Jensen, Victor L. et al. (2009) International Worm Meeting "The novel cilia protein DAF-25 is required for DAF-11 cilia localization."

Riddle, Donald L., Bishop-Hurley, Sharon, Bialas, Nathan J., Jensen, Victor L., Leroux, Michel R.

[International Worm Meeting, 2009]

Four alleles of daf-25 were isolated from screens for new temperature-sensitive Daf-c (dauer-constitutive) mutants. At restrictive temperature, daf-25 mutants form dauer larvae even when food is abundant. The Daf-c phenotype can be rescued by maternally supplied daf-25(+). Compared to N2, daf-25 adults failed to avoid sucrose and high concentrations of NaCl, indicating that they are defective in sensing osmotic gradients. There is also a reduction of brood size when worms are shifted to the restrictive temperature (25 deg C) at L4, despite normal levels of progeny at the permissive temperature (15 deg C). The Daf-c phenotype of daf-25 is suppressed by daf-10/IFT122 but not daf-6/PTCHD3. This puts DAF-25 function downstream of DAF-6 but upstream of DAF-10. This implies that DAF-25 function requires cilia because DAF-10 is required for proper cilia development, whereas DAF-6 is required for proper formation of the receptor channel and exposure of the ciliated neurons to the environment. DAF-25 does not appear to be required for ciliogenesis, because there is a normal pattern of DiI staining in all stages, including the dauer. A rescuing DAF-25::GFP fusion protein is expressed in ciliated neurons, as well as some interneurons, and shows sub-cellular localization to the cilia. Because the phenotype, epistatic order and expression profile of daf-25 are similar to daf-11 (encoding a membrane-bound guanyl cyclase), we thought there may be an effect on DAF-11 in a daf-25 mutant background. Indeed, DAF-25 is required for proper DAF-11::GFP localization to the cilia. This may be a specific interaction because daf-25 mutations do not affect the cilia localization of GFP translational fusions for TAX-4/CNGA1 or OSM-9/TRPV4. We show that DAF-25 is a novel cilia protein. We are currently exploring the possible role of the mammalian ortholog of DAF-25 in human disease.

Jensen, Victor L. et al. (2009) International Worm Meeting "RNAi screen of DAF-16/FOXO target genes links dauer formation and innate immunity."

Park, Donha, Jensen, Victor L., Simonsen, Karina T., Riddle, Donald L., Lee, Yu-Hui

[International Worm Meeting, 2009]

The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway for dauer formation. To identify novel downstream genes, we screened previously identified candidate genes putatively regulated by DAF-16 using RNAi. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi and constitutive dauer formation (Daf-c). Among 515 genes screened, 22 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. Two of these genes, srh-99 and hpd-1, were previously identified SynDaf genes, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1 are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, knockdown of two of these genes, lys-1 and clc-1 causes C. elegans to be more susceptible to infection by the pathogen Staphylococcus aureus. clc-1 may represent a novel defense mechanism in C. elegans as it has been shown to function in epithelial cohesion, and its expression is up-regulated upon infection. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and wild-type N2 (at 27 deg C) were all enhanced by exposure to pathogenic bacteria. We conclude that knockdown of a gene required for innate immunity increases infection by pathogens, and this led to increased dauer formation in our screen. We propose that formation of the non-feeding dauer dispersal stage is a behavioral response to pathogens.

Jensen, Victor L. et al. (2013) International Worm Meeting "Analysis of developmental RNA-Seq libraries reveals signature profile for cilia-related genes."

Jensen, Victor L., Leroux, Michel R., Li, Chunmei, Timbers, Tiffany A., Morin, Ryan D.

[International Worm Meeting, 2013]

Ciliogenesis in C. elegans hermaphrodites occurs during late embryogenesis, with most cilia being formed within hours of each other. This makes C. elegans well suited to expression analyses to identify novel cilia genes. Using the available RNA-Seq libraries we identify a signature expression pattern specific to cilia-related genes, which includes a large spike in expression at late embryo with a reduction at mid-L1 (first larval stage) and low levels of expression at all other stages. P-values for Pearson correlation for each gene in the genome are compared to 41 known ciliary genes and each gene is then assigned the minimum calculated p-value. Using a 1E-5 cutoff and only including genes with human orthologues, we identify 635 genes with the specific pattern. Comparisons to CilDb (an online database of previously published ciliary genomics/proteomics experiments) reveals an 15-fold enrichment of genes with greater than seven or more cilia references. Further clustering of this list based on individual expression patterns increases the enrichment by greater than 40 times. Extending this expression analysis method, we also find a shared-but different from the above-expression pattern among serpentine olfactory G-protein-coupled receptors, many of which are known to be targeted to cilia. Using this approach, we identify evolutionarily-conserved proteins that function within three distinct ciliary regions or modules. One, a recently-characterized protein linked to retinitis pigmentosa, is found in the proximal-most segment of the axoneme, which is implicated as a ciliary gate needed for effective signalling. Another protein is present at the basal body, which is required for the formation of cilia. Finally, a third protein represents a previously undescribed component of the intraflagellar transport (IFT) machinery, which plays a critical role in ciliogenesis and delivering cargo (e.g. signalling proteins) to cilia.

Jensen, Victor L. et al. (2017) International Worm Meeting "Role of metazoan dynein in transition zone formation, ciliary maintenance, and processive intraflagellar transport."

Mohan, Swetha, Timbers, Tiffany A., Leroux, Michel R., Cai, Jerry, Jensen, Victor L.

[International Worm Meeting, 2017]

Cilia are ubiquitous microtubule-based organelles found at surface of metazoan cells. Non-motile (sensory/primary) cilia act to receive signals from the environment, and motile cilia either move cells or the surrounding fluid. Cilia are built and maintained using a cargo-trafficking intraflagellar transport (IFT) machinery powered by kinesin (anterograde) and dynein (retrograde) molecular motors. How IFT-dynein contributes mechanistically to ciliary processes in metazoans remains poorly studied, in part because null mutants exhibit a severe terminal phenotype with IFT protein accumulations in the bulbous tips of highly truncated cilia. We carried out a screen for retrograde IFT transport defects in C. elegans, and identified the first temperature-sensitive IFT mutant in a metazoan. Using this strain, we show that the IFT-dynein (CHE-3) heavy chain is essential for cilium maintenance and correct formation of the transition zone (TZ) ciliary gate. Cilia resorb upon shift to restrictive temperature (inactive IFT-dynein), and are restored upon return to permissive temperature (active IFT-dynein). Importantly, this resorption and regrowth is observed for primary cilia in terminally differentiated, sensory neurons. We next investigated how IFT-dynein enables processive IFT, in contrast to the bidirectional tug-of-war mechanism of transport used by kinesin and dynein elsewhere in the cell. We find that the IFT-dynein heavy, intermediate and two light chains are trafficked by the IFT-subcomplex B, unlike the light intermediate chain, which is transported independent of the canonical IFT complex. This differential transport provides an effective mechanism by which the retrograde transport machinery is maintained in an inactive state during anterograde transport. Together, our findings reveal that IFT-dynein is required for ciliary maintenance and TZ formation, and regulates the processivity of anterograde IFT by spatial separation and thus inactivation of its components.

Victor L. Jensen et al. (2006) European Worm Meeting "A Potential DAF-16 Target Involved in Longevity"

David L. Baillie, Victor L. Jensen, Donald L. Riddle

[European Worm Meeting, 2006]

Victor L. Jensen1, David L. Baillie2, and Donald L. Riddle3. Genes differing in steady-state RNA levels between N2 and daf-2 adults and N2 dauer larvae were identified using Serial Analysis of Gene Expression (SAGE) databases. One gene, a putative transcription factor, was isolated from a screen for genes involved in longevity. The outcrossed knockout allele was shown to have extended longevity, ~3% Him and ~2% Vab phenotypes. The 1 kb of genomic sequence 5? of the confirmed transcription start site contains several possible DAF-16 binding sites. By using a series of truncated promoter sequences to drive GFP expression, possible in vivo activity for these DAF-16 binding sites was observed. One consensus site DBE (DAF-16 Binding Element) may act to repress transcription. This is in agreement with the SAGE data which shows down regulation of this transcription factor in the daf-2 background. In daf-2 mutants DAF-16 is activated and is required for extended longevity. Transcriptional targets and the DNA binding motif for the transcription factor will be elucidated, and its role in longevity will be characterized.

Victor L Jensen et al. (2005) International Worm Meeting "A potential DAF-16 target involved in oxidative stress and longevity"

Donald L Riddle, Victor L Jensen

[International Worm Meeting, 2005]

Genes differing in steady-state RNA levels between N2 and daf-2 adults, and N2 dauer larvae were identified using Serial Analysis of Gene Expression databases. RNA interference (RNAi) was performed in an RNAi hypersensitive background. RNAi for a gene encoding a putative transcription factor, which may be involved in oxidative stress response, was shown to reduce lifespan. The RNAi-treated animals were sensitive to juglone, an inducer of oxidative stress, but increased sensitivity was not observed in daf-16 mutants. The 1 kb of genomic sequence 5' of the predicted start codon contains several possible DAF-16 binding sites. By using a series of truncated promoter sequences to drive GFP expression, in vivo activity for these DAF-16 binding sites was confirmed. One DAF-16 binding motif enhances GFP expression, whereas another inhibits expression. Transcriptional targets and the DNA binding motif for the transcription factor will be elucidated, and its role in oxidative stress will be characterized.

Victor L Jensen et al. (2008) European Worm Meeting "DAF-16/FOXO target genes that regulate dauer formation include those for innate immunity."

Victor L Jensen, Donald L Riddle, Yu-Hui Lee

[European Worm Meeting, 2008]

The DAF-16/FOXO transcription factor is the major downstream output of the. insulin/IGF1R signaling pathway in C. elegans for lifespan and dauer. formation. DAF-16 activity is required for dauer formation in N2 and. insulin/IGF1R pathway mutants. To determine the effects of individual DAF-. 16 target genes on dauer formation, we tested previously identified. candidate genes putatively regulated by DAF-16. We used RNAi in a. sensitized background [eri-1(mg366); sdf-9(m708)], which enhances RNAi and. constitutive dauer formation (Daf-c). Among 515 genes we tested, 22 have a. synthetic Daf-c (SynDaf) phenotype with sdf-9. Two of these genes are. previously identified SynDaf genes. Five other genes are involved in. processes known to affect dauer formation. Two of the 22 genes are known. to participate in innate immunity, and six more are predicted to be. involved. The latter result suggests that the immune response may. contribute to the dauer decision, and we are currently testing a model for. this interaction.

Victor L Jensen et al. (2008) European Worm Meeting "The maternal-effect Daf-c gene daf-25 is expressed in ciliated neurons and functions in neurosecretion"

Victor L Jensen, Sharon Bishop-Hurley, Marco Gallo, Donald L Riddle

[European Worm Meeting, 2008]

We isolated four alleles of daf-25 in screens for new temperature-sensitive. Daf-c (dauer-constitutive) mutants. At restrictive temperature, daf-25. mutants form dauer larvae even when food is abundant. The Daf-c phenotype. can be rescued by maternally supplied daf-25(+). Compared to N2, daf-25. worms failed to avoid sucrose and high concentrations of NaCl, indicating. that they are defective in sensing osmotic gradients. There is also a. reduction of brood size when worms are shifted to the restrictive. temperature (25degreesC) at L4, despite normal levels of progeny at the. permissive temperature (15degreesC). Epistasis results put DAF-25 function. downstream of DAF-6, but upstream of DAF-10. This implies that DAF-25. function requires cilia because DAF-10 is necessary for proper cilia. production. DAF-6 is required for proper formation of the amphid channel,. which connects the ciliated neurons to the environment. Indeed, a daf-. 25p::GFP reporter is expressed in the ciliated neurons among other tissues.. Because daf-25 dauer larvae are defective in dauer recovery, similar to. unc-31 dauers, we analyzed dense core vesicle secretion. Using a paex-. 3::ANF::GFP (gift from Erik Jorgensen), we observed a secretion defect in a. deletion mutant that affects both isoforms of daf-25. By contrast, a. deletion mutant that only affects one of the isoforms, and has a weaker Daf-. c phenotype, has no defect in ANF::GFP secretion. A model for the role of. DAF-25 in dauer formation will be presented.

Hunter CP et al. (1991) International C. elegans Meeting "NON-AUTONOMY OF HER-1 FUNCTION IMPLICATES CELL INTERACTIONS IN C. ELEGANS SEX DETERMINATION."

Hunter CP, Wood WB

[International C. elegans Meeting, 1991]

Activity of the her-l gene is required for normal male development in C. elegans. Loss-of-function her-l mutations cause feminization of XO animals (normally male) to produce fertile hermaphrodites, while gain-of-function her-l mutations cause masculinization of XX animals ( normally hermaphrodite) to produce pseudo-males. Genetic analysis indicated that her-l functions early in the hierarchy of regulatory interactions among the major sex determination genes (1). We have analyzed her-l genetic mosaics to determine which cells must express her-l for wild-type male development. If her-l functions cell- autonomously, then in XO animals her-l (+) cells will follow male fates, and her-1(-) cells will follow hermaphrodite fates. If her-l or a her-l regulated activity functions non-autonomously, then the her-l genotype of a cell may not determine its sexual phenotype. The phenotypes of XO her-l genetic mosaics are variable, but clearly show that both her-1(-) and her-l (+) cells can follow either hermaphrodite or male fates. We conclude that her-l is neither necessary nor sufficient to cell-autonomously determine male sexual phenotypes and, therefore, that her-l or a her-l regulated activity must be able to function non-autonomously. The observed patterns of non-autonomous effects suggest that many or all cells express and respond to her-l activity. These findings implicate cell interactions in C. elegans sex determination. Earlier genetic analysis indicated that her-l might directly control tra-2 activity (1). Molecular characterization of the gene products of her-l and tra-2 (see abstracts by M. Perry et al. and P. Kuwabara et al) is consistent with a model in which a secreted ligand encoded by her-l interacts with a cell-surface receptor encoded by tra-2. We will discuss the possible role of such an interaction in coordinating the sex determination decision.

Ellis RE et al. (1991) International C. elegans Meeting "PROGRESS IN CLONING fog- 1"

Kimble JE, Ellis RE

[International C. elegans Meeting, 1991]

The fog-l gene plays an important role in the decision of germ cells to differentiate as either spermatocytes or oocytes (Doniach 1986, Barton and Kimble 1990). In fog-l mutants, all germ cells develop into oocytes. This is true for both males and hermaphrodites. However, these fog-l mutations do not appear to affect other aspects of sexual development. Furthermore, the fog-l mutant phenotype is not suppressed by mutations in any of the other genes known to control sex- determination. Thus fog-l might function to initiate spermatogenesis in response to other genes that regulate general sexdetermination. There are 42 fog-l mutations. These mutations arise at a frequency typical for knockout mutations in other C. elegans genes, and four of these fog-l alleles were isolated from a screen capable of recovering deletions. Thus it is likely that these mutations cause a loss of fog- l function. In order to study the mechanisms by which fog-l functions, and to determine how fog-l expression is regulated by the genes that control sex-determination, we are now cloning this gene. The fog-l gene is located on the left arm of LGI, between sup-ll and unc-ll, a region spanned by a large contig of about one megabase in size. By mapping DNA polymorphisms with respect to fog-l, we have narrowed down the region in which fog-l must be located, and are now injecting YAC DNA that covers this region in an attempt to rescue fog-l mutants.