Neil Umbreit et al. (2007) International Worm Meeting "C. elegans for inquiry-based genetics in high schools."

Neil Umbreit, Brian Dirienzo, Lew Jacobson, Alison Slinskey Legg

[International Worm Meeting, 2007]

Genetics in high school biology is often taught by lecture and paper exercises, methods which typically fail to stimulate student interest. The Gene Team Curriculum Roundtable (see abstract by ASL and LJ, this meeting) is developing a module for high school labs using C. elegans for an inquiry-based approach to some core principles of Mendelian genetics. The current version uses unc-76 V and dpy-13 IV mutants in crosses to allow students to "discover" dominant, recessive and partial-dominant behavior of alleles, the principle of independent assortment of alleles during meiosis, and the concepts of sex determination. Students manipulate the worms, make observations, form hypotheses, collect quantitative experimental data, and use these data to support or refute their hypotheses. The exercise takes 8-10 class periods over more than 3 weeks to complete, but is designed with flexible alternatives so that teachers can choose to implement only parts of the full sequence while maintaining the focus on scientific inquiry. To aid in implementation, we provide equipment (including Leica S6E dissecting scopes), materials and staff support at the schools. Among the pitfalls and solutions we have identified: (a) Students need at least one day of worm-picking practice before starting crosses. (b) Many students and teachers have difficulty learning to recognize males. It helps greatly to show them, using a trinocular dissecting scope with digital camera output to an LCD display or projector. (c) It is confusing that mating generates both cross-progeny and self-progeny, and that the ratio of these is unpredictable. If teachers use Punnett squares to explain this, the quantitative issues are confusing. (d) High-school textbooks invariably use single-letter gene symbols, so both teachers and students become confused by worm gene nomenclature. (e) Inquiry-based learning takes time. Even simple experiments may take more class days than teachers are willing to accept. The module has been tested with the 2006 Gene Team participants, with a University-level Pre-Biology class, and in various versions in 5 area high schools in academic year 2006-07. Baseline measurements of instructional effectiveness are underway. The hands-on nature of the inquiry and its perceived relevance to modern biological research seems to hold much more appeal for students and to excite public interest. Supported by a Science Education Partnership Award (SEPA) from the National Center for Research Resources, NIH.

Voegtlin, Thomas et al. (2011) International Worm Meeting "An Open-Source Neuromechanical Model of C. Elegans Locomotion."

Cohen, Netta, Voegtlin, Thomas

[International Worm Meeting, 2011]

A neuromechanical model of locomotion in C. elegans was recently proposed by Jordan H. Boyle [1]. One of the main results is that both swimming and crawling can be generated by a single neural circuit, reflexively modulated by the environment. This supports the known experimental results showing that different forms of C. elegans forward locomotion (e.g., swimming and crawling) can be described by a modulation of a single biomechanical gait [2]. The modelling result illustrates the importance and the potential of neuromechanical simulations for the analysis of the worm's behaviour. In order to continue this work, and to make it usable by a broader audience, we have developed a similar neuromechanical model of the worm using CLONES. CLONES (Closed Loop Neural Simulation) is an open source framework for neuromechanical simulations. CLONES implements a communication interface between a neural simulator, called BRIAN [3], and a physics engine for biomedical applications, called SOFA [4]. BRIAN and SOFA are open-source simulators that are easy to use and provide high performance. Our implementation of the worm's locomotion reproduces the neural model described in [1]. However, there are two key differences between the original physical model and our implementation. Firstly, Boyle's model considers that the body of the worm has zero mass (a low Reynolds number approximation). In contrast, the SOFA simulator allows us to integrate equations with mass and inertia. Secondly, the original model uses rigid rods of fixed length orthogonal to the body axis (approximating the incompressibility of the body due to high internal pressure). In SOFA rigid rods are modeled as springs of very high stiffness. The physical system simulated in SOFA is described using a XML syntax. The neural network model interpreted by BRIAN is written in Python, using MATLAB-like syntax. Thus, the model is completely interpreted, and it is possible to visualize/interact with the simulation during runtime. Physical environments containing obstacles or chemical concentration gradients can be defined easily. References 1. Boyle JH: C. elegans locomotion: an integrated approach. PhD thesis, university of Leeds, 2009 2. Berri S, Boyle JH, Tassieri M, Hope IA and Cohen N, Forward locomotion of the nematode C. elegans is achieved through modulation of a single gait HFSP J 3:186, 2009; 3. Goodman DF, Brette R: Brian: a simulator for spiking neural networks in Python. Front Neuroinform 2:5, 2008 4. Allard J, Cotin S, Faure F, Bensoussan PJ, Poyer F, Duriez C, Delingette H, Grisoni L: SOFA - an Open Source Framework for Medical Simulation. Medicine Meets Virtual Reality (MMVR'15), pp. 13-18, 2007.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Paramita Ray et al. (2004) Mid-west Worm Meeting "ceh-28 is required for feeding in C. elegans"

Peter G Okkema, Paramita Ray

[Mid-west Worm Meeting, 2004]

NK-2 family homeobox genes are important developmental regulators in many organisms. The C. elegans genome contains 4 members of this gene family ( ceh-22, ceh-24, ceh-27 and ceh-28 ). ceh-22 is expressed in the pharyngeal muscles where it regulates gene expression with the pan-pharyngeal factor PHA-4, and we are determining if additional NK-2 family members function similarly in other pharyngeal cell types. Brian Harfe and Andy Fire have previously shown a ceh-28::gfp transcriptional fusion is expressed exclusively in the M4 pharyngeal neuron. We have similarly found a ceh-28::gfp translational fusion containing 3.7 kb of 5' flanking DNA is expressed in M4. Expression initiates during embryogenesis in two cells, and we speculate these cells are M4 and its sister cell, which dies during normal development. We are currently verifying the expression pattern of the endogenous ceh-28 gene using anti-CEH-28 antibodies. M4 is a motor neuron essential for pharyngeal isthmus peristalsis and feeding. To functionally characterize ceh-28 , we have isolated a deletion mutant ceh-28(cu11) . This deletion removes much of the ceh-28 homeobox and is likely a null allele. Homozygous ceh-28(cu11) strains can be maintained, although these mutants appear starved and exhibit slow growth and partially penetrant larval lethality. Similar phenotypes were observed in ceh-28(RNAi) animals. All ceh-28 mutant worms have a stuffed pharynx phenotype similar to animals in which M4 has been killed by laser ablation (Avery and Horvitz, 1987), suggesting M4 is defective. We are currently examining M4 differentiation, morphology and function in ceh-28(cu11) mutants to understand the role of ceh-28 in pharyngeal development.

Wilhelm, Thomas et al. (2015) International Worm Meeting "A C.elegans RNAi screen identifies novel genes with antagonistic pleiotropic effects."

Medina, Rebeca, Wilhelm, Thomas, Richly, Holger, Byrne, Jonathan

[International Worm Meeting, 2015]

In 1957, George C. Williams published the antagonistic pleiotropy hypothesis of aging, which was later mathematically supported by Brian Charlesworth. This theory predicts that some genes mediate beneficial effects early in life when natural selection is strong, but are detrimental late in life when natural selection is weak. Natural selection favors these beneficial effects on fitness early in life at the expense of enriching harmful effects late in life; hence leading to the evolution of aging.We screened 800 chromatin regulators and transcription factors in order to identify novel genes that exhibit antagonistic pleiotropy. Genes were inactivated in the post-reproductive phase by the use of RNAi from day nine of adulthood onwards. The screen led to the identification of several genes that significantly extend lifespan upon late-life inactivation, while shortening lifespan when inactivated early in life.The forkhead box (FOX) A transcription factor pha-4 represents a top candidate of our screen. Late-life inactivation of pha-4 resulted in a strong lifespan extension of 33%. In contrast, inactivation of pha-4 from the first day of adulthood reduced C.elegans lifespan. This is of particular interest, as pha-4 has been shown to mediate diet-restriction and germline-less induced longevity. Our findings indicate that, depending on the age, pha-4 is either needed for normal lifespan and longevity or exhibits detrimental effects. We are currently aiming to identify and to characterize the biological processes underlying the observed negative effects of pha-4 late in life.Generally, genes that behave according to the antagonistic pleiotropy hypothesis may represent promising drug targets to fight age-associated diseases. Our findings emphasize that timing needs to be addressed to fully understand how genes and gene networks impinge on the aging process.

Hammarlund M et al. (1997) International C. elegans Meeting "A Genetic Screen For Downstream Effectors of ICE"

Hammarlund M, Maricq AV

[International C. elegans Meeting, 1997]

CED-3 and its human homolog, Interleukin-1-beta Converting Enzyme (ICE), are known to play a central role in a cell's decision to undergo apoptotic death. However, the downstream effectors of these cysteine proteases are not well understood. In an effort to understand the processes by which cysteine protease activation leads to apoptotic death, we are conducting screens for supressors of ICE-induced cell death in C. elegans. In previous work with Cori Bargmann (UCSF), we fused the glr-1 promoter to the human ICE sequence and integrated this construct. glr-1::ICE animals are touch-defective due to the ICE-induced apoptotic death of interneurons. We mutagenized this strain with EMS and screened for suppression of the touch phenotype. Four suppressor strains were isolated from this screen. These strains are currently being characterized. Since the pathway that leads from ICE to apoptosis is likely to be extensively branched, we are taking a parallel approach that will boost our screening efficiency. We obtained the ceh-28 promoter from Brian Harfe and Andrew Fire, who showed that ceh-28::gfp is expressed only in the M4 pharyngeal neuron. Leon Avery has shown by laser ablation that M4 is required for survival beyond L1. M4 innervates the posterior half of the isthmus; when M4 is killed, the isthmus remains closed and the animal starves. Avery also found that M4-ablated animals are viable on media containing arecoline, a drug which depolarizes M4's post-synaptic target. We have fused the ceh-28 promoter to the human ICE sequence. We expect ceh-28::ICE animals to be non-viable on normal media due to apoptotic death of M4, but viable on arecoline. This will allow us to select for suppressors of the M4-ablated phenotype by growing ceh-28::ICE animals on arecoline, mutagenizing, and then moving F2's to normal media. This selection is likely to yield several broad classes of genes. In addition to downstream effectors of ICE, we expect to find mutations that allow ceh-28::ICE animals to survive by depolarizing the isthmus.

Paramita Ray et al. (2005) International Worm Meeting "ceh-28 is required for M4 neuronal differentiation and function in C. elegans"

Peter Okkema, Paramita Ray

[International Worm Meeting, 2005]

The M4 pharyngeal neuron is essential for peristalsis of ingested bacteria through the pharyngeal isthmus, and, when it is ablated, animals arrest with a characteristic stuffed pharynx. Brian Harfe and Andy Fire have previously shown a gfp reporter regulated by upstream sequences from the NK-2 family homeobox gene ceh-28 is exclusively expressed in M4. We are characterizing the role of ceh-28 in M4 development. We have constructed a ceh-28::gfp reporter containing 3.7 kb of 5-flanking DNA and found it is expressed in the M4 cell from late embryogenesis through adulthood. However, in contrast to Harfe and Fires fusion, our ceh-28::gfp reporter is expressed in two cells in younger embryos. In wild-type embryos, the sister of M4 (MSpaaaaap) undergoes programmed cell death in mid embryogenesis, and we hypothesize the two gfp expressing cells in early embryos are M4 and MSpaaaaap. Consistent with this hypothesis, we observe 2 ceh-28::gfp expressing cells in the pharynx throughout the life cycle in mutants defective in programmed cell death. We have isolated the deletion mutant ceh-28(cu11) and found these animals exhibit partially penetrant larval lethality and a stuffed pharynx similar to animals missing M4. These phenotypes are also found in ceh-28(tm1258) mutants. ceh-28(cu11) mutants are rescued by transformation with a wild-type ceh-28 genomic DNA fragment, indicating these phenotypes result from loss of ceh-28 function. In ceh-28(cu11) mutants, M4 is present and expresses ceh-28::gfp and other markers of M4 differentiation, but this M4 appears morphologically abnormal. In addition, we occasionally observe an extra ceh-28::gfp expressing cell in ceh-28(cu11) mutants, which may be MSpaaaaap. Thus, ceh-28 is necessary for development and function of the M4 neuron, and may play a role in MSpaaaaap cell death. Interestingly, ceh-28(cu11) mutants also exhibit abnormalities in the pharyngeal g1 gland cell processes. These processes extend near the M4 axons and cell body, and we suggest M4 is necessary for normal growth of the g1 cell processes.

Takashi Hirose et al. (2006) Development & Evolution Meeting "A Screen For Mutants Defective in the Specification of M4 Sister Cell Death"

Takashi Hirose, Bob Horvitz

[Development & Evolution Meeting, 2006]

In C. elegans, 131 somatic cells undergo programmed cell death during wild-type hermaphrodite development. The process of programmed cell death includes four distinct steps: cell-death specification, execution, engulfment and degradation. While many genes involved in cell-death execution, engulfment and degradation have been identified, the process of cell-death specification is poorly understood. The M4 motor neuron regulates muscle contraction in the pharynx, while the M4 sister undergoes programmed cell death in embryonic development. The reporter Pceh-28::gfp (kindly provided by Brian Harfe and Andrew Fire) is expressed in the M4 of wild-type animals and in both the M4 and the M4 sister of mutants defective in programmed cell death. We constructed strains carrying a Pceh-28::4XNLSgfp reporter. These strains have fluorescence tightly localized in the nucleus and allowed us to efficiently screen for mutants defective in M4 sister cell death by using a dissecting microscope equipped with fluorescence optics. Screens of 71,820 mutagenized haploid genomes yielded at least 75 independent mutations that cause extra gfp-expressing cells. By complementation tests, we identified at least 34 alleles of ced-3, 11 alleles of ced-4 and four alleles of egl-1. Five other isolates defining at least three genes had an extra gfp-expressing cell inside the pharynx. We do not know if the extra gfp-expressing pharyngeal cell in each of these mutants is the M4 sister cell. We are currently mapping the unidentified mutations in which one extra gfp-expressing cell is inside the pharynx. One, n4796, has been located to a 180 kbp interval on LG V. Rescue experiments are currently in progress. We also mapped a mutant in which one or two extra gfp-expressing cells are outside the pharynx. n4784 is located within a 86 kbp interval on LG X, and the mutant phenotype could be rescued by two overlapping cosmids. In the overlapping region, there is a single predicted gene, F49E10.5, which is most similar to adenovirus E1A C-terminal binding protein, a transcriptional corepressor. We have identified mutations in F49E10.5 in n4784 animals and in five other isolates that showed a similar phenotype. Further phenotypic characterization is in progress.

Grussendorf, Kelly et al. (2011) International Worm Meeting "The EXC-1 GTPase Is Required for Maintenance of Tubule Shape in the Excretory Canals."

Buechner, Matthew, Riss, Amanda, Grussendorf, Kelly

[International Worm Meeting, 2011]

Tubulogenesis involves the formation of tubule shape and diameter along both apical (lumenal) and basal sides. Once the tubule is formed, this structure needs to be regulated and maintained. The excretory canals provide a simple model to study these processes. The excretory canal cell, located near the terminal bulb of the pharynx, extends two hollow processes to the left and right lateral side of the worm, where they bifurcate and extend anteriorly and posteriorly to form an H-shaped structure. The set of EXC proteins maintain the structure of the apical surface of the canal. Mutations in the exc genes allow formation of fluid-filled cysts in the lumen of the canal. The Exc-1 phenotype exhibits canal cysts of variable size and number that are frequently located at the distal ends of the canals. Cysts can be very small, or as large as the entire diameter of the worm. We have cloned the exc-1 gene via fosmid and gene rescue, RNAi phenocopy, expression pattern, and allele sequencing, and found that this gene encodes a homologue of the family of mammalian Interferon-Inducible GTPases (IIGP), proteins that include a Ras GTPase domain. Overexpression of exc-1 causes the canal cell to form normal-diameter tubules, but with a defective basal surface. This phenotype also occurs through overexpression of other exc genes. exc-1 shows genetic interactions with other members of the exc family; the results imply that EXC-1 acts downstream of the LIM-domain protein EXC-9, but upstream of the guanine exchange factor EXC-5 (See poster by B. Mattingly). We have crossed exc-1 (rh26) mutants to marker strains that show the position of subcellular organelles within the canals. exc-1 mutants appear to have the same effect on subcellular organelles as does loss of EXC-5, a buildup of apical early endosomes with concomitant loss of recycling endosomes, leading to blockage of recycling of material to the apical membrane and presumed weakening of the apical surface. Current work is investigating possible direct protein-protein interactions between EXC-1 and EXC-5, and EXC-1's role in the tubular amphid sheath cells. We are grateful for marker constructs and advice supplied by Barth Grant, John White, Brian Ackley, Erik Lundquist, and Monica Driscoll, as well as support from NINDS R03-NS067323 and the Inez Jay Fund of the University of Kansas.

Takashi Hirose et al. (2007) International Worm Meeting "CEH-34 is Required for M4 Sister Cell Death and Pharyngeal Development."

Takashi Hirose, Bob Horvitz

[International Worm Meeting, 2007]

In C. elegans, 131 somatic cells undergo programmed cell death during wild-type hermaphrodite development. While many genes involved in programmed cell death have been identified, it remains unclear how a particular cell is specified to survive or to die by programmed cell death. To identify genes involved in the specification of programmed cell death, we screened for mutants defective in the deaths of the sister of the pharyngeal M4 neuron. The M4 motor neuron is generated during embryonic development and survives to regulate muscle contraction in the pharynx, while the M4 sister undergoes programmed cell death. The reporter Pceh-28::gfp (kindly provided by Brian Harfe and Andrew Fire) is expressed in the M4 of wild-type animals and in both the M4 and the M4 sister of ced-3 mutants, which are defective in all somatic programmed cell deaths. Screens of 71,820 mutagenized haploid genomes yielded 75 independent mutations that result in at least one extra Pceh-28::gfp-expressing cell. One of the isolates, n4780, is mutated in pig-1, a par-1-like gene that encodes a serine/threonine kinase and affects asymmetric neuroblast divisions. We also identified two egl-1 alleles, n4820 and n4827, for which we could not find any mutations in a coding region. These results suggest that the processes of asymmetric cell division and the regulation of egl-1 expression affect M4 sister cell death. One of the other isolates, n4796, is not an allele of any known cell-death gene and affects the M4 sister cell death but not the NSM sister, RIM sister, RIC sister or VC neuron cell death. Using SNP mapping and transformation rescue, we found that n4796 is an allele of ceh-34, a Six class homeobox gene. CEH-34 is expressed in pharyngeal cells and anterior body wall muscles. A deletion mutant (kindly provided by Shohei Mitani) showed more severe pharyngeal morphological defects than n4796 animals, suggesting that ceh-34 controls a variety of pharyngeal cell fates, including the M4 sister cell death. In Drosophila, the Six family homeobox gene sine oculis is a component of a highly conserved transcriptional network that specifies eye development. This transcriptional network includes eyeless/vab-3, eyes absent/eya-1 and dachshund/dac-1. We examined M4 sister cell death in C. elegans vab-3, eya-1 and dac-1 mutants and found that eya-1 mutants also have an extra Pceh-28::gfp-expressing cell; vab-3 and dac-1 mutants do not. These results suggest that at least some of the components of this transcriptional network are required for M4 sister cell death in C. elegans.