie McGovern

[Development & Evolution Meeting, 2008]

We are studying the molecular and cellular basis of tumor formation in the germ line of C. elegans as a paradigmatic model for tumor seeding and its relationship to stem cell niches. Germ cells in the distal gonad remain undifferentiated in response to a signal produced by the Distal Tip Cell (DTC) (Kimble and White 1981). The DTC produces the DSL ligand LAG-2 that interacts with the GLP-1/Notch receptor in the germ line to maintain a self-renewing population of neighboring germ cells (Austin and Kimble, 1987; Yochem and Greenwald, 1989; Lambie and Kimble, 1991; Henderson et al., 1994). During larval stages, the DTC moves away from the midline, and this movement is critical for proper germline patterning. Two independent mechanisms underlie early DTC migration: an intrinsic migration program and the extent of germline proliferation. As a result of these two mechanisms, proximal-most germ cells eventually lie outside the influence of the DTC and differentiate, setting up the distal-proximal germline pattern. Proximal germline tumors (Pro phenotype) can form when cells in the developing germ line fail to differentiate at the appropriate time (Pepper et al., 2003(a,b); Killian and Hubbard, 2004, Voutev et al., 2006). We have found three different genetic/cellular defects that cause proximal tumor formation, each of which results in a delay in germline differentiation. This delay inappropriately juxtaposes undifferentiated germ cells in the proximal oviduct with certain somatic gonad cells of the proximal sheath lineage. Based on cell ablation studies (Killian and Hubbard, 2005), we showed that the Pro phenotype occurs in response to a latent proliferation-promoting signal produced by the proximal sheath lineage. To determine the molecular basis for this latent proliferation-promoting signal, we examined the other 9 putative DSL ligands in the genome. We found that apx-1 is expressed in the correct cells of the somatic gonad, and that reduction of apx-1 activity can suppress the Pro phenotype in three different mutant conditions, each of which have a different anatomical focus of activity but share the same delay in germline differentiation. We further found that apx-1 and glp-1 are required continuously to maintain the proximal germline tumors. These results suggest a novel general mechanism for tumor formation.

Berry LW et al. (1995) International C. elegans Meeting "CHARACTERIZATION OF A NOVEL glp-1(gf) ALLELE"

Berry LW, Westlund B, Schedl TB

[International C. elegans Meeting, 1995]

GLP-1, a putative transmembrane receptor, is required for several cell fate decisions throughout development. In the germ line, present evidence suggests GLP-1 transduces a proliferative signal from the distal tip cell (DTC) to the distal stem cell population (Austin and Kimble, 1987; Yochem and Greenwald, 1989). In glp-1(lf) mutants, all germ cells prematurely enter the meiotic pathway resulting in a germ-line proliferation defective phenotype (Austin and Kimble, 1987). Our current research focuses on the role of GLP-1 in the germ line through characterization of a glp-1(gf) allele, oz112, with the opposite germ-line phenotype; hermaphrodites and males have a semi-dominant tumorous phenotype. Several results suggest the cellular defect associated with the tumorous germ-line phenotype is a failure of germ cells to leave the mitotic cell cycle (or enter the meiotic cell cycle). First, the normal distal (mitotic) - proximal (meiotic) germ-line polarity is not established in glp-1(oz112gf) hermaphrodites at 25oC. Second, there is no evidence that proximal germ cells enter the meiotic cell cycle during L4, as observed in wild-type animals. Finally, removal of the DTC with a laser microbeam during L1 and L3 in glp-1(oz112gf) animals results in continued proliferation, instead of entry into meiosis as seen in wild-type DTC-ablated animals (Kimble and White, 1981). Current evidence suggests the glp-1(oz112gf) receptor is signal independent. Double mutant analysis shows glp-1(oz112gf) is epistatic to lag-2, which encodes the proliferative ligand from the DTC (Tax et al., 1994; Henderson et al., 1994). In addition, the oz112gf molecular lesion has been localized to the glp-1 extracellular domain in a region where constitutively activating mutations are found in the closely related lin-12 receptor (Greenwald and Seydoux, 1990). The lag-1 gene is also involved in the glp-1 signal transduction pathway (Lambie and Kimble, 1991) and appears to act downstream of glp-1(oz112gf). Both weak (glp-1(lf)-like) and strong (L1 lethal) lag-1 alleles are epistatic to glp-1(oz112gf). Two somatic defects are associated with glp-1(oz112gf) mutants. First, both hermaphrodites and males exhibit an incompletely penetrant multivulva phenotype. Second, a small percentage of glp-1(oz112gf) animals die during L1. We are characterizing these defects using antibodies and lineage analysis.

Katsuyuki K Tamai et al. (2005) International Worm Meeting "MIG-24, a novel bHLH transcription factor, required for distal tip cell migration in C. elegans"

Kiyoji Nishiwaki, Katsuyuki K Tamai

[International Worm Meeting, 2005]

The C. elegans hermaphrodite gonad is bilobed and the U-shaped morphology of the anterior and posterior gonad arms is determined by directional migration of gonadal distal tip cells (DTCs) each located at the distal-most ends of the gonad primordium. To identify the molecules that regulate DTC migration, we have isolated many mutants defective in gonad morphogenesis due to abnormal DTC migration (mig). The DTCs in mig-24 (k168) mutants prematurely terminate migration, leading to thick and short gonadal arms and partial sterility. mig-24 was positionally cloned and found to encode a novel bHLH transcription factor, also designated as hlh-12. The RNAi knockdown of mig-24 resulted in the same phenotype as was observed the mig-24 (k168) original mutants. The mig-24 promoter and Venus fusion gene was expressed specifically in DTCs from the L2 to the young adult stages, during which DTCs migrate. Since many bHLH proteins function in heterodimers, we examined the possibility that MIG-24 also forms heterodimers. Among 35 bHLH proteins in C. elegans, HLH-2 was examined, because RNAi of hlh-2 phenocopies the DTC migration defects observed in mig-24 (k168) (Karp and Greenwald, Dev. Biol., 2004). To assess the physical interaction between MIG-24 and HLH-2, co-immunoprecipitation assay was performed. As a result, it was shown that MIG-24 physically interacts with HLH-2. To understand further the biological function of MIG-24, we attempted to identify its transcriptional target genes. Two proteins, LAG-2, a Delta/Serrate-like protein (Henderson et al., Development, 1994) and GON-1, an ADAM-TS family metalloprotease (Blelloch and Kimble, Nature, 1999), are known to be expressed in the DTCs. We examined the expression of fusion genes for lag-2 or gon-1 promoters and the reporter GFP gene. We found that the expression of gon-1p::GFP, but not lag-2p::GFP, was significantly weakened in the mig-24 compared to that in the wild-type background, suggesting that gon-1 is a downstream target of the mig-24 transcription factor. Furthermore, the chromatin immunoprecipitation (ChIP) assay revealed that MIG-24 can bind to the gon-1 promoter region, implying the possibility that MIG-24 directly regulates the gon-1 expression. We propose a model that the MIG-24/HLH-2 heterodimer activates the transcription of gon-1 in DTCs to remodel the components of the gonadal basement membrane, thereby regulating the migration of DTCs.

Syuichi Takano et al. (2007) International Worm Meeting "Gene expression changes in response to warm and cool temperatures is augmented in daf-2 mutants."

William H. Boylston, Wendy Lee, Simon Tavare, Syuichi Takano, Christina Curtis, Pamela L. Larsen

[International Worm Meeting, 2007]

Cool temperature is an environmental factor that has been associated with increased life span for many species. The genetic pathways and mechanisms controlling cool longevity are unknown. In C. elegans, wild-type life span at 15C is twice as long as it is at 25C. daf-2 mutants fail to show the same change in life span at the two temperatures. This defect suggests that the daf-2 gene functions in the response to different cultivation temperatures. DAF-16 is not required for the change in life span due to temperature because daf-16 mutants show a magnitude of change like wild type. DAF-16 is necessary but not sufficient for daf-2 longevity since over-expression of daf-16 results in a life span extension of ~12-50% (Lee et al., Current Biology 2001; Henderson & Johnson, Current Biology 2001; Lin et al., Nature Genetics 2001), whereas daf-2 mutations yield an increase of 200-300%. Our working model is that two mechanisms contribute to the extreme daf-2 mutant longevity observed at 25C compared to wild type. One mechanism is DAF-16-dependent while the other is DAF-16-independent and related to temperature. A biological response to environmental conditions can occur at the transcriptional level; for instance adaptive transcriptional changes have been shown in response to hypoxia. To study the homeostatic response to each cultivation temperature, we used Affymetrix microarrays to assess gene expression at 15C and 25C for wild type and five daf-2 mutant alleles. Approximately 800 genes are differentially expressed at 25C compared to 15C in N2. In the mutants m577, m41, m579 and e1370 the number of differentially expressed genes increases by 2.2, 3.4, 4.4 and 6.7 fold, respectively. Thus, the daf-2 mutants appear more responsive to a change in temperature. In each case, roughly half of the differentially expressed genes are at higher and half at lower levels. This rules out the simple model that slower life at 15C leads to lower expression levels in general. It is possible that the moderate alteration in gene expression in m577 and m41 is predominately beneficial and that the large number of alterations in e1370 becomes both beneficial and risky. There are many pleiotropic phenotypes in e1370 animals at 25C. Also, wild type fares less well at 25C, so the expectation is that some of the changes are detrimental and others are beneficial. Sorting of the functional outcome of expression changes is underway.

Megha B Satyanarayana et al. (2003) International Worm Meeting "Transcriptional Control of hil-1,a linker histone that functions in C. elegans longevity."

Megha B Satyanarayana, James A Waddle

[International Worm Meeting, 2003]

Reduced DAF-2/Insulin growth factor receptor signaling doubles adult lifespan [1], a benefit absolutely dependent on the forkhead transcription factor DAF-16 [1,2]. An important extension of these studies is to identify DAF-16 target genes that initiate changes required for increased lifespan. hil-1 encodes a linker histone that is upregulated in response to starvation/dauer arrest [3] and may play an important role in mediating the benefits of increased DAF-16 activity (Ilkayeva and Waddle, unpublished). In well-fed worms, HIL-1 is found at low levels in the cytoplasm and nucleus of the pharynx, somatic gonad, vulva, and certain cells in the head and tail. Upon starvation, HIL-1 accumulates in most somatic nuclei in the animal. The distribution pattern of DAF-16 during the fed and starved states [4] mirrors that of HIL-1. Long-lived adults, such as daf-2 mutants [1], show nuclear HIL-1 accumulation. HIL-1 accumulation in long-lived mutants and starved animals is blocked by daf-16 mutations (Ilkayeva and Waddle, unpublished). Forced expression of HIL-1 under a DAF-16-independent, heterologous promoter is sufficient to increase the lifespan of otherwise wild-type animals (Ilkayeva and Waddle, unpublished), suggesting that HIL-1 enriched chromatin may contribute to the gene expression profile that promotes longevity. To understand how hil-1 is regulated in response to reduced DAF-2 signaling and starvation, we are analyzing the hil-1 promoter by deletion and mutation analysis. A search for the preferred DAF-16 binding element (DBE) (TTGTTTAC [5]) in the sequence of hil-1 yields a DBE at 1067 nt upstream from the ATG (The DBE in the C. briggsae hil-1 homolog is conserved). We predict that the hil-1 DBE may be a direct target of DAF-16. Mutation of the 8-bp DBE to a restriction site (TGGGCCCC) blocks starvation-induced hil-1-GFP expression in wild-type animals. hil-1-GFP expression in starved daf-16 null animals is also blocked. To determine whether hil-1 is a direct target of DAF-16, we will assay recombinant GST-DAF-16 and DAF-16-containing worm extracts for their ability to cause a mobility shift of wild-type and DBE-mutant hil-1 promoter fragments. Other tests are underway to determine whether the hil-1 DBE is required for HIL-1 induction in daf-2 mutant adults and other long-lived mutants. 1. C. Kenyon,et al.(1993).Nature.366.461 2. S. Ogg,et al.(1997).Nature.389.994 3. S. J. Jones,et al.(2001).Genome Res.11.1346 4. S. T. Henderson and T. E. Johnson(2001).Curr Biol.11.19755. 5. T. Furuyama,et al.(2000).Biochem J.349.629