David Michaelson et al. (2006) Development & Evolution Meeting "Soma-germline Interactions Required for Robust Larval Germline Proliferation"

Rachael Lader, E. Jane Hubbard, David Michaelson, Dina Esposito, Chris Ryan, John Maciejowski, Jessica Dy-Johnson

[Development & Evolution Meeting, 2006]

Proper control of cell proliferation is critical for normal development. While the molecular basis for the proliferation/differentiation decision is relatively well-characterized in certain developmental contexts, less is known about cell-cell interactions that control of the extent of proliferation within a developing organ. The C. elegans germ line offers a tractable model system for exploring this problem. Previous work (McCarter et al., 1997; Killian and Hubbard, 2005), indicates that the distal pair of somatic gonadal sheath cells (Sh1) promote robust larval germline proliferation. We are taking several approaches to identify the molecular basis for this interaction. Forward genetic screens, molecular analysis and directed RNAi studies identified a critical role for optimal ribosome biogenesis in the sheath (Killian and Hubbard, 2004; Voutev et al., submitted) for its germline proliferation-promoting activity. To identify additional players, a genome-wide RNAi screen and candidate screens are underway. One screening strategy is based on the observation that reduced larval germline proliferation inhibits gonad arm extension, delays meiotic entry, and thereby uncouples the coordination between somatic gonad and germline development. As a result, under certain conditions, reducing larval germline proliferation can enhance proximal germline tumor formation (Pro phenotype; Killian and Hubbard, 2005; see also abstract by Maciejowski and Hubbard). Thus we are screening for soma-autonomous RNAi-induced enhancers of Pro. In addition, candidate screens suggest that the insulin pathway contributes to robust larval germline proliferation.

Aric Rogers et al. (2007) International Worm Meeting "Post-transcriptional regulation downstream of the insulin-like signaling pathway in C. elegans."

Gordon Lithgow, Pankaj Kapahi, Di Chen, Aric Rogers, Gawain McColl, Alan Hubbard

[International Worm Meeting, 2007]

The insulin-like signaling (ILS) pathway plays a major role in the regulation of lifespan, metabolism, and neurodegenerative phenotypes in C. elegans. These phenotypes are modulated by gene expression downstream of the forkhead transcription factor DAF-16. A number of studies have used CHIP, SAGE, and microarrays to identify downstream mediators of DAF-16 at the genome-wide level. These studies have begun to resolve the complicated transcriptional network regulated by DAF-16 that determines certain phenotypes associated with ILS mutants. Additional elucidation may involve a better understanding of post-transcriptional regulation, as some studies in yeast and mammalian cells have reported a lack of correlation between the transcriptome and the proteome (Gygi et al. 1999, Ideker et al. 2001). Furthermore, deregulation of post-transcriptional processing and mRNA translation is now known as a crucial determinant in aging and age-related diseases including cancer, diabetes, and neurodegeneration. Despite intense study of ILS by numerous researchers, little is known about how DAF-16 and its direct as well as indirect targets affect post-transcriptional regulation of gene expression. We examined post-transcriptional changes in gene expression at the genome-wide level using a method referred to as translational state array analysis (TSAA). To measure the translation state of the mRNA, transcripts bound by ribosomes (polysomes) were separated using sucrose density gradient centrifugation. Resolved polysomes were fractionated according to low or high numbers of ribosomes bound per transcript. Unbound (untranslated) mRNA was also collected and all three fractions were analyzed using standard microarray techniques. We report that a number of genes previously shown to regulate lifespan and fat storage exhibit differences in post-transcriptional processing in a comparison of daf-2 and daf-2:daf-16 C. elegans mutants. Furthermore, the expression of numerous RNA-binding and mRNA translation regulating genes involved in post-transcriptional processing show changes in gene expression as assessed by mRNA ribosomal load. Preliminary lifespan analyses using RNAi-mediated gene suppression suggests that this method has been highly successful in identifying novel genes that determine lifespan in this pathway. Together, these results are the first to demonstrate vast post-transcriptional control by the ILS pathway in C. elegans. A better understanding of post-transcriptional processing within the ILS pathway will provide more insight into the basic mechanisms that determine longevity and metabolism.

Nariya, Hardik et al. (2017) International Worm Meeting "An investigation of the effects of artificial light at night on C. elegans offspring production and lifespan."

Buchanan, Bryant, Wise, Sharon, Zhushma, Rosa, Nariya, Hardik, Shinn-Thomas, Jessica

[International Worm Meeting, 2017]

Artificial light at night (ALAN) has many broad-scale and global implications for ecosystems and wildlife that have evolved under a 24-h circadian cycle. With increased urbanization, artificial light at night has directly altered natural photoperiods and nocturnal light intensity. Artificial light at night can disrupt behavioral patterns such as foraging activity and mating in animals. Disturbances in natural light and dark cycles also affect melatonin-regulated circadian and seasonal rhythms in Drosophila. We investigated the impact of ecologically relevant levels of light pollution on an important invertebrate model, Caenorhabditis elegans, as the impact of night lighting at these light levels is currently unknown. In this study, we exposed worms to artificial light at four intensities: 10-4 lx (control, comparable to natural nocturnal darkness), 10-2 lx (comparable to full-moon lighting and a low level of light pollution), 1 lx (comparable to dawn/dusk or intense light pollution), and 100 lx (dim daylight level comparable to extreme light pollution) on a 12L:12D photoperiod (100 lx treatments experienced constant light). We measured the impact of these light treatments on offspring production in hermaphroditic C. elegans. We grew worms for 2 generations in each light treatment, and then recorded the lifespan and counted the number of hatched offspring produced in the F3 generation. Our data show no significant differences among light levels for lifespan or offspring production suggesting that at least for these life history traits, ALAN does not affect these soil nematodes. Future directions include measuring additional life history traits and circadian gene expression for worms exposed to ALAN.

Diana Dalfo et al. (2008) Development & Evolution Meeting "RNAi screening to identify enhancers of the germline tumor phenotype in C. elegans"

E. Jane Hubbard, Diana Dalfo

[Development & Evolution Meeting, 2008]

There is significant interplay between the control of cell fate decisions and cell proliferation during development, yet the mechanisms underlying this interplay are not well understood. We are using the C. elegans germ line to investigate these mechanisms. C. elegans, germline polarity is established when a subset of previously undifferentiated germ cells differentiate and enter meiosis at a distinct developmental time and position. We have isolated mutants that disrupt the pattern of germline development such that the initial meiotic onset is delayed. The ultimate consequence of this delay is a large mass of proliferating germ cells in the proximal germ line of the adult. This phenotype, proximal proliferation (Pro), is observed with certain alleles of glp-1 (Pepper et al., 2003(a,b)). glp-1 is a receptor of the LIN-12/Notch family, and its activity promotes the proliferative or undifferentiated state and/or inhibits differentiation (meiosis) in the germ line (Austin and Kimble, 1987; Yochem and Greenwald, 1989). We are using a glp-1(Pro) allele as a starting point to identify additional factors that affect early germline proliferation and initial meiotic entry. Our previous data indicate that the distal pair of somatic gonadal sheath cells influence germline proliferation and act as an additional, non-DTC-mediated, mechanism to promote robust larval germline proliferation (Killian and Hubbard, 2005). We also observed that ablation of these cells enhances the glp-1(Pro) phenotype (Killian and Hubbard, 2005). Therefore, to identify the sheath-autonomous proliferation-promoting activity, we are performing candidate and genome-wide RNAi screens for enhancers of the Pro phenotype.

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.

Shownkeen R et al. (1995) International C. elegans Meeting "THE CAENORHABDITIS ELEGANS PHYSICAL MAP"

Chissoe SL, Waterston RH, Fraser A, Sulston JE, Shownkeen R, Coulson AR

[International C. elegans Meeting, 1995]

Twelve contigs of cosmids and yeast artificial chromosomes (YACs) span more than 95Mb of the 100Mb C.elegans genome. 650 markers link the physical and genetic maps.Hybridisation of tag-sequenced cDNA clones to a map-representative set of YACs indicates that the map incorporates in excess of 99.8% of genes. The map is accessible in ACeDB. We (S.C.) are investigating the representation by bacterial artificial chromosomes (BACs) of regions of the genome not represented by cosmids. Two grids of YACs, of 958 clones ('Poly2') and 223 clones ('Suppoly') are available on request. The latter represents regions of the genome that have been characterised or better defined since the selection of clones for the former. Cosmid clones and YAC grids are available from the Sanger Centre (requests to alan@sanger.ac.uk; FAX 01223 494919). YAC clones and 'cm' series cDNA clones are available from the Sanger Centre or Washington University (rw@nematode. wustl.edu; FAX 314 362 2985).

Dalfo, Diana et al. (2009) International Worm Meeting "RNAi-based identification of genes involved in germline proliferation."

Dalfo, Diana, Albert Hubbard, E. Jane

[International Worm Meeting, 2009]

Precise control of cell proliferation is necessary to create organs of the correct size and shape. We are interested in understanding the molecular basis for the control cell proliferation in vivo, and we are using the C. elegans germ line as a model. In C. elegans, as in other organisms, germline proliferation is a key step in the development of a reproductively competent individual. Previously, we isolated mutants that disrupt the pattern of germline development and ultimately produce a large ectopic mass of proliferating germ cells (a germline tumor) in the proximal part of the gonad arm (Pepper et al., 2003a,b; Killian and Hubbard, 2004; Voutev et al., 2006). This phenotype, proximal proliferation (Pro), is observed with certain mutant alleles of glp-1 (Pepper et al., 2003). glp-1 encodes a receptor of the LIN-12/Notch family, and its activity promotes the proliferative or undifferentiated state and/or inhibits differentiation (meiosis) in the germ line (Austin and Kimble, 1987; Yochem and Greenwald, 1989). We are performing a genome-wide RNAi screen for enhancers of the Pro phenotype to look for factors that affect early germline proliferation and initial meiotic entry.

Roumen Voutev et al. (2005) International Worm Meeting "Pro mutants, germline tumors, sheath cells, and ribosome biogenesis"

Roumen Voutev, E Jane Albert Hubbard, Darrell J Killian, James Hyungsoo Ahn

[International Worm Meeting, 2005]

Increasing evidence suggests that general cellular processes influence development in specific ways, likely reflecting tissue-specific differences in cellular demands. Gonadogenesis is proving an excellent model system to explore the intersection of ribosome biogenesis and development. Our data suggest that certain somatic gonad functions that influence germline proliferation are very sensitive to perturbations in ribosome biogenesis.Our analysis of pro (PROximal PROliferation) mutants led us to consider the intersection of development and ribosome biogenesis. The Pro phenotype is characterized by a proximal germline tumor. Our results support a model whereby the pro-1(na48) Pro phenotype stems from a cascade of events starting with an early reduction in germline proliferation due to functional defects in the distal sheath. This germline proliferation defect retards gonad arm extension, which in turn, causes a delay in meiotic entry. The delay in meiotic entry inappropriately juxtaposes mitotic germ cells with cells of the proximal sheath lineage and thereby allows these germ cells to respond to a latent proliferation-promoting activity of the proximal sheath lineage. Thus, due to developmental asynchrony between the somatic gonad and germ line, a proximal germline tumor eventually develops in the initially under-proliferated germ line (Killian and Hubbard, 2005).Several lines of evidence suggest that ribosome biogenesis is a limiting process for the germline proliferation-promoting activity of the distal sheath. PRO-1 is a conserved WD-repeat protein that acts in the sheath lineage of the somatic gonad (Killian and Hubbard, 2004). The budding yeast ortholog of PRO-1, IPI3, has been implicated in several processes including pre-rRNA processing (Peng et al., 2003; Krogan et al., 2004). Several C. elegans orthologs of direct IPI3 interactors that are also involved in ribosome biogenesis cause a Pro phenotype when depleted by RNAi. Reduction of ncl-1 increases rDNA transcription (Frank and Roth, 1998; Frank et al., 2002) and suppresses the pro-1 Pro phenotype. Reduction of lin-35/Rb in the soma also suppresses the Pro phenotype. This result is particularly intriguing since Rb negatively regulates rDNA transcription in mammals. In addition, the relative levels of rRNA processing intermediates are altered in pro-1(na48) mutants. Finally, pro-2, as defined by pro-2(na27), likely encodes an ortholog of yeast NOC2 which is implicated in nucleolar/nucleoplasmic transport of the large ribosomal subunit.

Chan S et al. (1991) International C. elegans Meeting "SEEKING UNC-40."

Culotti JG, Su M-W, Chan S, Hedgecock EM

[International C. elegans Meeting, 1991]

unc-40 is required with unc-6 to guide ventral migrations on the nematode epidermis (Hedgecock et al., Neuron 4, 61-85, 1990). We have positioned unc-40 genetically by 3-factor crosses with dpy-5 and bli-4 as flanking markers and we have isolated 5 new alleles of unc-40 (2 spontaneous, 2 gamma, and 1 EMS) making a total of 12. DNAs from cosmids covering this region (obtained from Alan Coulson and John Sulston) are being used to probe DNA and RNA from the mutants and DNA from strains carrying duplications that break to either side of unc-40 (obtained from Anne Rose's laboratory). We are also injecting cosmid DNAs into gonads to look for rescue by germline transformation. The brood sizes of various unc-40 alleles are very small, so we have resorted to injecting the wild type in order to create a series of transformed lines, each carrying an extrachromosomal array of a different cosmid. Each array will be passed into appropriately marked unc-40 animals to ask if any can rescue the mutant phenotype. Mosaic experiments are also underway to determine whether unc-40 acts in migrating cells.

Maine EM et al. (1997) International C. elegans Meeting "ROLE of ego GENES IN GERMLINE DEVELOPMENT"

Maine EM, Stacey SC, Spoerke JM, Qiao L

[International C. elegans Meeting, 1997]

Germline proliferation in C. elegans is accomplished via an inductive mechanism whose components include the LAG-2 signal, GLP-1 receptor, and LAG-1 effector. To identify pathway targets and additional pathway components, we are studying genes that interact genetically with glp-1. We will report our latest findings with several genes, including ego-3 and ego-6. ego-3(om40) was recovered as an enhancer of glp-1 and ego-3(om118) by noncomplementation with om40. om40 enhances lag-1 and emb-5, another Ego gene whose mutant phenotype (described by J. Hubbard) is similar in many ways to ego-3. Mutations in both genes produce a (transient) arrest in germline mitosis at L3 stage and maternal effect embryonic lethality. om118 is also associated with a tightly linked zygotic lethality. The om40 and om40/om118 germline and Mel phenotypes, as well as the om118 Let phenotype, can be rescued using an ~8 kb DNA fragment predicted to contain a gene with a "novel" product. We are now confirming that this gene is ego-3. Mutations in ego-6 enhance lag-1 and fail to complement mutations in ego-1. ego-6 and ego-1 mutants have similar defects in germline mitosis and early meiotic prophase. Both genes map between unc-13 and gld-1, but appear to be distinct based on Df and three-factor mapping. To determine the nature of the gene products and show definitively that ego-1 and ego-6 are distinct, we are looking for DNA polymorphisms associated with six ego-6 alleles and relevant Df breakpoints.