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[
International C. elegans Meeting,
1999]
The O/E family of rHLH transcription factors has been implicated in neurogenesis, axonal pathfinding, muscle formation and B cell maturation. The murine O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis and are expressed in olfactory neurons throughout development where they may regulate components of the olfactory signaling cascade. We have identified a C. elegans O/E homologue (CeO/E) that is encoded by the
unc-3 locus. Unc-3 mutants display an uncoordinated phenotype attributed to a severe defasiculation and miswiring of the ventral cord (VNC). Using a GFP fusion to the
unc-3 promoter and specific antibodies, we observed that CeO/E is expressed transiently in the excitatory VNC motor neurons and throughout development in a pair of chemosensory neurons (ASI amphid neurons). Several observations suggest that the protein may have distinct roles in the two cell types. Although the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI,
unc-3 mutants enter the dauer pathway under inappropriate conditions. Although CeO/E possesses highly conserved domains associated with DNA binding in the mammalian homologue, identification of DNA binding sites for the C. elegans protein has not been achieved. We have shown that CeO/E protein can homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. Several
unc-3 target genes (
daf-7,
unc-17/cha-1,
unc-4 ) have been identified by other laboratories and each contains a mammalian binding site in the promoter region. Remarkably, we have been unable to demonstrate CeO/E binding at these sites. We are generating chimeras of O/E-1 and CeO/E to understand the DNA binding specificity of the CeO/E protein and utilizing a yeast-2-hybrid screen with both O/E-1 and CeO/E to identify interacting proteins in C. elegans . These studies should reveal the mechanism of CeO/E transcriptional activation and target gene regulation.
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Derr, KD, Rice, William J., Politi, Kristin A., Fisher, Kevin, Crocker, Chris, Hall, David H.
[
International Worm Meeting,
2009]
The plasma membrane of the nematode hypodermis features a unique set of stacked membrane invaginations that we presume to be associated with cuticle deposition, although there is still no experimental evidence to prove it. These structures were first noted by TEM in C. elegans by Sam Ward in the early 1970s, and briefly mentioned in WormBook I by John White (1). Here we present electron microscopic evidence for their organization in the wild type adult, combining thin sections, freeze fracture and electron tomography to view their organization in groupings along the body wall. The so-called "Ward body" resembles a Golgi body that has been rotated on end so that each succeeding membrane leaflet can fuse directly to the plasma membrane. Ward bodies are often clustered nearby to one another, but the orientation of each Ward body seems to be independent of its neighbors, and seemingly has no fixed angle with respect to the body axis. In some cases their leaflets lie virtually parallel to the plasma membrane. Similar membrane infoldings have also been seen in the excretory duct''s luminal membrane, but are not known to occur in seam cells or other epithelia in the nematode. We hypothesize that large extended proteins, such as collagens, may co-assemble within the protected environment of a Ward body leaflet prior to deposition in the cuticle. Supported by NIH RR 12596 to DHH. 1. W. Wood (Ed), The nematode C. elegans. Chapter 4, "The Anatomy", Fig 2a, 1988.
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[
International C. elegans Meeting,
2001]
Cell division results in the precise halving of genetic material. In yeast, such partitioning requires the protease separin 1 . This protease acts at the metaphase to anaphase transition to degrade sister chromatid cohesion, which in turn permits chromosomes to segregate to opposite poles of a dividing cell . In addition to facilitating chromosome segregation, separin promotes spindle elongation in S. cerevisiae through the activity of a calcium-binding domain 2 . Separin activity also depends heavily on the anaphase inhibitor securin, which localizes separin to both the nucleus and spindle mid-zone, and inhibits its protease activity until anaphase. Here, we report a requirement for the C. elegans homologue of yeast separin during oocyte meiosis. RNAi of this homologue, which contains both the protease and calcium binding domains of S. cerevisiae separin, results in multi-nucleated one-cell embryos with multiple spindles. In these embryos, the chromosomes are disorganized and the embryos fail to produce polar bodies. Taken together, these results suggest a role for C.elegans separin in chromosome segregation and cytokinesis in the early embryo. Currently, we are investigating if C. elegans separin also plays a role in exit from M-phase of the cell cycle. Additionally, we hope to identify protein partners of C.elegans separin by using the protein as bait in a yeast 2-hybrid screen. This screen may prove the most effective way of identifying a C. elegans securin homologue, as all of the known securins from other organisms demonstrate no amino acid conservation. Also, to date, no separin interacting proteins other than securin have been identified. Results of the screen will be discussed at the meeting. 1.Uhlman, F., Wernic, D., Poupart, M., Koonin, E., and K. Nasmyth. 2000. Cleavage of Cohesin by the CD Clan Protease Separin Triggers Anaphase in Yeast. Cell 103:375-386. 2.Jensen, S., Segal, M., Clarke, D., and S. Reed. 2001. A Novel Role of the Budding Yeast Separin Esp1 in Anaphase Spindle Elongation: Evidence that Proper Spindle Associatioin of Esp1 is Regulated by Pds1. J.Cell Biol. 152:27-40.
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[
International Worm Meeting,
2011]
C. elegans enter dauer under conditions of limited food, high temperature, and high concentrations of dauer pheromone. Signaling through heterotrimeric G proteins regulates sensitivity to food and dauer pheromone1,2. We are interested in whether the specific G proteins, EGL-30 (Gaq) and GOA-1 (Gao) are important for sensing the environmental signals that regulate dauer entry because it has been suggested that GOA-1 may regulate dauer formation3. EGL-30 signaling antagonizes GOA-1 signaling, suggesting that EGL-30 may also somehow regulate dauer formation.
By separately controlling pheromone, temperature, and food levels we have begun to study the role of EGL-30 and GOA-1 in dauer formation and recovery. We assayed dauer formation in response to different environmental cues in
egl-30 and
goa-1 mutants. We found that EGL-30 and GOA-1 were not necessary for normal dauer formation at 25 deg C. Because some proteins are only required for dauer entry at higher temperatures4, we wanted to assay dauer formation in
egl-30 and
goa-1 mutants at 27 deg C. However, animals with
egl-30 and
goa-1 mutations were not viable at 27 deg C, therefore we could not determine whether these proteins are necessary for dauer formation in response to high temperatures. We determined that GOA-1 and EGL-30 were required for normal sensitivity to dauer-related ascarosides. Further studies are underway to determine the role of these specific heterotrimeric G proteins in relaying food related dauer signals.
1. Dong MQ, Chase D, Patikoglou GA, Koelle MR. (2000). Genes Dev. 14(16): 2003-14.
2. Zwaal, RR, Mendel, JE, Sternberg, PW, and Plasterk, RHA. (1997). Genetics. 145: 715-727
3. Keane and Avery. (2003). Genetics. 164(1): 153-62.
4. Ailio, M and Thomas, JH. (2000). Genetics. 156: 1047-1067.
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Fisher, Kevin, Nguyen, Ken C.Q., Hall, David H., Crocker, Chris, Derr, KD, Rice, William J., Politi, Kristin A., Gunther, Leslie
[
International Worm Meeting,
2009]
C. elegans has been studied intensively using serial section reconstruction for several decades, so that every cell type is now known in considerable detail. Nevertheless, limitations of the serial section technique (notably the poor resolution of detail within the depth of each 50 nm "thin" section) have made it difficult to model the shapes of fine details in many organelles, such as the cristae of a mitochondrion, the canaliculi of the excretory canal, membrane ruffles in many cell types, or the constituents at a chemical synapse. High pressure freeze fixation and freeze substitution (1) are a required element in preserving smaller structures that generally escaped our notice in conventional TEM imaging. Modern electron microscopes using higher energy electrons now offer much better resolution by collecting multiple images in a tilting series through comparatively thick sections (150 nm) using the SerialEM program (2). The Protomo software package (3) is used to compute a 3D tomographic reconstruction that offers the same level of detail in any dimension (roughly 2 nm resolution). Annotation and modeling is done using IMOD (4) and/or Amira. This permits us to take a new look at many familiar objects in the anatomy of C. elegans, to identify missing parts of the whole anatomy, and potentially, to detect smaller anatomical defects in a variety of mutant backgrounds. Here we will introduce the tomographic procedure, and share finished 3D models of some typical intracellular organelles at high resolution. Supported by NIH RR 12596 to DHH. 1.Weimer, R.M. (2006) Methods Mol. Biol. 351: 203-221. 2.Mastronarde, D.N. (2005) J. Struct. Biol. 152: 36-51 3.Winkler, H. and Taylor, K.A. (2006) Ultramicroscopy 106: 240-254. 4.Kremer, J.R., Mastronarde, D.N. and McIntosh, J.R. (1996) J. Struct. Biol. 116: 71-76.
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[
International Worm Meeting,
2005]
The Center for C. elegans Anatomy holds an archive of over 200,000 electron micrographs generated by the C. elegans community over the last 30 years including those used in such landmark studies as the reconstruction of the hermaphrodite *(White et al., 1986; Hall et al., 1991). We have been developing a digital image database that will make this material readily available to the community at large. This web-based searchable database will allow C.elegans researchers to efficiently retrieve high resolution TEM images (and later, images from other types of microscopy) over internet. We are using MySQL as a relational database management system, and JAVA, Java Server Page (JSP) and Java Database Connectivity(JDBC) technology to develop the data access and web interface layers. We are scanning archival prints and negatives for wild type and mutants at various magnifications and covering most tissues. Image database features include: 1.Advanced search tools let the user choose an image by creating a query to the database according to sex, age, genotype, body region and tissue type. 2. A browse list that sorts the retrieved images by age, body region and worm name. 3. For each image there are three sizes and associated archival data such as source, age, genotype, fixation method, microscopy method, tissue type, color code etc. stored in the database. Thus the user can scan thumbnails, inspect a medium resolution view, or assess the archival data before downloading the highest resolution image. 4. Data sharing function: an online feedback form allows the user to add their own comments for an individual image; if the user chooses to make it public, comments will be posted as added archival data for that image; alternately they can save it as private with limited access to a selected audience. The project is part of Wormatlas:
http://www.wormatlas.org and is supported by NIH RR 12596. *We are grateful for donation of many images from MRC/LMB, U. Wisconsin, U. Missouri, Mt. Sinai Res. Institute, Caltech and others. We welcome further donations of EM images from wild type and mutant studies to our collection.
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Crossley, Merlin, Liu, Chu-Kong, Lun, Aaron, Tan, Melinda S-Y, Setiyaputra, Surya, Mackay, Joel, Kant, Sashi, Nicholas, Hannah R.
[
International Worm Meeting,
2011]
Mammalian members of the C-terminal binding protein family of transcriptional repressors are recruited to promoters through interactions with DNA-bound transcription factors that contain amino acid motifs of the form PXDLS. Although similarly able to interact with PXDLS-containing transcription factors, we have found that the sole C. elegans member of this protein family, CTBP-1, also contains intrinsic DNA binding capacity in the form of a THAP domain. We have identified additional THAP domain-containing CtBPs in the nematode, echinoderm and cephalochordate lineages. The distribution of these lineages within the animal kingdom suggests that the ancestral form of the animal CtBPs may have contained a THAP domain that was subsequently lost in the vertebrate lineage.
Since determining the structure of the THAP domain of CTBP-1 by nuclear magnetic resonance spectroscopy, we have used a variety of biophysical approaches to define the DNA contact surface of this domain and to assess the affinity of binding to an 11 bp consensus binding site derived from site selection experiments. Using the CisOrtho program1 we have identified promoters that contain putative CTBP-1-THAP binding sites, representing candidate CTBP-1 target genes. With reference to both our own and published2 microarray datasets comparing transcripts from wild type animals with those from
ctbp-1 mutants, and to expression pattern data, we have defined a sub-set of these as likely in vivo targets of CTBP-1-mediated repression.
Given the reported role of CTBP-1 in the regulation of lifespan and stress resistance2, and other investigations implicating CTBP-1 in aspects of neuronal development (D. Yucel, unpublished), our identification of CTBP-1 target genes will make an important contribution to understanding the function of this transcriptional regulator in a range of contexts.
1.Bigelow HR, Wenick AS, Wong A, Hobert O. 2004. BMC Bioinformatics 5: 27
2.Chen S, Whetstine JR, Ghosh S, Hanover JA, Gali RR, et al. 2009. Proc Natl Acad Sci U S A 106: 1496-501.
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[
International C. elegans Meeting,
1997]
A hallmark of fertilization is a transient increase in the level of free calcium in the newly fertilized oocyte. This calcium "spike" has been demonstrated during in vitro fertilization in a number of organisms. We are interested in whether C. elegans oocytes also exhibit a similar calcium spike at fertilization. Since the oocyte can not be removed from the ovary and remain viable we have circumvented this problem by imaging fertilization and early development in utero. Calcium indicator dyes [Ca-green dextran (10K) and Ca-crimson dextran (10K), Molecular Probes, Inc., Eugene, OR] were injected directly into oocytes. The worms with injected oocytes were subsequently anesthetized in 0.1% tricaine and 0.01% tetramisole to immobilize them for the purpose imaging. The all-solid-state multi-photon imaging system developed at the IMR was used to image the oocytes and embryos in timelapse. Both the fluorescence signal and the transmitted IR brightfield image were collected simultaneously. We detected fluctuations in the levels of cytoplasmic calcium during fertilization, second meiotic division and mitosis. We could not determine exactly when the sperm actually fertilizes the oocyte however, we observed both morphological as well as physiological changes that appear to be closely associated with the fertilization event. We detected pin point flashes of fluorescence in the cytoplasm which appear to occur just prior to the fertilization event. These flashes may indicate changes in the oocyte capacitance for fertilization. Later there was a rise in cytoplasmic calcium that appeared to be global. It is thought that this rise is a direct result of fertilization and corresponds to the calcium "wave" seen in other organisms. Immediately after this rise in calcium, the oocyte changes from a cylindrical to an oval shape and within a few seconds passes into the spermatheca. The elevations detected during meiosis and mitosis appear to be localized to specific regions or structures in the embryo such as the pronuclei and the mitotic centrosomes. Supported by NSF IBN-9117559 (S.S.) and NIH Biomedical Research Technology Grant RR 00570 (IMR, J.W.)
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[
Development & Evolution Meeting,
2006]
We have developed two online resources to allow users to learn the anatomy of C. elegans based upon electron microscopic images of the animal, and light micrographs of gfp-labelled animals to illustrate all tissues and cell types. WormAtlas (www.wormatlas.org) is a text-based website that offers several means to study the animal, including on a tissue-based Handbook, a wholistic approach (Slideable Worm), a comprehensive Glossary, and online access to key anatomical publications. One can also explore the cell lineages, the neuronal wiring diagram, and individual pages for each neuron that summarize their anatomy, synaptic interactions and receptors. Most portions of the website have direct links to relevant portions of WormBase (www.wormbase.org) so that one can quickly compare anatomical, molecular and genetic information about a particular cell or tissue. New in 2006 is an improved search function covering the whole website.
WormImage (www.wormimage.org) is an online database that can retrieve archival electron microscope data from several laboratories. Many key thin section series are available from the MRC, Missouri and AECOM collections, including male and hermaphrodites, embryos, adults dauer, and larval stages. The site has more than 5000 digital images, most with hand annotations to mark identified cell types. More images are added weekly. The user can search this database by animal name, age, sex, or by regional information (head, midbody, tail) or by tissue types to identify potential images of interest. The program first presents small thumbnail versions so that one can quickly sort among more interesting choices. By clicking on a thumbnail, larger thumbnail versions are quickly retrieved, along with information explaining the source of the image and how the specimen was prepared. Click again on this image, and a still larger image can be retrieved. Full size digital scans are also available on request.
We are very grateful to many laboratories that have contributed data to our collections for presentation on these two websites, and to peer reviewers who have helped to check them for accuracy. We welcome your comments on how to improve these resources. This work is funded by NIH RR 12596.
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
We have developed two online resources to allow users to learn the anatomy of C. elegans based upon electron microscopic images of the animal, and light micrographs of gfp-labelled animals to illustrate all tissues and cell types. WormAtlas (www.wormatlas.org) is a text-based website that offers several means to study the animal, including on a tissue-based Handbook, a wholistic approach (Slideable Worm), a comprehensive Glossary, and online access to key anatomical publications. One can also explore the cell lineages, the neuronal wiring diagram, and individual pages for each neuron that summarize their anatomy, synaptic interactions and receptors. Most portions of the website have direct links to relevant portions of WormBase (www.wormbase.org) so that one can quickly compare anatomical, molecular and genetic information about a particular cell or tissue. New in 2006 is an improved search function covering the whole website.
WormImage (www.wormimage.org) is an online database that can retrieve archival electron microscope data from several laboratories. Many key thin section series are available from the MRC, Missouri and AECOM collections, including male and hermaphrodites, embryos, adults dauer, and larval stages. The site has more than 5000 digital images, most with hand annotations to mark identified cell types. More images are added weekly. The user can search this database by animal name, age, sex, or by regional information (head, midbody, tail) or by tissue types to identify potential images of interest. The program first presents small thumbnail versions so that one can quickly sort among more interesting choices. By clicking on a thumbnail, larger thumbnail versions are quickly retrieved, along with information explaining the source of the image and how the specimen was prepared. Click again on this image, and a still larger image can be retrieved. Full size digital scans are also available on request.
We are very grateful to many laboratories that have contributed data to our collections for presentation on these two websites, and to peer reviewers who have helped to check them for accuracy. We welcome your comments on how to improve these resources. We especially thank Igor Antoshechkin (WormBase, Caltech) for his help in upgrading the search function on WormAtlas. This work is funded by NIH RR 12596