Jordan, James M. et al. (2017) International Worm Meeting "Development of germline tumors after extended starvation during L1 arrest."

Hubbard, E. Jane Albert, Chitrakar, Rojin, Jordan, James M., Baugh, L. Ryan, Guzman, Ryan, Webster, Amy K.

[International Worm Meeting, 2017]

Environmental stress during development can have persistent effects on adult physiology. C. elegans has a variety of attributes that make it an ideal model for such developmental origins of adult disease. We found that extended starvation during L1 arrest results in the formation of germline tumors in adults. We performed RNAseq at egg-laying onset, and our analysis suggested a role for glp-1/Notch signaling, germline apoptosis, insulin-like signaling (IIS) and fatty acid metabolism in tumor formation. We found that disruption of gld-1/Quaking, car-1/LSM14, cgh-1/DDX6 and cep-1/p53 significantly enhance tumor formation after L1 starvation and that glp-1/Notch is epistatic to L1 starvation. SYTO12 staining also suggests reduced germline apoptosis in tumorous gonads. Together these results are consistent with disruption of germ cell differentiation and apoptosis following L1 starvation, resulting in tumor formation. We hypothesize extended L1 starvation causes a "latent niche" to form due to aberrant germ cell-gonadal sheath communication, which increases glp-1/Notch signaling activity and drives the observed effects on germ cell differentiation and apoptosis. We also found that reducing IIS in the soma during recovery from starvation suppresses tumor formation in daf-16/FoxO-dependent fashion, confirming a role of IIS as suggested by RNA-seq. RNA-seq suggests reduced IIS alters regulation of fatty acid metabolism, including down-regulation of enzymes involved in fatty acid synthesis, desaturation, oxidation in peroxisomes and mitochondria, and storage as triacylglycerides. We found that supplementation with the monounsaturated fatty acid oleic acid suppresses tumor formation. Furthermore, RNAi of acs-17, an IIS-regulated acyl-CoA synthetase, enhances tumor formation alone and prevents tumor suppression by reduced IIS. Taken together, this suggests IIS-mediated fatty acid metabolism is tumorigenic following L1 starvation, and acs-17 and oleic acid act downstream of IIS to promote a less oncogenic mode of fatty acid metabolism. We are working to more specifically define the effects of L1 starvation and IIS on fatty acid metabolism, and to link those effects to glp-1/Notch signaling in order to explain how early-life nutrient stress causes adult germline pathology.

Jordan, James M. et al. (2015) International Worm Meeting "Extended starvation during L1 arrest appears to reduce fitness in the exposed animals but cause potentially adaptive effects in their descendants."

Sandrof, Moses A., Jordan, James M., Jobson, Meghan A., Baugh, L. Ryan, Hibshman, Jon D.

[International Worm Meeting, 2015]

Embryos hatched in the absence of food arrest development in the first larval stage. Larvae can survive this so-called "L1 arrest" for weeks and resume grossly normal development upon feeding. L1 arrest provides a model for nutritional control of development, and it is routinely used to synchronize cultures and freeze strains. However, the long-term phenotypic consequences of L1 arrest, including epigenetic or pathological effects, have not been described. We characterized the effects of extended L1 arrest on a variety of life-history traits in the exposed animals and their descendants. Larvae recovered from 1 week of arrest grow slowly, taking longer to become reproductive, and are smaller as adults. They also display reduced reproductive potential, with the smallest individuals most severely affected. Pharyngeal pumping and grinding are reduced, perhaps contributing to slow growth and reduced fertility. Adults tend to hold their eggs, and many of them die as a bag of worms, but this appears to be due to physiological regulation as opposed to defective development. However, protruding and exploded vulvae are observed, though relatively rare. The progeny of starved animals are also affected: abnormally shaped embryos with reduced hatching efficiency are common, F1 larvae are smaller upon hatching and after 48 hr growth, the smallest individuals have decreased fertility and there is an increased incidence of males. Larvae that were starved for 1 week are no more resistant to heat or starvation. In fact, larvae recovered from starvation are more sensitive to starvation when exposed later in development, arguing against a "thrifty" phenotype in the narrow sense of the exposed generation. However, the progeny and grandprogeny of starved larvae are more resistant to starvation as L1 larvae. In addition, the progeny, grandprogeny and great-grandprogeny (F3) are more resistant to heat, suggesting epigenetic inheritance of acquired stress resistance. Taken together, our results suggest that extended starvation during early larval development has a variety of ecologically-relevant phenotypic effects. The results are inconsistent with a relatively simple fitness trade-off in the exposed animals but instead suggest a combination of potentially maladaptive and adaptive effects that play out over generations.

Butler JA et al. (1994) Worm Breeder's Gazette "Looking For Extracellular Matrix Proteinases in C. elegans"

Kramer JM, Butler JA

[Worm Breeder's Gazette, 1994]

LOOKING FOR EXTRACELLULAR MATRIX PROTEINASES IN C. ELEGANS. James A. Butler and James M. Kramer, Northwestern University Medical School. Department of CMS Biology, Chicago IL 60611

Wissmann AK et al. (1994) Worm Breeder's Gazette "Characterization of the let-502 Gene"

Mains PE, Wissmann AK, McGhee JD

[Worm Breeder's Gazette, 1994]

Characterization of the let-502 gene Andreas Wissmann, James D. McGhee and Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Webster, Amy K. et al. (2017) International Worm Meeting "Transgenerational effects of long-term dauer arrest."

Hibshman, Jonathan D., Chitrakar, Rojin, Webster, Amy K., Jordan, James M., Baugh, L. Ryan

[International Worm Meeting, 2017]

Long-term effects of extended starvation during L1 arrest have been shown to persist to the F3 generation, suggesting transgenerational epigenetic inheritance. Though effects on lifespan and gene expression have been reported (Rechavi et al), we have only observed relatively subtle transgenerational effects on L1 starvation survival and heat resistance (Jobson, Jordan et al). Furthermore, we found that a minority of starved worms transmitted these effects, requiring phenotype-based sorting of the starved population to detect transgenerational effects among their descendants. In contrast to L1 arrest, larvae can survive dauer arrest for months and the majority of C. elegans in the wild are found as dauers. Persistent effects of dauer arrest have been reported for adults (Hall et al), but transgenerational effects have not been reported. We reasoned that dauer arrest is likely a more ecologically relevant and robust model for transgenerational effects of starvation. Here, we used a liquid culture system carefully controlling worm density and food availability to cause virtually 100% of N2 larvae to enter dauer arrest. These worms remain dauers for months and become reproductive adults when allowed to recover on plates. By assessing various life-history traits, we found that worms subjected to long-term dauer arrest exhibit apparent fitness costs upon recovery, including maternal effects in the F1 generation. However, L1 starvation survival was increased population-wide in the F3 generation (without phenotypic sorting of ancestors), suggesting potentially adaptive transgenerational effects of long-term dauer arrest. This phenotypic effect depended on the length of time the P0 generation spent in dauer arrest, suggesting that dauer formation alone is not sufficient. Notably, the transgenerational effects occurred in the absence of detectable gene expression changes, as if the presumed epigenetic effect on gene expression is distributed over many genes with a small effect on each. This work suggests that experiencing a stress such as starvation can initially be costly, but future generations may exhibit increased fitness in particular circumstances. References: Hall, S.E. et al., A cellular memory of developmental history generates phenotypic diversity in C. elegans. Current Biology, 2010. 20(2): p. 149-155. Jobson, M.A., Jordan J.M. et al., Transgenerational Effects of Early Life Starvation on Growth, Reproduction, and Stress Resistance in Caenorhabditis elegans. Genetics, 2015. 201(1): p. 201-12. Rechavi, O. et al., Starvation-Induced Transgenerational Inheritance of Small RNAs in C. elegans. Cell, 2014. 158(2): p. 277-287.

Moore, Brad T et al. (2013) International Worm Meeting "WormSizer: Image-based analysis of nematode size and shape."

Jordan, James M, Baugh, L Ryan, Moore, Brad T

[International Worm Meeting, 2013]

The fundamental phenotypes of growth rate, size and morphology are the result of complex interactions between genotype and environment. We developed a high-throughput software application, WormSizer, which computes size and shape of nematodes from brightfield images. Existing methods for estimating volume either coarsely model the nematode as a cylinder or assume the worm shape or opacity is invariant. Our estimate is more robust to changes in morphology or optical density as it only assumes radial symmetry. This open source software is written as a plugin for the well-known image-processing framework Fiji/ImageJ. WormSizer is designed to be used by biologists, and works with images from a standard dissection microscope. We evaluated the technical performance of this framework, and we used it to analyze growth and shape of several canonical Caenorhabditis elegans mutants in a developmental time series. We confirm quantitatively that a Dumpy (Dpy) mutant is short and fat and that a Long (Lon) mutant is long and thin. We show that daf-2 insulin-like receptor mutants are larger than wild-type upon hatching but grow slow, and WormSizer can distinguish dauer larvae from normal larvae. We also show that a Small (Sma) mutant is actually smaller than wild-type at all stages of larval development. WormSizer works with Uncoordinated (Unc) and Roller (Rol) mutants as well, indicating that it can be used with mutants despite behavioral phenotypes. We used our complete data set to perform a power analysis, giving users a sense of how many images are needed to detect different effect sizes. Our analysis confirms and extends on existing phenotypic characterization of well-characterized mutants, demonstrating the utility and robustness of WormSizer.

Wypijewska A et al. (2012) Biochemistry "7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity."

Wypijewska A, Darzynkiewicz E, Davis RE, Jemielity J, Bojarska E, Lukaszewicz M, Stepinski J

[Biochemistry, 2012]

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

O'Connell EM et al. (2015) J Infect Dis "Targeting Filarial Abl-like Kinases: Orally Available, Food and Drug Administration-Approved Tyrosine Kinase Inhibitors Are Microfilaricidal and Macrofilaricidal."

Nutman TB, O'Connell EM, Bennuru S, Steel C, Dolan MA

[J Infect Dis, 2015]

BACKGROUND: Elimination of onchocerciasis and lymphatic filariasis is targeted for 2020. Given the coincident Loa loa infections in Central Africa and the potential for drug resistance development, the need for new microfilaricides and macrofilaricides has never been greater. With the genomes of L. loa, Onchocerca volvulus, Wuchereria bancrofti, and Brugia malayi available, new drug targets have been identified. METHODS: The effects of the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib on B. malayi adult males, adult females, L3 larvae, and microfilariae were assessed using a wide dose range (0-100 M) in vitro. RESULTS: For microfilariae, median inhibitory concentrations (IC50 values) on day 6 were 6.06 M for imatinib, 3.72 M for dasatinib, and 81.35 M for nilotinib; for L3 larvae, 11.27 M, 13.64 M, and 70.98 M, respectively; for adult males, 41.6 M, 3.87 M, and 68.22 M, respectively; and for adult females, 42.89 M, 9.8 M, and >100 M, respectively. Three-dimensional modeling suggests how these tyrosine kinase inhibitors bind and inhibit filarial protein activity. CONCLUSIONS: Given the safety of imatinib in humans, plans are underway for pilot clinical trials to assess its efficacy in patients with filarial infections.

Wood WB (1976) Worm Breeder's Gazette "maternal effects among ts zygote-defective (zyg) mutants."

Wood WB

[Worm Breeder's Gazette, 1976]

We have studied maternal effects in 23 zyg ts mutants to estimate the times of expression of genes whose products are required in embryogenesis. We have used the following three tests, called arbitrarily A, B, and C. A test: Heterozygous (m/+) L4's are shifted to 25 C and allowed to self-fertilize. If 100% of their eggs yield larvae (25% of which express the mutant phenotype as adults), then the mutant is scored as maternal (M). If 25% of the F1 eggs fail to hatch, then the mutant is scored as non-maternal (N). An M result indicates that expression of the + allele in the parent allows m/m zygotes to hatch and grow to adulthood. A result of N indicates the opposite: that the + allele must be expressed in the zygote for hatching to occur. Out of 23 zyg mutants tested, 3 were scored N and 20 were scored M in the A test. Therefore, for most of the genes defined by these mutants, expression in the parent is sufficient for zygote survival, even if the gene is not expressed in the zygote. B test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with N2 (+/+) males. If eggs fail to hatch at 25 C, but mated hermaphrodites shifted to 16 C produce cross progeny to give proof of mating, then the mutant is scored M. If cross progeny appear in the 25 C mating, then the mutant is scored N. An M result indicates that expression of the + allele in the zygote is not sufficient to allow m/+ progeny of an m/m hermaphrodite to survive. Conversely an N result indicates either that zygotic expression of the + allele is sufficient for survival, or that a sperm function or factor needed for early embryogenesis can be supplied paternally (see C test below). Out of the 23 zyg mutants tested, 11 were scored M and 12 were scored N. The combined results of A and B tests and their simplest interpretation are as follows. Ten mutants are M,M; the genes defined by these mutants must be expressed in the hermaphrodite parent for the zygote to survive. Ten mutants are M,N; these genes can be expressed either in the parent or in the zygote. Two mutants are N,N; these genes must be expressed in the zygote. One mutant is N,M; this gene must be expressed both in the maternal parent and in the zygote. C test: Homozygous (m/m) hermaphrodites reared at 25 C are mated with heterozygous (m/+) males. If rescue by a +/+ male in the B test depends on the + allele, then only half the cross progeny zygotes of a C test mating (m/+ male x m/m hermaphrodite) should survive. However, if rescue depends on a function or cytoplasmic component from the male sperm, then all the cross progeny zygotes in a C test should survive. Of the 10 M,N mutants, 6 have been C tested; one exhibited paternal rescue independent of the + allele. The A and B tests also were carried out on 16 mutants that arrest before the L3 molt (acc mutants). In the A test on 2 of these mutants, all m/m progeny of m/+ parents grew to adulthood at 25 C. Therefore, parental contributions are sufficient to overcome a progeny mutational block as late as the L2 stage. All 16 acc mutants scored N in the B test.

Ali Khan L et al. (1994) Worm Breeder's Gazette "cej-1 Encodes a Novel Protein With Poly-Threonine Motif"

Siddiqui SS, Fukushige T, Ali Khan L, Tsukita Sh, Tabish M, Itoh M

[Worm Breeder's Gazette, 1994]

cej-1 Encodes a Novel Protein with Poly-Threonine Motif M. L. A. Khanl, M. Tabish, T. Fukushigel1 S. Tsukita2, M. Itoh , Sh. Tsukita , and S. S. Siddiqui. (1): Lab. of Molecular Biology, Dept of Ecological Engg. Toyohashi Univ. Technology, Toyohashi 441, and (2). National Institute for Physiological Sciences, Okazaki 444, Japan.