Politi, Kristin A. et al. (2009) International Worm Meeting "Three dimensional structure of the Ward body."

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.

Shoenfeld, Julien et al. (2011) International Worm Meeting "Determining the role of heterotrimeric G proteins in dauer entry."

Danzi, Bryan, Shoenfeld, Julien, Myers, Edith

[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.

Hall, David H. et al. (2009) International Worm Meeting "Electron tomography: a new tool for exploring C. elegans cellular anatomy."

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.

Huawei Weng et al. (2005) International Worm Meeting "A web-based searchable image database for C.elegans anatomy."

Huawei Weng, Toni Long, Tylon Stephney, Erik Hyrkas, David H Hall

[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.

Nicholas, Hannah R. et al. (2011) International Worm Meeting "Defining target genes of the transcriptional repressor protein CTBP-1."

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.

Centonze VE et al. (1997) International C. elegans Meeting "FREE CALCIUM LEVELS FOLLOWING FERTILIZATION IN MUTANTS OF C. ELEGANS."

Strome S, Centonze VE, White JG, Polinko E

[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.)

Zeynep Altun et al. (2006) Development & Evolution Meeting "Studying Worm Anatomy Online: WormAtlas And WormImage"

Laura Herndon, Huawei Weng, Zeynep Altun, David Hall, Robyn Lints

[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.

Zeynep Altun et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Studying Worm Anatomy Online: WormAtlas And WormImage"

Laura Herndon, Robyn Lints, Zeynep Altun, Huawei Weng, David Hall

[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

Gamss, Sandy et al. (2009) International Worm Meeting "Chemotherapeutic activation of CEP-1-dependent and CEP-1-independent Cell Death in Caenorhabditis elegans."

Gamss, Sandy, Bargonetti, Jill, Melendez, Alicia

[International Worm Meeting, 2009]

The p53 tumor suppressor protein is a regulator of apoptosis, growth arrest and autophagy. In response to DNA damage, functional p53 activates transcription of genes involved in cell cycle arrest, autophagy and apoptosis. Not surprisingly, p53 is mutated or deleted in over 50% of all mammalian tumors. We are investigating p53-independent cell death pathways in order to identify cell death mechanisms that can be induced in human cancer cells without functional p53. The C. elegans ortholog of p53, cep-1, functions during normal meiotic chromosome segregation and germ line cell death. We would like to identify chemotherapeutic drugs that induce cell death in animals that lack cep-1 activity. g-irradiation and UV-C have been shown to induce an increase in germ cell death in wild-type and not in cep-1 mutant worms. However, whether the same occurs for standard chemotherapeutics has not been examined. We are testing whether different DNA damaging drugs activate the CEP-1 pathway. We detected activation by quantitative PCR of the egl-1 target gene. Previously we have shown that 10-decarbomyl mitomycin C (DMC) can kill human cancer cells independently of p53 activity by a mechanism of action that includes the down-regulation of Chk1. Etoposide, mitomycin C (MC) and Nutlin-3 require an active p53 checkpoint pathway to elicit cell death. Interestingly, we have found that etoposide, mitomycin C, DMC and Nutlin-3 can elicit CEP-1 mediated activation of egl-1 transcription, similar to what has been shown with g-irradiation and UV-C. In addition, etoposide, MC and DMC exposure decreased the brood size of wild-type worms but only DMC exposure decreased the brood size of cep-1 mutants. We are also investigating whether autophagy is a possible mechanism of p53 independent cell death. Interestingly, exposure to the mitomycins and Nutlin-3 treatment in autophagy deficient animals activated egl-1, but exposure to etoposide did not. These results suggest that autophagy participates in a specific DNA damage signaling pathway. We will report on our experiments on germ cell death induced by the compounds in wild type and autophagy deficient animals using Nomarski optics and cell death markers. This work was supported by a NIH SCORE Grant (1SC1CA137843-01) and was facilitated by a NIH Research Centers in Minority Institutions award from the Division of Research Resources (RR-03037).

Kelley, Melissa et al. (2015) International Worm Meeting "A novel regulatory network in C. elegans embryos contributes to epidermal structural integrity during development."

Fay, David, Kelley, Melissa

[International Worm Meeting, 2015]

During development, biomechanical forces contour the body and provide shape to internal organs. We have discovered a regulatory network that is required to maintain epidermal architecture in response to biomechanical forces in C. elegans embryos. Embryos and larvae that are doubly mutant for mec-8; sym-3 or mec-8; sym-4 have defective anterior morphology, called the Pharynx Ingressed or Pin phenotype. Using a microarray approach, we have discovered that the expression of C. elegans fbn-1, a fibrillin-related protein, is regulated by a conserved splicing factor, MEC-8/RBPMS, which is required for the correct processing of fbn-1 mRNA. FBN-1 is secreted at the apical surface of epidermal cells as a component of the embryonic sheath. Acting in a parallel pathway to MEC-8 are two conserved proteins, SYM-3/FAM102B and SYM-4/WDR44, which localize to vesicles at or near the plasma membrane. Our data indicate that SYM-3 and SYM-4 function with the RAB-11 GTPase, a known regulator of endocytic recycling and exocytosis. We have also identified VHA-20/ATP6-ap2/(P)RR, a multifunctional protein involved in vesicle acidification and planar cell polarity, as enhancing the Pin defect in the sym-4 mutant background and being critical in RAB-11 recycling endosome localization. Thus, SYM-3-SYM-4-RAB-11 and VHA-20 may promote the correct targeting of FBN-1 or other extracellular matrix proteins to the embryonic sheath. In addition to providing resistance of the epidermis to a pharyngeal pulling force, FBN-1 stabilizes the epidermis during embryonic elongation, when circumferential actomyosin bundles arrayed along the body axis undergo contraction. C. elegans FBN-1 has two integrin-binding RGD domains. We found that mutations in sym-3 and sym-4 enhance the notched-head phenotype seen in integrin pathway mutants, suggesting that SYM-3/4 may be important in proper integrin localization. Correspondingly, inhibition of alpha and beta integrins enhances the Pin phenotype in sym-3, sym-4, and fbn-1, indicating that integrins help to maintain structural integrity against biomechanical forces. RNAi studies have revealed that spectrins and cadherins are also a part of this regulatory network to maintain epidermal structure and integrity. Taken together, we have identified a novel network that is required to maintain epidermal architecture in response to a variety of biomechanical forces during embryogenesis.