Vincent Galy et al. (2006) European Worm Meeting "MEL-28, a Novel Nuclear Envelope and Kinetochore Protein Essential for Zygotic Nuclear Envelope Assembly"

Carmen Lopez-Iglesias, Vincent Galy, Iain W. Mattaj, Cerstin Franz, Peter Askjaer

[European Worm Meeting, 2006]

Vincent Galy, Peter Askjaer, Cerstin Franz1, Carmen Lopez-Iglesias5, and Iain W. Mattaj1. The nuclear envelope (NE) of eukaryotic cells mediates nucleo-cytoplasmic transport and contributes to control of gene expression. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. Assembly of nuclear pore complexes (NPCs) and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here we report the identification and characterization of MEL-28, a large conserved protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. Light microscopy and transmission electron microscopy analysis demonstrate that MEL-28 localizes at NPCs during interphase, at kinetochores in early to mid mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early assembling, stable NE component required for all aspects of NE assembly. Based on its dynamic localization, we suggest that MEL-28 may link chromatin to the assembling NE.

Van Buskirk, Cheryl (2017) International Worm Meeting "A forward genetic screen for mutants defective in recovery sleep."

Van Buskirk, Cheryl

[International Worm Meeting, 2017]

In response to damaging conditions such as noxious heat or UV exposure, C. elegans enters a period of behavioral quiescence known as stress-induced sleep or recovery sleep (RS), during which sensory responses are dampened and feeding and movement cease1. Recovery sleep is mediated by activation of EGF receptors on the peptidergic ALA interneuron and subsequent release of a collection of neuropeptides1-3. At this time it is not known how cellular damage leads to the initiation of EGF signaling, and gaps remain in our understanding of signal transduction events within ALA as well as in the target tissues affected by ALA peptides. In order to uncover genes required for recovery sleep we have initiated an EMS screen for sleepless F2 animals, using the pore-forming toxin Cry5B as our damage-inducing agent. Progeny of sleepless candidates are tested for responses to other known sleep-inducing stressors, such as heat and UV light. Mutants that are found to be generally defective in recovery sleep are kept for SNP mapping, complementation as needed, and eventual whole-genome sequencing. We will present our detailed methods, some obstacles that have been overcome in our mapping of sleep mutants, and information on candidate identity if available. We also describe how this project has been implemented within the context of an undergraduate laboratory course called BIOL447 FIRE: Full Immersion Research Experience. 1. Hill AJ, Mansfield R, Lopez JMNG, Raizen DM, Van Buskirk C. 2014. Cellular Stress Induces a Protective Sleep-like State in C. elegans. Curr. Biol. 24, 2399-2405. 2. Nelson MD, Lee KH, Churgin MA, Hill AJ, Van Buskirk C, Fang-Yen C, Raizen DM. 2014. FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans. Curr. Biol. 24:2406-2410. 3. Nath RD, Chow ES, Wang H, Schwarz EM, Sternberg PW. 2016. C. elegans stress- induced sleep emerges from the collective action of multiple neuropeptides. Curr. Bio.l 26:2446-2455.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Braukmann, Fabian et al. (2015) International Worm Meeting "Extracellular vesicle (ecv)RNA in C. elegans."

Braukmann, Fabian, Miska, Eric

[International Worm Meeting, 2015]

In all domains of life, non-coding RNAs regulate gene expression1. This mechanism called RNA interference has been a powerful technique in basic research over the last two decades. Today, RNAi enters the hospital and RNAi therapeutics have the potential to revolutionize drug development 2. One of the key challenges for RNAi therapeutics remains efficient RNA delivery. RNAi effectors move from cell to cell and spread through tissues in invertebrates, plants and fungi 3. In the mammalian system, extracellular vesicles (ECVs) shuttle RNA between cells 4. Recently, Wang et al. showed that C. elegans secrets ECVs in to the environment, possibly delivering RNA from animal to animal 5. However, the presents of RNA in ECVs and the molecular mechanism of mobile RNA between individuals is not known in any system. Here, I propose a purification protocol for extracellular vesicle (ecv)RNA released by the model organism C. elegans. Using this protocol, we sequenced small RNAs and RNAs from males, hermaphrodites and males deficient in vesicle release. A subset of small non-coding RNAs and messenger RNAs were enriched in male ECVs. This work characterizes the ecvRNA profile of C. elegans and may help to elucidate the social role of RNAs and has the potential to inform new approaches to RNAi therapy.1. Cech, T. R. & Steitz, J. A. The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94 (2014).2. Ozcan, G., Ozpolat, B., Coleman, R. L., Sood, A. K. & Lopez-Berestein, G. Preclinical and clinical development of siRNA-based therapeutics. Adv. Drug Deliv. Rev. (2015). doi:10.1016/j.addr.2015.01.0073. Sarkies, P. & Miska, E. A. Molecular biology. Is there social RNA? Science 341, 467-468 (2013).4. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654-659 (2007).5. Wang, J. et al. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr. Biol. 24, 519-25 (2014).

Mansfield, R. et al. (2015) International Worm Meeting "Disruption of cellular proteostasis drives sleep behavior in C. elegans."

Mansfield, R., Van Buskirk, C.

[International Worm Meeting, 2015]

Sleep is widely regarded to be a restorative state, and the physiological perturbations associated with sleep deprivation are extensive. Despite intensive study, none of these perturbations has in turn been shown to drive sleep. Recently our lab has shown that in C. elegans, environmental stressors such as heat, cold and toxins induce a sleep-like state (Hill et al., 2014). As noxious environmental stressors are known to interrupt normal proteostasis, the existence of a subsequent sleep state suggests that sleep serves to assist in the restoration of normal proteostasis. This idea is supported by our observation that sleepless animals have impaired survival following severe stress (Hill et al., 2014). Further, we have found that chaperone response defective mutants display exaggerated sleep responses. These mutants include animals lacking the stress-induced transcription factors hsf-1/HSF-1, daf-16/FOXO and a component of the endoplasmic reticulum stress-response pathway hsp-4/BiP. We are testing site-of-action for this effect by examining strains with tissue-specific rescue of HSF-1. In addition, we are using pharmacological inhibitors of the proteasome to test whether direct inhibition of the proteostasis machinery can trigger sleep. Last, we are performing RNAi against both positive and negative regulators of the heat shock transcriptional response, and screening for sleep phenotypes. The results of these efforts will be presented.Hill AJ, Mansfield R, Lopez JMG, Raizen DM, Van Buskirk C (2014) Cellular stress induces a protective sleep-like state in C. elegans. Curr Bio 24:1-7.Zimmerman JE, Naidoo N, Raizen DM, Pack AI (2008) Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 31:371-6.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.

Kiontke, Karin C. et al. (2009) International Worm Meeting "New Caenorhabditis species: phylogeny and evolution."

Fitch, David H. A., Felix, Marie-Anne, Kiontke, Karin C.

[International Worm Meeting, 2009]

Recently, nine new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to nineteen, eleven of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10 and 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Although none of them is the sister species of C. elegans, C. sp. 5 and C. sp. 9 are close relatives of C. briggsae. C. sp. 9 can hybridize with C. briggsae in the laboratory. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. Three of these species, C. sp. 7, C. sp. 9 and C. sp. 11 have been chosen for genome sequencing. Four further new species branch off before C. japonica within a monophyletic clade which also comprises C. sp. 3 and C. drosophilae. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and seven from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. Other characters, like the shape of the stoma and the male tail, introns, susceptibility to RNAi and genome size are being evaluated in the context of the phylogeny. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last few years. There is no indication that we are even close to knowing all species in this genus.

Xiaodong Wang et al. (2003) International Worm Meeting "Evolution of a novel gene expression pattern in C. elegans"

Xiaodong Wang, Helen M Chamberlin

[International Worm Meeting, 2003]

Previous studies have shown that C. elegans ovo-related gene lin-48 expresses in a small number of cells including the excretory duct cell. In the related species C. briggsae, the expression is conserved in all cells except the excretory duct. This lin-48 expression difference affects excretory duct morphogenesis. In C. briggsae, as well as in C. elegans lin-48(sa496) mutants, the excretory duct is more anterior than in C. elegans wild type. This indicates that C. elegans lin-48 (Ce-lin-48) is involved in duct morphogenesis and positioning, but this gene function is absent in C. briggsae (1). We have made reporter transgenes composed of the lin-48 regulatory sequences from C. elegans or C. briggsae driving expression of green fluorescent protein (GFP). Tests of these clones in each species showed that only the Ce-lin-48 is expressed in excretory duct cell in C. elegans animal. These results indicate that there are differences in both cis-regulatory sequences and trans-acting proteins between the two species. By creating chimeric reporter transgenes including C. elegans and C. briggsae regulatory sequences, we have found that one difference between the two species is the presence of regulatory sequences in Ce-lin-48 that respond to the bZip protein CES-2 (1). The lin-48 gene expression differences between C. elegans and C. briggsae could result from loss of excretory duct expression in the C.briggsae lineage or acquired expression in the C. elegans lineage. To distinguish between these possibilities, we have analyzed three additional Caenorhabditis species (C. remanei, C. sp. CB5161 and C. sp. PS1010). We found these species have a duct morphology similar to C. briggsae indicating the C. elegans morphology is unique to this species. For comparison to C. elegans and C. briggsae, we have isolated the lin-48 gene from C. remanei and C. sp. CB5161. Alignment of the lin-48 regulatory sequences reveals that the sequences are more conserved among C. briggsae, C. remanei and C. sp. 5161. Several conserved domains are absent from C. elegans, whereas the previously identified CES-2 binding sites are absent from the other species. Currently, we are creating lin-48::gfp reporter transgenes for each species to observe the gene expression patterns. Further experiments with these transgenes will allow us to test whether the differences between C. elegans and the other species result from a loss of repressor elements or gain of activator elements in the C. elegans gene. (1)X. Wang and H. M. Chamberlin (2002) Genes & Development 16: 2345-2349.