Wang, John et al. (2009) International Worm Meeting "Life is not fair: larger chromosomes are transmitted to males in C. elegans."

Herr, Winship, Chen, Pei-Jiun, Keller, Laurent, Wang, John, Wang, George

[International Worm Meeting, 2009]

The genome is typically viewed as a collection of cooperating genes whose alleles, in sexual eukaryotes, are segregated fairly during sexual reproduction. However, underneath the veneer of the harmonious and egalitarian genome, conflicts among genes exist and the lottery of gene transmission is sometimes biased. Aside from transposable elements and sperm competition, these topics have been underexplored in C. elegans. We decided to investigate if genetic inheritance in C. elegans is also biased. We tested whether chromosomal aberrations in a heterozygous state are transmitted unequally to offspring using strains carrying transgene insertions or deficiencies. For 4/4 integrated transgene strains tested, we found that heterozygote males (GFP/+) transmitted the insertion-bearing chromosome preferentially to male progeny. The magnitude of the transmission skew was large and differed among the lines (ranging from 2:1 to 6:1 ratio of GFP:non-GFP males). In parallel, we observed a skew of equivalent magnitude in transmission of the non-GFP bearing chromosome to hermaphrodites. We also tested two large deficiencies (tDf1 and nDf24). For both, heterozygote males transmitted the wild-type and deficiency alleles preferentially to the male and hermaphrodite progeny, respectively. These results show that the genetic inheritance in C. elegans is indeed biased, with the larger chromosomes preferentially transmitted to males. To examine the sensitivity of the mechanisms responsible for this transmission skew, we then tested if smaller chromosomal aberrations would exhibit the same phenotype. Thus far, we have tested single gene deletions in unc-47 (gk192) and unc-63 (gk234). For both we observed small (~5%) but significant biases in transmission. This suggests that the skewing mechanism may be able to detect very small chromosomal differences in the range of a few kb. As an entry to understanding transmission skew, we first asked if the skew is sex-specific. We examined transgene transmission in XO her-1 mutants and found that sperm, but not oocytes, display transmission skew. This suggests that transmission skew may happen during spermatogenesis. Transmission skew has important implications and poses interesting questions. This effect could play an important role in genome evolution and reveals an unexpected genetic linkage between autosomes and the X chromosome. More interestingly, the insertion-bearing chromosomes could serve as a model for birth of Y-chromosomes and for understanding how rapid evolution of sex determination systems occurs.

John Wang et al. (2001) International C. elegans Meeting "Global Analysis of Gene Expression Patterns in the Dauer Larvae of Caenorhabditis elegans"

Stuart K Kim, John Wang

[International C. elegans Meeting, 2001]

We are using the full-genome microarrays to profile gene expression differences associated with exit from dauer larvae. In principle, by examining gene expression differences on the whole-genome level, we will be able to illuminate the complete set of genes that are implicated for dauer-specific attributes. Identification of dauer-enriched genes would provide a framework for understanding dauer-specific characteristics, such as altered energy metabolism, certain aspects of aging, stress resistance, and the coordinated execution of a complex morphological change. To identify genes involved with dauer exit, we performed DNA microarray experiments with RNA isolated at different time points after addition of food to a dauer population. Each RNA sample was compared to a common reference RNA, and multiple RNA samples were prepared for each time point. Since some of the genes regulated upon dauer exit might be in response to the introduction of food rather than a dauer-specific developmental response, we also examined the feeding of starved L1's over a similar timecourse. We have identified and begun to analyze about 2700 and 1900 genes that are reproducibly altered during dauer exit and L1 feeding, respectively. Examination of the kinetic profiles of these genes reveals several temporally distinct expression groups, defining a developmental cascade of events that occur during dauer exit or L1 feeding. We find about 400 genes that have similar expression patterns in both timecourses and that constitute a common feeding response. Approximately 900 genes have different expression profiles in the two timecourse. Most are regulated only in the dauer timecourse. These likely represent the dauer-specific developmental response. We have separated the genes, identified above, into different groups based on their expression patterns by hierarchical clustering. Since genes that function together in a common process tend to be placed in the same cluster, we are currently pursuing in-depth analysis of the different clusters to ascertain what types of processes occur during dauer exit and L1 feeding. We found that stress genes (such as heat shock proteins and superoxide dismutases) are highly expressed in the dauer larvae. We also find that detoxification genes are enriched in the dauer larvae. Another interesting and unexpected discovery is that some Major Sperm Proteins (MSPs) are expressed in dauers. MSPs were previously reported to be only expressed in males or L4 and young adult hermaphrodites and to function only as components of sperm. Expression of MSPs in the dauer larvae may reveal an alternative function for a subset of the MSPs.

Le, Son Tho et al. (2013) International Worm Meeting "Testing for non-Mendelian assortment of chromosomes in Caenorhabditis."

Le, Son Tho, Kao, Chia-Yi, Wang, John

[International Worm Meeting, 2013]

Eukaryotic genome sizes range over 10,000-fold with a correlation between larger genomes and greater organismal size and complexity. In part, population genetic principles can explain this observation. Smaller organisms typically have larger population sizes which are more efficient at purging weakly deleterious mutations such as transposons and other insertions resulting in smaller genome sizes; larger organisms are the opposite. In contrast to the population genetic predictions, the reverse is observed for the species with known genome sizes within the Elegans group of the Caenorhabditis genus. Gonochoristic (male-female) species have larger genomes than hermaphroditic species despite the former predicted to have larger populations sizes than the latter. Why is this? Interestingly, Mendel's law of random chromosome assortment is violated in C. elegans males that are heterozygous for autosomal chromosomes of differing sizes whereby sons inherit the longer chromosome while the hermaphrodite daughters inherit the shorter chromosome, in a phenomenon which we call skew. Because a single hermaphrodite can start a new population, skew could explain how genomes of hermaphroditic species evolve to be smaller than the ancestral gonochoristic species. For this to be true, skew would be predicted to be a general property of the Caenorhabditis species. To this end, we are testing for the presence of skew in other Caenorhabditis species. We are also interested in understanding the mechanisms underlying skew and have initiated a forward genetic screen for genes that suppress skew.

Zhang, Liangyu et al. (2019) International Worm Meeting "The auxin-inducible degradation (AID) system: updates."

Dernburg, Abby, Cheng, Ze, Ward, Jordan, Wang, John, Zhang, Liangyu

[International Worm Meeting, 2019]

Spatial and temporal control of protein activity is essential for investigation of biological regulatory mechanisms. However, established approaches for protein depletion in C. elegans have primarily relied on disrupting genes or inhibiting their expression. To address this issue, we have adapted the auxin-inducible degradation (AID) system to C. elegans for conditional protein depletion. During the last few years, this versatile approach has become a powerful tool for spatiotemporal regulation and analysis of protein function in C. elegans. This system relies on expression of a plant-specific F-box protein, TIR1, which interacts with endogenous Skp1 and Cul1 proteins in worms to form a functional SCF ubiquitin ligase complex in the presence of auxin (indole acetic acid, or IAA). This complex recognizes and polyubiquitinates a short (44-amino acid) degron sequence (derived from plants) fused to a protein of interest internally or terminally. Upon exposure of the intact worms with TIR1 expressed in a specific tissue/cells to auxin, rapid (as fast as 20 minutes) and tissue-specific degradation can be robustly achieved. Many advantages of the AID system make it easily be adapted to one's specific project: various TIR1 strains are now available through CGC, high efficient CRISPR/Cas9 enables insertion of such a small degron into C. elegans genome without any pain, and the compound auxin is cheap and easy to apply to worms. We recently found the AID system can be used to degrade multiple degron tagged proteins simultaneously. It can also be used together with RNAi to increase the penetration to a single target or to deplete multiple targets at the same time. By using a low concentration of auxin, the AID system can be repurposed for partial depletion of a target to various extents, which facilitates study of dosage dependent functions. We are currently testing a few ways to enhance the performance of the AID system and to repurpose the AID system for other applications. We will update our findings at the meeting. Reference: L Zhang, JD Ward, Z Cheng, AF Dernburg, The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans, Development 142 (24), 4374-4384

Liu, Chenshu et al. (2021) International Worm Meeting "Nuclear lamina cooperates with inner nuclear membrane proteins to counteract LINC-mediated forces during oogenesis in C. elegans"

Wang, John, Dernburg, Abby F., Liu, Chenshu, Wu, Fan, Lung, Zoe

[International Worm Meeting, 2021]

The nuclear lamina is essential to protect genome integrity from mechanical stress. This requirement is more stringent in some tissues and developmental events. During oogenesis, meiotic nuclei are situated in a stressful environment filled with cytoskeletons. The nuclear lamina, consisting of the lamin proteins, is a conserved component of the nuclear envelope that can confer mechanical rigidity to the meiotic nuclei. C.elegans expresses a single lamin protein, LMN-1, which is similar to mammalian B-type lamin. Loss of LMN-1 results in near-complete sterility, with hypercondensed chromatin observed in many germline nuclei. The exact functions of LMN-1 in meiotic nuclei during oogenesis, however, remains unclear. Using the auxin-inducible degradation system, we found that acute depletion of LMN-1 in C.elegans germline recapitulated nuclear collapse seen in lmn-1 homozygotes during late stages of meiotic prophase. LMN-1 depletion also led to persistent DNA double strand breaks and elevated apoptosis, but germline apoptosis is neither sufficient nor required for nuclear collapse. We further observed prolonged and excessive clustering of the LINC complex proteins SUN-1 and ZYG-12 at the nuclear envelope (NE) upon LMN-1 acute depletion. Importantly, co-depletion of SUN-1 or ZYG-12, or inhibition of dynein-mediated forces, rescued the nuclear collapse triggered by acute LMN-1 depletion. By contrast, co-depletion of the inner nuclear membrane proteins EMR-1/LEM-2 or SAMP-1 rendered nuclei susceptible to collapse even earlier, at the time of meiotic entry. Live imaging demonstrated that shrinkage of the NE preceded chromosome hypercondensation during nuclear collapse, and that before NE shrinkage happened, LINC complex asymmetrically redistribute to one side of the NE in a dynein-dependent manner. Finally, the connection between the pairing center regions of the chromosomes and the NE, albeit being important for LINC complex function during homolog pairing, is dispensable for nuclear collapse caused by LMN-1 depletion. Together our results suggest that lamin cooperates with additional inner nuclear membrane proteins to protect meiotic nuclei from collapse by antagonizing forces exerted by dynein and transmitted through the LINC complex during oogenesis. Our work has also established an inducible system for modeling laminopathy.

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.

Kyung Won Kim et al. (2005) International Worm Meeting "RNP-8 interacts with the GLD-2 cytoplasmic poly(A) polymerase and regulates its polyadenylation activity"

Kyung Won Kim, Judith Kimble, Liaoteng Wang

[International Worm Meeting, 2005]

Polyadenylation is critical for mRNA stability and translational activation. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs promotes their translation. The C. elegans gld-2 gene is required for multiple steps in germline development, including the mitosis/meiosis decision. GLD-2 is a cytoplasmic poly(A) polymerase (PAP) that lacks an RNA recognition motif (1); similar PAPs have been identified in virtually all eukaryotic organisms (2). A yeast two-hybrid screen using GLD-2 as bait isolated GLD-3, RNP-8 and LARP-1 (1; L. Wang and J. Kimble, unpublished). GLD-2 has little PAP activity on its own, but it is stimulated in vitro by GLD-3 (1). We have now focused on RNP-8 to ask whether this GLD-2 interactor also stimulates GLD-2 activity and to determine whether RNP-8 has a major role in germline development. Preliminary data reveal that rnp-8(RNAi) and gld-2 mutants have similar defects during oogenesis. Both gld-2 and rnp-8 mRNAs are abundant in embryos, L4 larvae, and adults, and rnp-8 mRNA is enriched in the germ line. To test whether RNP-8 stimulates the GLD-2 PAP activity, we performed assays for PAP activity in vitro. Preliminary data suggest that RNP-8 can indeed stimulate GLD-2 PAP activity. Taken together, we suggest that RNP-8 stimulates GLD-2 activity to control germline development.1. Wang, L., Eckmann, C.R., Kadyk, L.C., Wickens, M., and Kimble J. (2002), A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316 2. Kwak, J.E., Wang, L., Ballantyne, S., Kimble J. and Wickens, M. (2004), Mammalian GLD-2 homologs are poly(A) polymerases. PNAS 101: 4407-4412

Zhang, Liangyu et al. (2021) International Worm Meeting "Meiotic cell cycle progression requires adaptation to a constitutive DNA damage signal"

Hollingsworth, Nancy, Stauffer, Weston, Wang, John, Zhang, Liangyu, Dernburg, Abby, Ziesel, Andrew, Yu, Zhouliang

[International Worm Meeting, 2021]

Defects in crossover formation lead to chromosome missegregation during meiosis. The mechanisms that ensure crossover formation and coordinate crossover designation with meiotic progression remain poorly understood. In C. elegans, defects in homolog pairing, synapsis, or recombination delay meiotic progression and prolong the activity of CHK-2, a meiosis-specific ortholog of the canonical DNA damage checkpoint kinase that plays essential roles during early prophase. We have now found that CHK-2 activity is both necessary and sufficient to inhibit crossover designation. CHK-2 is normally inactivated at mid-pachytene, but persists under conditions that prevent or delay the establishment of crossover precursors on one or more chromosomes. The pathway that mediates CHK-2 inactivation in wild-type or crossover-deficient conditions has not been established. We find that CHK-2 is inactivated and destabilized through inhibitory phosphorylation by Polo-like kinases (PLKs), a mechanism previously implicated in adaptation to DNA damage checkpoint signaling in proliferating cells. Recruitment to the synaptonemal complex and establishment of crossover precursors activate PLKs to phosphorylate CHK-2 and drive meiotic progression. Additionally, we find that stepwise reduction of CHK-2 activity enables the rapid, concerted designation of crossover sites on all chromosomes at mid-pachytene. These findings reveal that a key transition during the meiotic cell cycle occurs through adaptation to a "constitutive" DNA damage response pathway that is implemented at meiotic entry. Evidence from budding yeast suggests that the Polo-like kinase Cdc5 may similarly promote inactivation and degradation of Mek1, a CHK-2 ortholog, and data from mice are also consistent with such a mechanism. Our work further clarifies the signaling network that coordinates chromosome dynamics, double-strand break formation, and recombination during meiotic prophase.

Kendall Wu et al. (2004) West Coast Worm Meeting "Identification of Genetic Biomarkers and Regulators of Aging in C. elegans"

James Lund, Min Jiang, John Wang, Yueyi Liu, Stuart Kim, Kendall Wu

[West Coast Worm Meeting, 2004]

Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the age-1 and daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.

Sternberg, Paul et al. (2015) International Worm Meeting "WormBase 2015."

Berriman, Matt, Howe, Kevin, Kersey, Paul, Stein, Lincoln, Harris, Todd, Sternberg, Paul, Schedl, Tim

[International Worm Meeting, 2015]

WormBase has existed for 15 years and has evolved in many ways. The new website is fully operational and has made the process of adding new data types, displays, and tools easier. Behind the scenes we are piloting an overhaul of the underlying database infrastructure to allow us to handle the ever increasing data, have the website perform faster, and allow more frequent updates of information. This is a critical time for the project, as we face considerable pressure from two directions. The first is that our funders really want us to do more with less. We are responding to this by leading the way in making curation (the process of extracting information from papers and data sets into computable form) more efficient using a new version of Textpresso (to be released later this calendar year); by discussing with other model organism information resources ways to work together to be more efficient and inter-connected; and by seeking additional sources of funding. The second, delightful, pressure is an increase in data and results generated by the C. elegans and nematode communities. While we are handling this increase by changes in our software for curation, the database infrastructure, and the website, we do need your help. Many of you have helped us over the last few years to identify data in your papers or by sending us data directly. We now need you to help with a few types of information by submitting the data via specially designed, user-friendly forms that ensure good quality and the use of standard terminology. In particular, we have a large backlog of uncurated information associating alleles with phenotypes. We pledge to make this process as painless as possible, and to improve WormBase's description of phenotypes with your feedback, starting at this meeting at the WormBase booth, workshops and posters. With your help, continual improvement of our efficiency, and additional sources of funding, we are optimistic that we can do much more with even somewhat less effort.Consortium: Paul Davis, Michael Paulini, Gary Williams, Bruce Bolt, Thomas Down, Jane Lomax, Todd Harris, Sibyl Gao, Scott Cain, Xiaodong Wang, Karen Yook, Juancarlos Chan, Wen Chen, Chris Grove, Mary Ann Tuli, Kimberly Van Auken, D. Wang, Ranjana Kishore, Raymond Lee, John DeModena, James Done, Yuling Li, H.-M. Mueller, Cecilia Nakamura, Daniela Raciti, Gary Schindelman.