Harvey, L-M. et al. (2019) International Worm Meeting "Identification and characterization of new actors of the RNAi pathway."

Harvey, L-M., Simard, M.J., Kakumani, P.K.

[International Worm Meeting, 2019]

The RNA interference pathway (RNAi) uses either ectopic (exo-RNAi) or endogenous (endo-RNAi) short non-coding RNA molecules known as short interfering RNA (siRNA) to regulate gene expression. To this day, knowledge on this regulation pathway was mainly obtained with studies made in plants and nematodes where it has been described as important for the genome protection and stability against pathogens. In contrast, our understanding of this regulatory pathway in higher eukaryotes is underwhelming. In mammals, a strong presence of these endogenous siRNA (endo-siRNA) has been detected in embryonic stem cells (ESCs), but in opposition to plants and nematodes, their function is yet to be fully understood. In an effort to address this question, we recently identified specific proteins associated to the short interfering RNA induced silencing complex (siRISC) in mouse ESCs using a 2'-O-methyl pulldown of an endo-siRNA followed by mass spectrometry. Beside Argonaute proteins, we identified 10 proteins associated with the mouse siRNA that are also encoded in the C. elegans genome, suggesting that they may play a conserved role in the endo-siRNA pathway. Therefore, we initiated the characterization of these new siRNA interactors in C. elegans, a biological system in which the tools and reagents needed to study components of the endo-siRNA pathway are well established in contrast to mammalian cells. Several molecular and genetic approaches are currently used to quickly understand the implication of these new factors in the RNAi pathway. During this meeting, we will present our recent progress on the characterization of some of the identified proteins. At term, this characterization will contribute to define the role of new conserved actors in the endo-siRNA pathway and lead to a better understanding of the pathway in mammals.

Harvey, A. M. et al. (2017) International Worm Meeting "Investigating the role of early double strand DNA break repair dynamics on repair pathway and partner choices during meiosis."

Libuda, D. E., Harvey, A. M.

[International Worm Meeting, 2017]

Meiosis is the specialized cell division utilized by sexually reproducing organisms to produce haploid gametes, such as sperm and eggs. During meiosis, programmed double strand DNA breaks (DSBs) are introduced in the genome of developing gametes and must be repaired with high fidelity to maintain genomic integrity as well as promote proper chromosome segregation. Repair of meiotic DSBs using homologous recombination is critical, as it establishes a crossover between each homolog pair. Interhomolog crossovers establish a physical link between chromosome pairs which ensures faithful chromosome segregation during the first meiotic division. Although each meiotic DSB undergoes both a template choice (homolog vs. sister chromatid) and a repair pathway choice (crossover vs. noncrossover) that are essential for achieving specific repair outcomes, the mechanisms underlying these meiotic DSB repair decisions are not well understood. The highly conserved recombinase RAD-51 is an early stage repair protein required for all meiotic homologous recombination events regardless of repair template or repair pathway choice. Cytological appearance of RAD-51 as a focus indicates the site of a DSB at an early stage of repair, while RAD-51 focus disappearance indicates progression of DSB further down a repair pathway. To observe early DSB repair dynamics, we created a functional GFP-tagged version of RAD-51 in Caenorhabditis elegans, where all of the stages of meiotic prophase can be visualized simultaneously in a single germ line. Live imaging of GFP::RAD-51 in whole worms revealed that RAD-51 forms two distinct classes of foci at DSB sites: 1) bright, static and long-lived; and, 2) flickering, transient, and short-lived. These data revealed that despite being at the same early stage of repair, DSBs can display distinct early DSB repair dynamics. Further, this finding suggests that DSB repair template and pathway decisions may be established at an early stage of repair. To determine whether these different classes of foci are associated with distinct repair template or pathway choices, our ongoing investigations are assessing the dynamics of RAD-51 foci in: 1) specific mutant backgrounds that lack specific repair outcomes; 2) defined phases of meiotic prophase I that are known to utilize specific repair templates; and, 3) precise regions of the genome where we can induce a single DSB and track its repair outcome. Overall, these studies will elucidate the relationship between early DSB repair dynamics and repair template and pathway choices.

A. Shorto et al. (2006) European Worm Meeting "Dauer Larvae Development and Fitness: Does it All Depend on your Environment?"

A. Shorto, S. C. Harvey, M. E. Viney.

[European Worm Meeting, 2006]

?. A. Shorto, S. C. Harvey and M. E. Viney. Natural isolates (including N2 and DR1350) of the free-living nematode Caenorhabditis elegans vary in their phenotypic plasticity of dauer larvae development. For example, some lines appear to be highly sensitive to dauer inducing conditions while others are less so. We have sought to investigate the causes and consequences of this plasticity.. To determine the fitness consequences of this plasticity of dauer development we have investigated how lifespan, total fecundity, reproductive schedule and population growth vary in N2 and DR1350 and in N2 x DR1350 recombinant inbred lines (RILs). We have found that the plasticity of dauer larvae development is positively correlated with the population growth rate (as measured by population size after 8 days of growth). Differences in population growth appear not to be dependent on lifetime fecundity or reproductive schedule (number of eggs laid per day) per se, but rather due to how the reproductive schedule changes in response to reduced food availability. Overall, these results suggest that there may be different reproduction and dauer formation strategies in response to environmental change.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

[Development & Evolution Meeting, 2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Nirav M Amin et al. (2005) International Worm Meeting "fozi-1, a gene required for proper development of the postembryonic mesoderm."

Jun Liu, Nirav M Amin, Kejin Hu, Dino Terzic

[International Worm Meeting, 2005]

Our lab is studying mesodermal patterning and cell fate specification by using the C. elegans M lineage as a model system. The M lineage arises from a single precursor cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic divisions to form 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). The SMs then migrate to the vulval region and further divide to give rise to uterine and vulval muscles. In a screen for mutants affecting the proper development of the M lineage (mesodermal lineage specification or mls mutants), we isolated four mutations of the same complementation group on chromosome III, cc607, cc608, cc609, and cc610. All four mutant alleles resulted in a terminal phenotype of missing M-derived CCs and extra SMs. Preliminary lineage analysis using M lineage specific GFP markers on the strongest allele cc609 revealed patterning defects after the third division of M. We are currently further characterizing these defects. Using a combination of recombination mapping, cosmid rescue, and RNAi, we identified the corresponding wild-type gene as K01B6.1. All four of our mutant alleles contained either point mutations or a deletion in the coding region of K01B6.1, further confirming this identity. K01B6.1 encodes a putative transcription factor with a glutamine rich region, a C2H2 zinc finger, and a large C-terminal FH2 (Formin Homology 2) domain. Thus we have named this gene fozi-1, standing for formin zinc finger protein-1. We are currently in the process of analyzing the expression pattern of fozi-1 by using both antibodies and GFP fusion constructs. In addition to its function in the M lineage, FOZI-1 also plays a role in other processes during C. elegans development, as a fozi-1 allele was identified in a screen by the Hobert lab for mutants that affect ASEL/ASER specification (personal communication).

J Liu et al. (1999) International C. elegans Meeting "Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages"

J Liu, AZ Fire

[International C. elegans Meeting, 1999]

Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery: mab-5 is expressed during the entire M lineage, but mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage. 1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)

Marisa Foehr et al. (2007) International Worm Meeting "Dorsoventral patterning of the postembryonic mesoderm requires both LIN-12/Notch and TGF-beta signaling."

Marisa Foehr, Jun Liu

[International Worm Meeting, 2007]

The C. elegans post-embryonic mesodermal lineage arises from a single precursor cell, the M mesoblast, which will diversify to generate distinct dorsal and ventral cell types. The dorsal daughter of M gives rise to a subset of body wall muscles and two non-muscle coelomocytes, whereas the ventral daughter of M gives rise to two sex myoblasts in addition to a subset of body wall muscles. Mutations in the C. elegans Schnurri homolog sma-9 cause ventralization of the M lineage. We have previously shown that SMA-9 antagonizes the Sma/Mab TGF-beta signaling pathway to promote dorsal M lineage fates (Foehr et al., 2006). Interestingly, loss-of-function mutations in the Notch receptor homolog lin-12 cause dorsalization of the M lineage (Greenwald et al., 1983), an exact opposite phenotype of sma-9 mutants. We have found that while LIN-12 protein is present in both the dorsal and ventral M lineage cells, the ligands for LIN-12, LAG-2 and APX-1, are asymmetrically localized in cells adjacent to ventral M-derived cells, and they function redundantly in promoting ventral M lineage fates. To investigate how LIN-12/Notch signaling interacts with SMA-9 and the Sma/Mab TGF-beta pathway in regulating M lineage patterning, we generated double and triple mutant combinations among lin-12, sma-9 and dbl-1 (the ligand for the Sma/Mab TGF-beta pathway) and examined their M lineage phenotypes. Our results suggest that the LIN-12/Notch pathway and the Sma/Mab TGF-beta pathway function independently in regulating dorsoventral patterning of the M lineage, with LIN-12/Notch required for ventral M lineage fates, and SMA-9 antagonism of TGF-beta signaling required for dorsal M lineage fates. Our work provides a model for how combined Notch and TGF-beta signaling regulates the developmental potential of two equipotent cells along the dorsoventral axis.

Anita U Rao et al. (2007) International Worm Meeting "Genetic Dissection of Heme Pathways in C. elegans."

Brian Le, Iqbal Hamza, Anita U Rao

[International Worm Meeting, 2007]

Heme serves as a cofactor for a number of proteins involved in key metabolic processes. In eukaryotes, heme synthesis occurs in the mitochondria by an evolutionarily conserved multi-step pathway. Hemes are hydrophobic and thus insoluble in the aqueous environment of the cell. Moreover, free heme is cytotoxic because of peroxidase activity. We therefore hypothesize that intracellular pathways exist for trafficking of heme from the site of synthesis in the mitochondria to various cellular destinations. However, identification of these heme transport pathways has been difficult because heme synthesis is regulated by multiple effectors and is tightly coordinated with apo-protein synthesis. We have previously shown that C. elegans and related helminths do not make heme albeit requiring exogenous heme for normal metabolic processes. Importantly, C. elegans show a biphasic response for heme; worms are growth-arrested at 1.5 <font face=symbol>m</font>M and at 800 <font face=symbol>m</font>M heme. These results suggest that although worms are obligate heme auxotrophs they are likely to have all the pathways for heme utilization beyond the point of heme synthesis. To identify pathways for heme transport in C. elegans, we exploited their biphasic response to heme by screening for mutants that could survive heme toxicity. We screened 300,000 haploid genomes and isolated 13 mutants at 800 <font face=symbol>m</font>M heme in liquid axenic medium. Based on the mutants growth profile in medium containing low and high heme, we categorized the mutants into three broad phenoclusters: class A, class B and class C. Class A mutants grew robustly under low and high (800 and 1000<font face=symbol>m</font>M) heme, Class B mutants grew exceptionally well under low heme, moderately well at 800 <font face=symbol>m</font>M heme, and not at all at 1000 <font face=symbol>m</font>M heme, and Class C mutants grow moderately well under high heme (800<font face=symbol>m</font>M), but exhibit normal growth under low heme. The mutants were further sub-clustered by using gallium protoporphyrin (GaPP), a toxic heme analog. Complementation analyses revealed that these 13 mutants fall into five complementation groups. Genetic mapping by recombination localized the mutants from each complementation group to chromosome III. We are now producing a high resolution map to define a genetic interval and pinpoint the exact nature of the molecular lesion in these mutants.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.