Ho, Vincy Wing Sze et al. (2013) International Worm Meeting "Defining regulatory pathway coupling cell division timing and cell fate differentiation in C. elegans by automated lineaging ."

He, Kan, Chan, Leanne, Liao, Jinyue, Chen, Long, Ho, Vincy Wing Sze, An, Xiaomeng, Zhao, Zhongying, Shao, Jiaofang, Yan, Hong, Chow, King, Huang, Xiaotai, Xie, Dongying, Wong, Ming-Kin

[International Worm Meeting, 2013]

Coordination of division pace among different cells is essential for proper formation of various tissues and organs during metazoan development. Failure in the coordination frequently leads to tumorous growth or abnormal cell death. How cell division paces are regulated to accommodate cell fate differentiation remains poorly understood. C. elegans embryogenesis provides a unique opportunity to address the issue due to its invariant development. To identify the regulatory proteins coupling cell division and fate determination, we performed a high-content screening of defects in embryonic cell lineage and cell fate determination for around 400 C. elegans genes using automated lineaging. We prioritized the gene list based on their conservation and potential defects during embryogenesis after inactivation. We inactivated each of the 400 genes through RNAi by injection and imaged three embryos per gene followed by lineaging analysis. The injected strain carries both a lineaging and a tissue marker, allowing us to simultaneously trace cell divisions and cell fate differentiation for every minute of embryogenesis. We curated embryonic cell lineage up to 350 cell stage with two replicates per gene. Preliminary analysis of cell division and/or expression phenotypes have not only confirmed function of genes with known function, but also helped assign functions to uncharacterized genes. Systematic analysis of defects in cell division and cell fate differentiation allows us to assemble regulatory pathway coupling the two biological processes. Our dataset provide an information-rich resource for further prediction of gene functions in C. elegans embryogenesis which likely have direct relevance to human biology. Methods for inference of gene network coupling cell division and cell fate determination based on our accumulated data will be presented.

Cao, Jianfeng et al. (2021) International Worm Meeting "Establishment of a morphological atlas of the Caenorhabditis elegans embryo using deep-learning-based 4D segmentation"

Yan, Hong, Zhao, Zhongying, Tang, Chao, Chan, Lu-Yan, Guan, Guoye, Cao, Jianfeng, Wong, Ming-Kin, Ho, Vincy Wing Sze

[International Worm Meeting, 2021]

The invariant development and transparent body of the nematode Caenorhabditis elegans enables complete delineation of cell lineages throughout development. Despite extensive studies of cell division, cell migration and cell fate differentiation, cell morphology during development has not yet been systematically characterized in any metazoan, including C. elegans. This knowledge gap substantially hampers many studies in both developmental and cell biology. Here we report an automatic pipeline, CShaper, which combines automated segmentation of fluorescently labeled membranes with automated cell lineage tracing. We apply this pipeline to quantify morphological parameters of densely packed cells in 17 developing C. elegans embryos. Consequently, we generate a time-lapse 3D atlas of cell morphology for the C. elegans embryo from the 4- to 350-cell stages, including cell shape, volume, surface area, migration, nucleus position and cell-cell contact with resolved cell identities. We anticipate that CShaper and the morphological atlas will stimulate and enhance further studies in the fields of developmental biology, cell biology and biomechanics (Cao†, Guan†, Ho†, et al. Nat. Commun., 2020, 11: 6254).

Zhao, Zhongying et al. (2021) International Worm Meeting "Genomic architecture of 5S rDNA cluster and its variations within and between species"

Ho, Vincy, Xie, Dongying, Ding, Qiutao, Chan, Lu-Yan, Li, Runsheng, Ren, Xiaoliang, Zhao, Zhongying, Ye, Pohao

[International Worm Meeting, 2021]

Ribosomal genes (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosome. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. Here, we generate ultra-long Nanopore and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform manual assembly of the cluster using the long reads that carry these units, which led to an assembly of rDNA cluster consisting of up to 167 5S rDNA units. The ordering and copy number of various rDNA units are indicative of separation time between strains. Surprisingly, we observed a drastically lower level of variation in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than C. elegans N2 strain, suggesting a unique mechanism in maintaining the rDNA cluster stability in the N2. Single-copy transgenes landed into the rDNA cluster shows the expected expression in the soma, supporting that rDNA genomic environment is transcriptionally compatible with RNA polymerase II. Delineating the structure and variation of rDNA cluster paves the way for its functional and evolutionary studies.

Okano H et al. (2000) Japanese Worm Meeting "The SWI/SNF complex is required for asymmetric cell division in C. elegans"

Kouike H, Sawa H, Okano H

[Japanese Worm Meeting, 2000]

In C. elegans, the polarities of many cells undergoing asymmetric divisions are regulated by LIN-44/Wnt and LIN-17/Frizzled. To identify more genes required for asymmetric division, we are screening for mutants affecting the asymmetric division of the T cell in the tail that is regulated by lin-17 and lin-44. We have so far identified 17 new mutants corresponding to 13 different genes. Lineage analyses have shown that the T cell division can be symmetric in mutants of at least 7 of the genes (psa-1 through psa-7). Using temperature-sensitiveness of the psa-1 and psa-4 mutant, we found that these genes are required during the mitosis of the T cell. We have cloned psa-1 and psa-4 to found that they encode homologs of SWI3 and SWI2 of yeast, respectively. These proteins are known to be components of the complex called SWI/SNF. The complex can remodel chromatin structure by destabilizing histone-DNA interaction. These results suggest that the chromatin structure is altered by the SWI/SNF complex in one of the daughter cells during the T cell division. In budding yeast, the SWI/SNF complex is required for the asymmetric expression of HO endonuclease in the mother but not in the daughter cell. HO catalyzes switching of the mating types in the mother cell. Therefore, our findings demonstrate that at least some aspects of the mechanisms of asymmetric cell division are conserved between yeast and a multicellular organism.

Williams, Kyle C. et al. (2011) International Worm Meeting "Essential Functions of IFA-2 Domains in C. elegans Fibrous Organelles."

Plenefisch, John, Williams, Kyle C.

[International Worm Meeting, 2011]

The C. elegans fibrous organelle (FO) complex consists of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments (IFs). FOs are found most prominently in the epidermis where they overlie body wall muscle and serve to transmit force from the muscle to the cuticle. IFA-2, one of four epidermally expressed IFs whose loss results in epidermal fragility and failure of muscle-cuticle force transmission, localizes to FOs. Unlike IFB-1 and IFA-3 that are required embryonically, IFA-2, although expressed in the embryo, is not essential for FO function until postembryonic stages. This distinctive phenotype allows us to specifically explore the functions of IFA-2 domains, and map their interactions with other FO associated proteins. All IFs contain three domains: a central rod domain and globular head and tail domains. The rod is essential for assembly of IFs into mature filaments while less is known about the functions of the head and tail domains. Roles of the head and tail domains of IFA-2 were examined by expressing GFP-tagged IFA-2 variants in transgenic animals and examining their phenotype and localization in wild-type and null backgrounds. Mutant variants included IFA-2 deleted for the head domain (DH), deleted for the tail domain (DT), deleted for both (RO), and variants that contained only the head domain (HO) or tail domain (TO). The expression and function of DH is virtually indistinguishable from full-length IFA-2. DT results in a lowered incorporation of IFA-2 into FOs and is unable to rescue a null allele of ifa-2, suggesting the tail domain is essential to IFA-2 function. Though both HO and TO are expressed well in the hypodermis at all stages, incorporation into FOs is variable between individual animals. High levels of early-stage HO incorporation into FOs correlate with a dominant-negative muscle detachment phenotype, while low-level incorporation results in healthy animals. This suggests the head domain interacts with FO components essential to tissue integrity. To determine the protein components of the FOs that interact with the head and tail domains, yeast two-hybrid and co-localization experiments are in progress. This work should help to elucidate the role of IFA-2 in FO function and reveal the underlying defects leading to tissue fragility.

Shao, Jiaofang et al. (2013) International Worm Meeting "Independent Regulation of Metabolism but Coordinated Control of Tissue Development by Epidermis Specific Proteins in Caenorhabditis elegans ."

Stamatoyannopoulos, John, Ho, Vincy, He, Kan, Shao, Jiaofang, An, Xiaomeng, Zhao, Zhongying, Ren, Xiaoliang, Xie, Dongying, Wong, Ming-Kin, Wang, Hao, Yan, Bin

[International Worm Meeting, 2013]

Cell fate specification demands a hierarchy of regulatory events. Initial specification is typically achieved by a master regulator which is relayed by tissue-specific regulatory proteins usually transcription factors for further enforcement of cell identities, but how the factors are coordinated between each other to "finish up" the specification remains poorly understood. C. elegans epidermis specification is initiated by a master regulator ELT-1 which subsequently activates its targets NHR-25 and ELT-3, two epidermis specific transcription factors, thus providing a superior paradigm for illustrating how the tissue specific regulatory proteins work together to enforce cell fate specification. Here we addressed the question through contrasting genome-wide in vivo binding targets between NHR-25 and ELT-3 that are important for epidermis development but not required for its initial specification in C. elegans. We first identified in vivo binding targets of NHR-25 by ChIP-seq and then compared them with those of ELT-3, the result of which demonstrated apparently differential regulation of metabolism but coordinated control of epidermal development between the two. Functional validation of the targets demonstrated that both activating and inhibitory roles of NHR-25 in regulating its targets. We further showed differential regulation of specification of AB and C lineage derived epidermis. Our results provide insights into how tissue specific regulatory proteins coordinate with one another to enforce cell fate specification initiated by its master regulator. Combined with functional analysis, we assembled a comprehensive gene network underlying C. elegans epidermis development and physiology which are likely to be broadly used across species.

Vidal-Gadea A G et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Biogenic amines mediate transition between crawling and swimming in C. elegans"

Brauner M, Gottschalk A, Erbguth K, Kressin L, Vidal-Gadea A G, Topper S, Young L, Pierce-Shimomura J T

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

A wide variety of animals must quickly adjust their pattern of locomotion to successfully navigate through different environmental niches. Selection and execution of the appropriate locomotory pattern is therefore paramount to survival. Although C. elegans is capable of performing many adaptive behaviors, it has been controversial whether forward crawling and swimming represent distinct gait-like forms of locomotion or the modulation of a single form of locomotion [1-3]. Biogenic amines have been shown to mediate the transition between gait-like forms of locomotion across taxa as diverse as sea slugs, leeches, lampreys and humans. We previously reported that C. elegans crawls and swims with distinct kinematics and different patterns of muscle activity [2]. We now combine quantitative behavioral analysis, optogenetic tools and neuronal ablation to show that C. elegans uses biogenic amines to switch between crawling and swimming in a gait-like manner. As in other invertebrates, we find that serotonin mediates the smooth transition from crawling to swimming in C. elegans. Serotonin is further required to inhibit motor behaviors (e.g. foraging and pharyngeal pumping) during swimming that normally only accompany crawling. Mirroring the role of dopamine in other invertebrates, C. elegans uses dopamine to successfully initiate and maintain crawling when emerging from liquid. Over 600 million years of separate evolution notwithstanding, the highly conserved role played by biogenic amines such as dopamine and serotonin across taxa attests to how vital their function is to adaptive strategies for locomotion. Korta J, Clark DA, Gabel CV, Mahadevan L, Samuel AD. J. Exp. Bio. 2007 210:2383-9.Pierce-Shimomura JT, Chen BL, Mun JJ, Ho R, Sarkis R, McIntire SL. PNAS. 2008 105:20982-7.Berri S, Boyle JH, Tassieri M, Hope IA, Cohen N. HSFP J. 2009 3:186-93.

Maike Thamsen et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Peroxiredoxin-2 Plays a Crucial Role in the Oxidative Stress Resistance of C. elegans"

Maike Thamsen, Caroline Kumsta, Ursula Jakob

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Aging is a complex physiological process and numerous aging theories have been proposed. One of the leading models is the free radical theory of aging, which suggests that the accumulation of reactive oxygen species (ROS) is causally linked to aging and cell death. Aging cells have been found to accumulate proteins with oxidative modifications, including side chain carbonylation and thiol modifications. It is this oxidative damage to specific cellular proteins that is often proposed to constitute one of the major mechanisms that link oxidative stress to age-associated loss of critical physiological functions. We have now started to monitor and visualize the effects of oxidative stress treatment of C. elegans using 2D gel electrophoresis. This analysis revealed a number of proteins that show substantially increased levels of oxidative protein modifications. One of these proteins is the highly abundant PRDX-2, a 2-Cys Peroxiredoxin, which is responsible for the detoxification of peroxides. We found that short-term exposure of synchronized L4 larvae to sublethal concentrations of HO leads to the rapid over-oxidation of PRDX-2''s catalytic cysteine. This reversible overoxidation leads to the inactivation of PRDX-2''s peroxidase activity and apparently turns PRDX-2 into a molecular chaperone. Prdx-2 knockout worms show substantial delays in oxidative stress recovery and reveal a significantly shortened lifespan at 15degC. To dissect the peroxidase function of PRDX-2 from its chaperone activity, we began to analyze the role of PRDX-2 in sestrin knockout worms. Sestrins are a class of proteins, which have recently been shown in human colon carcinoma (RKO) cells to selectively reduce the overoxidized form of 2-Cys peroxiredoxins. Preliminary evidence suggests that sestrin knockout worms accumulate PRDX-2 in the overoxidized form, indicating that sestrin might be the dedicated PRXD-2 sulfinic acid reductase in C. elegans. Interestingly, both prdx-2 and sestrin knockout worms reveal similar phenotypes, indicating that it is indeed the peroxidase activity of PRDX-2 that plays the major lifespan determining role in C. elegans.

Ryan Doonan et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Disparate Effects of the Five C. elegans Superoxide Dismutases on Dauer Formation, Stress Resistance and Aging"

Patricia Back, Filip Matthijssens, David Gems, Jacques Vanfleteren, Glenda Walker, Ryan Doonan, Joshua McElwee

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

The superoxide free radical (O) can act as a secondary messenger in cellular signaling or as a chemical weapon in immune defense, and might also influence aging, either accelerating it by causing molecular damage, or slowing it by hormetic stimulation of stress responses. Cytosolic, extracellular and mitochondrial O pools are detoxified by different superoxide dismutase (SOD) isoforms. sod-1 and sod-5 encode cytosolic Cu/Zn SOD, sod-4 extracellular Cu/Zn SOD and sod-2 and sod-3 mitochondrial Mn SOD. We have used reverse genetic approaches to investigate the role of each class of SOD and, by inference, each O pool, on C. elegans life history traits, particularly lifespan. We have also characterized the expression of each sod gene in detail, using real time PCR, Western blotting, enzyme activity assays and GFP reporter studies. We report that sod-1 and sod-2 function in reproductive growth, whereas sod-3 and sod-5 function is largely restricted to the diapausal dauer larva stage. We tested the effect of deleting each sod gene on lifespan. Only sod-1(0) reduced lifespan, slightly and to a similar degree in wild-type and daf-2 mutant backgrounds. Moreover, over-expression of sod-1 slightly increased adult lifespan. Additional over-expression of catalase did not further increase lifespan. Interestingly, sod-4(0) enhanced daf-2 mutant longevity, and also the daf-2 dauer constitutive phenotype. This could imply that extracellular SOD generates HO which enters the cell and activates receptor tyrosine kinase signaling, as does occur in mammals. sod-2; sod-3 animals, with no Mn SOD, were hypersensitive to induced mitochondrial oxidative stress (hyperoxia) yet normal lived under normoxic conditions. This strongly implies that intra-mitochondrial superoxide does not contribute to aging in C. elegans. Our results imply that each O pool exerts a different effect on lifespan: the extracellular pool promotes longevity, the cytosolic pool promotes aging, and the intra-mitochondrial pool has no effect at all.

Niemuth N et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Investigating Tissue-specific Levels of Reactive Oxygen Species in C. elegans Aging"

Knoefler D, Jakob U, Niemuth N, Diederich A

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The nematode Caenorhabtidis elegans has been repeatedly demonstrated to be an excellent model system for aging as it is an easily cultivatable, genetically malleable, and short-lived organism. Moreover, the development and life cycle of C. elegans is understood down to the level of individual cells, and conserved mechanisms that regulate aging in higher species exist in and have even been discovered using this tiny nematode.The free radical theory of aging centers on superoxide (O2.-), a constant byproduct of respiration in mitochondria. Reactions of this radical anion produce other reactive oxygen species (ROS), including hydroxyl radicals (HO.), which are known to cause widespread damage to proteins, lipids, and DNA. Increased O2.- production with age has been repeatedly observed in rat and other animal models, which is taken as support for the free radical theory of aging. Recently, this conception of aging has been turned on its head by the observation that the lifespan extending effects of glucose restriction in C. elegans seem to be mediated by induction of mitochondrial respiration and a subsequent increase in ROS and that treatment of C. elegans with superoxide generating juglone can extend lifespan. Resolution of this apparent conflict and exploration of the role of ROS in aging requires tools for measuring levels of ROS over the lifespan of C. elegans, including in tissues acutely affected by aging such as the neurons and tissues responsible for detoxification of oxidative species such as the intestine.To determine the levels of ROS in specific tissues over the course of nematode lifespan, H2O2-sensitive yellow fluorescent protein HyPer [1] and glutathione redox sensor Grx1-GFP2 [2] were expressed in C. elegans under the control of the SNB-1 and GES-1 promoters for expression in the neurons and intestine respectively. Using fluorescent microscopy, the ratio of emissions of the fluorophore at 420 and 500 nm will be compared in larval and adult worms, and this ratio will be used to determine the extent of ROS present in each tissue at each life stage.Using this system insight will be gained into the role that oxidative species play in the process of agin g. 1. Belousov et al. 2006. Nat. Methods. 3, 281-286. 2. Gutscher et al. 2008. Nat. Methods. 5, 553-559.