Sarinay Cenik, Elif et al. (2019) International Worm Meeting "Maternal Ribosomes Are Sufficient for Tissue Diversification during Embryonic Development in C. elegans."

Hall, Richard Nelson, Tang, Ngang Heok, Cenik, Can, Jin, Yishi, Arribere, Joshua A., Fire, Andrew, Meng, Xuefeng, Sarinay Cenik, Elif

[International Worm Meeting, 2019]

Caenorhabditis elegans provides an amenable sys- tem to explore whether newly composed ribosomes are required to progress through development. Despite the complex pattern of tissues that are formed during embryonic development, we found that null homozygotes lacking any of the five different ribosomal proteins (RPs) can produce fully functional first-stage larvae, with similar developmental competence seen upon complete deletion of the multi-copy ribosomal RNA locus. These animals, relying on maternal but not zygotic contribution of ribosomal components, are capable of completing embryogen- esis. In the absence of new ribosomal components, the resulting animals are arrested before progression from the first larval stage and fail in two assays for postembryonic plasticity of neuronal structure. Mosaic analyses of larvae that are a mixture of ribosome-competent and non-competent cells suggest a global regulatory mechanism in which ribosomal insufficiency in a subset of cells triggers organism- wide growth arrest.

Yeung MH et al. (1997) International C. elegans Meeting "THE USE OF C. ELEGANS FOR INSECTICIDE DISCOVERY"

Hodgkin JA, Yeung MH, Robinson MP

[International C. elegans Meeting, 1997]

C. elegans can be used as a tool in the discovery of new insecticides. Lead Detection - Wild type C. elegans can be used for screening of unknown compounds. The ideal is to use the target species on its normal host plant. However, that may not be amenable to screening. C. elegans has its advantages: - short life cycle and ease of culturing means the C. elegans can be grown to a large number in a short time using inexpensive culture medium. It can be stored frozen between use. - Our initial study shows that C. elegans has a reasonably selective activity to a spectrum of insecticide standards. Mode Of Action (i) - a panel of C. elegans mutants (each with known resistance or sensitivity) can be used to define the mode of action of unknown compounds. - If a mutant responds in a characteristic way to a compound, it is indicative of a relevant mode of action. - If all mutants do not provide any characteristic response to a compound, it is indicative of a novel mode of action. - Behavioural symptoms can be very useful. - It can be useful for predicting resistance and cross-resistance. - It can be useful for grouping unknown compounds. Mode Of Action (ii) - the unknown compound with a putative novel mode of action can be used as selective agent after EMS mutagenesis to generate mutants. - It can be useful towards understanding the mode of action by carrying out further work on the mutant. - The existing extensive information on ACeDB will facilitate characterisation of the mutant. - The mutant can be screened against a spectrum of putative relevant mode of action insecticide standards to check for cross resistance.

Guo X et al. (2010) Biology of the C. elegans Male, Madison, WI "The Mechanism of Maintaining Mating Potency During Aging"

Guo X, Garcia R L

[Biology of the C. elegans Male, Madison, WI, 2010]

Every organism ages, and during aging, the function of the organism deteriorates including behaviors such as locomotion, mating and feeding. Many studies have focused on the molecular signaling, which can extend or shorten lifespan. But little is known about why a specific behavior deteriorates during aging. In our study, we are trying to figure out the mechanism by which C.elegans males mating behavior declines during aging. Through mating potency analyses, we find that wild-type C. elegans virgin males' mating behavior begins to decline at day 3 of adulthood. 80%, 70% and 40% males can mate at day 1, day 2 and day 3, respectively. Sir-2.1 null mutant males cannot mate well from day 2. 75% and 10% sir-2.1 null males can mate at day 1 and day 2, respectively. Since caloric restriction can extend the lifespan of C. elegans, we hypothesize that starvation can also prolong the mating potency of males. Indeed, when we starve L4 wild-type males for 20hrs and then re-fed them, those males mating potency can be prolonged. Then we ask if the caloric restriction works through SIR-2.1 to prolong mating potency. To our surprise, caloric restriction during L4 can actually rescue the sir-2.1 null males mating potency at day 2. Cycloheximide – a translational inhibitor can also extend the lifespan of C. elegans, so we check if cycloheximide can affect mating potency. It can prolong the mating potency of wild type, but not sir-2.1 null mutant males. Because the neuromuscular mating circuit of C. elegans males is mainly regulated by ACh signaling. We check the sensitivity of acetylcholine signaling during aging by exposing males to ACh agonist such as levamisole and arecoline, we find that males become more sensitive to Ach agonist when they are aged, which is a possible explanation for why aged males cannot mate well. Indeed, males that have decreased Ach signaling due to having one copy of functional ACh receptors genes unc-29, acr-16, acr-18 and gar-3, can mate better at day 3 than wild type. For sir-2.1 null mutant males, we find that they are more sensitive to ARE than age-matched him-5 males, which is consistent with their accelerated decline in mating behavior. Considering that all three factors that can affect mating potency can down-regulate gene expression: 1. SIR-2.1 is a histone deacetylase histone that can promote the chromosome condensation, thus attenuate gene transcription; 2. Starvation can reduce translation; And 3. Cycloheximide can inhibit elongation process during translation. We hypothesize that during aging more Ach receptors are expressed so that males become more sensitive to Ach agonist. And SIR-2.1, starvation, or cycloheximide can down-regulate the Ach receptors expression so that aged wild type males can mate more efficiently.

Manser J et al. (2000) West Coast Worm Meeting "Characterization of CAN cell and excretory canal defects in mig-10(ct41) mutants"

Manser J, Washington N

[West Coast Worm Meeting, 2000]

We have continued our characterization of mig-10 mutant defects using a ceh-23-gfp reporter strain (kyIs5 IV, kindly provided by Jennifer Zallen) which allows scoring of CAN cell bodies and axons (see Manser et al. WCWM 1998 abstracts, p.167). In the current studies, we have focused on the amber (putative null) mig-10(ct41) allele. Nearly 90% of CAN cell bodies (61/68) are significantly displaced anteriorward in mig-10(ct41);kyIs5 animals, compared to less than 8% (5/64) for the kyIs5 strain. Manser and Wood (1990) previously reported a penetrance of 60% for CAN cell body misplacement in mig-10(ct41). The higher penetrance observed using the gfp reporter probably reflects a synergy between mig-10 and ceh-23-gfp similar to that reported by Forrester et al. (1997) for other CAN migration mutants. A significant percentage of posteriorly directed CAN axons in mig-10(ct41);kyIs5 animals (23/67) terminate in positions significantly anterior to their normal target region near the anus. In contrast, all anteriorly directed CAN axons appear to extend to their normal target region in the head. This apparent directional bias is somewhat surprising given that mig-10(ct41) disrupts both anteriorward and posteriorward cell body migrations (Manser and Wood, 1990). One possibility is that the CAN axon defects are an indirect result of the shortening of the posterior excretory canals observed in mig-10(ct41) (Manser and Wood, 1990). Specifically, because CAN axons are closely associated anatomically with the excretory canals (CAN=canal associated neuron), perhaps the canals serve as guidance cues for axon outgrowth. To investigate this possibility, we have scored both CAN axon and posterior excretory canal defects in the same individuals. In more than 90% of mig-10(ct41);kyIs5 animals scored (28/31), the posteriorly directed CAN axon extended significantly beyond the point of termination of the posterior excretory canal. This set includes animals in which the posterior CAN axon extends to the anal region while the posterior excretory canal terminates significantly anterior to the vulval region. Due to the generally severe truncation of the posterior canals caused by mig-10(ct41), we have also observed animals in which the CAN cell body is positioned posterior to the canal terminus. In such animals, anteriorly directed CAN axons were observed to extend over regions of the anterior-posterior axis from which canals were missing. These observations do not support the view that the excretory canals serve as guidance cues for CAN axon outgrowth.

Tinya Fleming et al. (2001) International C. elegans Meeting "A possible repulsive role for FGF signaling in CAN migration"

Gian Garriga, Fred Wolf, Tinya Fleming

[International C. elegans Meeting, 2001]

Fibroblast growth factor (FGF) can act as an important guidance cue in attracting migrating cells to their appropriate destination. In Drosophila , FGF signaling directs cell migration during tracheal branching, while in C. elegans , FGF guides the migration of the sex myoblasts to the gonad. We have found that mutations in the C. elegans FGF homolog egl-17 and the FGF receptor egl-15 also influence the migration of the canal-associated neurons (CANs) from the head to the mid-body region, but only in a sensitized background. egl-17 and egl-15 mutants enhance the CAN migration defects of a vab-8 mutant. This enhancement suggests a role for the FGF pathway in CAN migration. In order to determine if FGF provides instructional or permissive information for CAN migration, we expressed a constitutively active form of egl-15 in a soc-2 mutant background and a soc-2 ; vab-8 mutant background. The soc-2 mutation does not affect CAN cell migration but rescues the lethality associated with constitutive EGL-15 activity. Expression of a constitutively active egl-15 construct in a vab-8 background enhances vab-8 CAN migration defects to a similar extent as an egl-15 loss-of-function mutation. This result is consistent with EGL-15 activity providing direction to CAN migration, since constitutively active EGL-15 could mask directional information provided by regulated EGL-15, possibly its asymmetric activation. Thus, the FGF pathway can likely act as a guidance mechanism for migrating CANs. To test if EGL-17 can act as an attractant or repellent to direct CAN migration, we misexpressed egl-17 in the head and mid-posterior region of C. elegans . Expression of egl-17 in the head from the lim-4 promoter partially rescues the defects of vab-8; egl-17 double mutants. In contrast, expression of egl-17 in the mid-posterior region from the mab-5 promoter enhances CAN migration defects of a vab-8 mutant. These results suggest that egl-17 can act as a repellent to direct CAN migration. The observation that an egl-17 promoter fused to GFP expresses in only two head cells during the time of CAN migration is also consistent with the model of egl-17 repelling the CANs. Our results implicate FGF in a new role as a repellent for migrating cells. Several molecules have been shown to act as both attractants and repellents, and our results suggest that FGF should be added to this growing list.

Ute Platzer et al. (2001) International C. elegans Meeting "Analyzing genetic networks that regulate the development of the early C. elegans embryo"

Ute Platzer, Hans-Peter Meinzer, Markus Gumbel

[International C. elegans Meeting, 2001]

The fate of a cell is determined by its "genetic state", by all proteins (or gene products) present in the cell. The genetic state of a cell can be represented by naming each gene and its current state, which can be either 1 ("on") or 0 ("off"). Cells change their states during development, according to the interactions between the genes. For example, end-1 is expressed as a result of the activity of the transcription factor SKN-1. A set of interactions is called a genetic network. Genetic networks can be modeled in (at least) two ways: First, as a matrix with as many rows and columns as there are genes in the network. The number in column i and row j represents the influence that gene no. i has on gene no. j . Second, as a boolean network: Each gene is assigned a logical operator, e.g. "AND" or "OR". Gene states serve as input values for other genes. We have developed a software system that allows the user to enter and analyze genetic networks. The analysis can be done using one of the methods mentioned above. The user can edit the network via a graphical interface. New genes can be added by simple mouse clicking, or they can be dragged with the mouse from a predefined "database"-network. Interactions between genes can also be added using the mouse. Alternatively, the user can directly specify interactions between genes by typing in the boolean function. The calculation of stable states of the network gives information about the number and kind of cell types that can be generated via this network. The user can thus check whether the genes he has entered so far are sufficient to obtain the different cell types observed in biological experiments. Furthermore, the user can enter data about the cells in the organism (e.g. radius and position), and he can define the initial states of all genes in every cell. Additional parameters, for example which gene is responsible for specifying the division ratio of a cell, can be fed in easily. The development of the worm embryo under control of the given network can then be simulated using the CellO program [1]. The cells reside within a 3-dimensional model of the egg shell. They divide according to the rules specified by the network. As the cells have a spatial relationship in this simulation, inductive interactions between cells are also possible. The user can define some genes in the network to be "external", meaning that they influence only genes in neighboring cells. It is also possible to set the cell positions as observed in wild type embryos [2]. This may be useful to study inductive interactions that depend on correct cell positions and spatial relations. To summarize, this piece of software can be used to (1) simply gather and sort information about genetic networks: a clear graphical view of the network is presented, (2) analyze networks: calculate stable states and watch cell and organism development, (3) make predictions about cell differentiation: by leaving out genes, the development of mutants may be predicted. We plan to implement a connection to internet databases, e.g. AceDB and WormBase, to import genetic data directly from the internet. As more and more data are collected, it may be possible to simulate the whole worm, not only the early embryo. References: [1] Gumbel M, Schnabel R, Meinzer HP (2000). 14th ESM Proceedings. SCS Publications, pp. 605-611. [2] Schnabel R, Hutter H, Moerman D, Schnabel H (1997). Dev Biol. 184(2), pp. 234-265.

Roumen Voutev et al. (2005) International Worm Meeting "Worms that FLP-out"

E Jane Albert Hubbard, Roumen Voutev

[International Worm Meeting, 2005]

In several non-worm model organisms, the FLP/FRT system (or related site-specific recombination systems) can be used to direct temporal and/or spatial control of gene expression. One way to achieve this control is by FLP-out: FLPase-catalyzed intra-molecular excision of DNA between tandem FRT sites. This excision can be engineered to alter the control of gene expression in several different ways. For example, gene expression can be turned on by reestablishing promoter control of gene expression after the FLP-out of intervening sequences originally separating the promoter from the gene. Alternatively, FLP-out can be used to eliminate gene expression by removal of gene sequences or by activating transcription of RNAi-inducing sequences. The FLP-ase itself can be expressed under a heat-shock promoter for temporal control or a tissue-specific promoter for spatial/temporal control. As a first step to harness this powerful technique in worms, we have succeeded in generating conditional FLP-out of FRT-flanked sequences present on extrachromosomal arrays. We will report further advances at the meeting.

Wei X et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Application of a Novel Repressible Binary Expression System (Q System) in C.elegans"

Shen K, Luo L, Wei X

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Spatial and temporal control of gene expression is an important tool for modern genetics studies in multi-cellular model organisms. One powerful genetic strategy is to use binary expression systems such as GAL4/UAS system in Drosophila. However the GAL4/UAS does not work very well in C.elegans. Alternative method is to use DNA recombinases such as FLP or Cre, but the expression level decreases very quickly in extrachromosomal array except being integrated and the expression cannot be suppressed. Recently, A novel repressible binary expression system, Q system, based on the regulatory genes from the Neurospora crassa qa gene cluster has been well established in Drosophila and mammalian cells. The transcriptional activator QA-1F (QF) can effectively activate expression of target genes under the control of the QF binding sites (Q-UAS); this expression can be repressed by transcriptional repressor QA-1S (QS) efficiently; the transcriptional suppression can be relieved by feeding animals with quinic acid. The Q system can provide precise spatial and temporal control of gene expression in Drosophila. We have successfully adjusted the Q system into C.elegans and proven its high specificity and sensitivity in backwards locomotion circuit. unc-4 is expressed in A-type neurons (DA neurons as well as VA neurons) in the ventral nerve cord. Under the control of unc-4 promoter, QF is expressed in DA and VA neurons and can activate the expression of Q-UAS::GFP in these neurons. The expression of GFP in A-type neurons can be suppressed by using unc-4 promoter to express suppressor QS . And the suppression can be relieved in 6 hours if feeding these transgenic animals on NGM plates containing quinic acid. Furthermore, using DA neurons specific promoter unc-4c to express QS to suppress the expression of GFP in DA neurons, we can label VA neurons specifically. It means that by using different combinations of tissue specific promoters driving expression of QF and QS, we can express a target gene in a more specific subset of cells. Moreover with the development of Mos1-induced single copy insertion technique to introduce FRT sites into genome, we can develop MARCM (Mosaic analysis with a repressible cell marker) in C.elegans to analyze single mutant cell in wild type animals.

FW Wolf et al. (1999) International C. elegans Meeting "Genes acting with vab-8 for the posterior-directed migration of the can cell."

G Garriga, P Yen, FW Wolf

[International C. elegans Meeting, 1999]

vab-8 functions cell autonomously to promote the posterior directed migrations of cells and axon growth cones. How this novel intracellular protein acts to guide migrations posteriorly remains to be determined. Analysis of gene interactions with misexpressed vab-8 and loss-of-function vab-8 mutants indicates a role for a limited set of genes in vab-8 dependent migrations. Misexpression of vab-8 in the ALM cell results in reorientation of ALM axon outgrowth from anterior directed to posterior directed. Loss-of-function unc-73 /TRIO and gain-of-function mig-2 /Rac mutations suppressed ALM axon reorientation without grossly altering vab-8 expression, suggesting that signaling through GTPases is essential for vab-8 function. Additionally, unc-73 but not mig-2 loss-of-function mutations enhanced hypomorphic vab-8 mutant CAN migration defects. mig-2 may normally act redundantly with other GTPases for CAN migration. egl-15 and enu-1 mutations enhanced the penetrance of ALM axon reorientation. Although egl-15 mutations alone did not disrupt CAN cell migration, they enhanced CAN migration defects of vab-8 . Mutations in egl- 17, which encodes an FGF ligand that may bind the EGL-15 FGFR, also enhanced vab-8 CAN migration defects. CLR-1 is a receptor tyrosine phosphatase that antagonizes EGL-15. clr-1(e1745ts) suppressed the CAN cell migration defects of a vab-8 hypomorph. Thus, egl-17 /FGF, egl-15 /FGFR, and clr-1 /RTP are required for proper CAN cell migration. Perhaps FGF signaling positions the CAN cells and the sex myoblasts act through similar mechanisms (Chen and Stern, TIG 14:322). enu-1 mutants have defects in axon pathfinding (Wightman et. al., Development 124:2571). enu-1(ev419) strongly enhanced uncoordination in both hypomorphic and null vab-8 mutants. Surprisingly, enu-1(ev419) suppressed the CAN cell migration defect of hypomorphic but not null vab-8 mutants, indicating that enu-1 is a negative regulator of vab-8 . ENU-1 may either reduce VAB-8 levels or VAB-8 activity. We are attempting to clone enu-1 .

Elise Walck et al. (2008) Development & Evolution Meeting "A Glazier-Granier-Hogeweg (GGH) Model For Caenorhabditis elegans Embryonic Development"

Scott Thatcher, Clayton Davis, Timothy Walston, Elise Walck

[Development & Evolution Meeting, 2008]

An understanding of the forces acting on cells in the early embryo can provide important information for how cells interact to determine their shapes, movements and fates. These forces can be both physical and genetic and can be intracellular or extracellular. The early Caenorhabditis elegans embryo provides an excellent environment to explore the forces acting during embryogenesis and to develop techniques that can be applied later to more advanced biological events. We report the development of a 4D GGH (Glazier-Granier-Hogeweg) model to simulate the 4-cell stage of embryogenesis. GGH modeling uses a Metropolis Monte Carlo algorithm to describe the evolution of systems based on the idea that many systems transform to minimize their overall energy. By using a fixed-lattice environment governed by the Hamiltonian, an overall energy equation, GGH modeling can simplify the description of cell-based phenomena. The early stages of embryogenesis offer an environment where components of the Hamiltonian can be identified and tested. Our Hamiltonian includes cell-cell adhesion, cell-shell contact, centromere rotation and elongation, and constraints of surface area and volume. Our GGH model allows for the incorporation of several biological components that have not be explored in previous models of C. elegans embryogenesis. In addition, several aspects of our model broaden the possible applications of GGH modeling.