17 Papers found (0.01 seconds)

Begley RR et al. (1998) West Coast Worm Meeting "A POTENTIAL METHOD FOR ENHANCER TRAPPING"

[

West Coast Worm Meeting,

1998]

Enhancer trap screens would be a powerful way to study gene function, as they could simultaneously report gene expression patterns, loss-of-function mutant phenotypes and sequence of the encoded gene products. We are currently developing an approach to perform enhancer trapping, using cul-1 as a marker to select for random integration of extrachromosomal arrays. cul-1 is a cell cycle repressor, and cul- 1(null) cells undergo excessive rounds of cell division. cul-1(null) animals expressing cul-1(+) from an extrachromosomal array have low brood sizes, because loss of the extrachromosomal array is common and generates a mosaic clone that has excessive cell growth and reduces brood size. When the cul-1(+) extrachromosomal array integrates into the chromosome, the cul-1(null) transgenic animals have much higher brood sizes because the array is attached to a chromosome and cannot be lost spontaneously. A large number of random integrants can be obtained by irradiating a transgenic line containing a cul-1 extrachromosomal array, and then selecting for integrants that rapidly outgrow other worms on a plate. We have used this approach to integrate a lacZ enhancer trapping vector (designed by Andy Fire) into random chromosomal sites. So far, we have generated 155 independant integrants, and 65 (42%) of these integrants express lacZ. Generally, the integrants express lacZ in a pattern that is similar among individuals from the same integrated line, but different between integrated lines. We used a PCR based assay to show that 3 of the integrated lines may be recessive lethal, and we are mapping and characterizing their lethal phenotypes. We are in the process of cloning the endogenous locus for these 3 enhancer traps with lethal phenotypes. Future experiments will show whether the lacZ expression patterns and mutant phenotypes for these three integrants match those of the endogenous genes that contain the enhancer trap insertion.

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.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[

International Worm Meeting,

2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

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]

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