Arthur Wetzel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "PSC-VB: A System for the Assembly, Visualization, Analysis and Sharing of Large Serial Section Datasets"

Richard Fetter, Cornelia Bargmann, Arthur Wetzel, David Deerfield, David Hall, Demian Nave, Stuart Pomerantz, Greg Hood

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Serial section electron microscopy is an essential source of image data for building 3D models of C. elegans anatomy with sufficient resolution to map neuronal development and synaptic connections. Despite continued advances in computing speed and capacity it is still difficult to store, visualize, and distribute large stacks of TEM images on PC and workstation computers. Achieving the proper mutual alignment and warping of image slices to simplify the segmentation and tracing of neuronal conections continues to be both computationally intensive and manually tedious. Developers at the Pittsburgh Supercomputing Center (PSC) have been working to extend the PSC Volume Browser (PSC-VB) to address these problems. PSC-VB is a networked client server system to interactively link users PC and Mac computers, running the PSC-VB graphical user interface, to high performance servers that provide large scale computing resources where the actual data storage and manipulation is done. The PSC-VB system, developed for use with the NLM Visible Human datasets and large MRI and CT time series studies of mouse cardiac function, was built to handle datasets to ~100 GBytes and is being extended to TByte scales. The system includes image processing toolkits, data upload services and the ability to import and export data volumes for use with other tools such as ImageJ. This poster describes the PSC-VB system and shows its use with original data from the MRC archives curated by David Hall and with a complete 3-fold embryo volume built from ~24 GBytes of TEM imagery captured by Richard Fetter in the Bargmann lab. A significant aim in both cases is to improve the speed and accuracy of segmentation and model construction by performing complete automated nonlinear slice alignment with minimal human intervention. We are working to extend the system to include additional data modalities and means for remote data sharing and collaborative multi-user work sessions.

HE Smith et al. (1999) International C. elegans Meeting "Genome-wide analysis of C. elegans sperm development"

S Ward, JF Nance, HE Smith, EB Davis

[International C. elegans Meeting, 1999]

Our lab is interested in the developmental processes that direct sperm formation in Caenorhabditis elegans . Prior genetic screens have identified 60 genes required for proper sperm development, and several of these fer (fertilization-defective) or spe (spermatogenesis-defective) genes have been cloned by cosmid rescue. However, analyzing the function of these genes has been hampered by the length of time required to clone each gene, and the lack of homology to genes of known function. Because all of the previously cloned fer/spe genes exhibit sperm-specific expression, the identification of those genes expressed only in the developing sperm would speed cloning and identify a subset of genes with homologs. The recent completion of the genome sequence offers the opportunity for genetic analysis on a global scale. Therefore, we are attempting to identify all of the estimated 400 genes expressed solely during sperm development by 1) sequencing clones from a sperm-specific subtracted cDNA library and 2) differential screening of microarrays (in collaboration with the laboratory of Stuart Kim). Both strategies utilize temperature-sensitive mutant hermaphrodites that produce only oocytes ( fem-1 ) or sperm ( fem-3gf ) at the restrictive temperature. Because the somatic tissues are unaffected, any difference in gene expression between these two mutants is due to differences between sperm and oocyte production. We have constructed two high-quality cDNA libraries from mRNA isolated from fem-1 and fem-3gf mutants. Both libraries have been normalized to equalize the relative representation of abundant vs. rare transcripts. The fem-1 library has been subtracted from the fem-3gf library to generate a library enriched to ~20% sperm-specific genes. Reverse Northern blots with fem-1 and fem-3gf probes have been used to identify sperm-specific clones, which are being sequenced by the Genome Sequencing Center at Washington University. By comparing these results to Stuart Kim's differential microarray screening with fem-1 and fem-3gf probes, we can assay the sensitivity and specificity of these two methods. We will also present data from our current attempts to improve germline expression of transgenic arrays as well as increased efficacy of germline RNA inactivation.

Pilgrim DB et al. (1991) International C. elegans Meeting "MOLECULAR ANALYSIS OF THE DAUER CONSTITUTIVE daf-7 GENE."

Johnsen RC, Pilgrim DB, Riddle DL

[International C. elegans Meeting, 1991]

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tcl tagging. On a chromosomal walk toward fem-2, one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9, which contains an insert of <30kb, was used to probe both a northern blot and a CDNA library constructed by Stuart Kim. The northern blot revealed a single hybridizing RNA, and ten cDNA clones of various sizes up to 2.4kb were isolated from the library. Hybridization results indicate that all these clones share common sequences. Probing restriction digested genomic DNA isolated from the Tcl-insertion mutant DR1039, daf-7 (m434) , revealed differences consistent with a 1.6kb Tcl insertion in DR1039 DNA when compared with nonmutant DNA. For example, a 2.2kb EcoRl fragment found only in mutant DNA hybridized to both DE9 and to Tcl. We intend to determine the sequence of a daf-7 cDNA in order to obtain information regarding its possible function, and possible relationship to the daf-l and daf-4 receptor protein kinases.

Beitel GJ et al. (1991) International C. elegans Meeting "CLONING AND SEQUENCING OF THE SYNTHETIC MULTIVULVAL GENE lin-9"

Beitel GJ, Horvitz HR

[International C. elegans Meeting, 1991]

A synthetic Multivulva phenotype can result from mutations in two separate genes, while each mutation alone results in an apparently wild-type phenotype (Ferguson and Horvitz, Genetics 123:109-121,1989). Mutations that can cause this synthetic Multivulva (syn Muv) phenotype have been grouped into two classes, A and B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. Iin-9(nll2) III is a class B mutation that results in an apparently wild-type phenotype by itself. We previously reported that ZK637 and overlapping cosmids can rescue lin-9 (WBG 11(2 ): p 29,1990). We have now identified an 8 kb subclone with rescuing activity. Using this 8 kb fragment to probe Stuart Kim's cDNA library, six positive plaques representing at least three independent clones were isolated. This 8 kb probe also detects a message of approximately 2.5 kb on Northern blots. The message level appears similar in eggs and mixed-stage populations. We are currently sequencing these cDNAs and looking for evidence that these transcripts do in fact represent the lin-9 gene. This work will be greatly aided by the genomic sequence provided by Molly Craxton, John Sulston and Alan Coulson, as ZK637 was fortuitously chosen as one of the first cosmids to be sequenced in the worm genome sequencing project.

Monica C Sleumer et al. (2005) International Worm Meeting "The Search for Regulatory Elements in C. elegans"

Obi L Griffith, Steven JM Jones, Monica C Sleumer, Xin Zhang, A Gordon Robertson, Mikhail Bilenky, Erin D Pleasance

[International Worm Meeting, 2005]

The availability of large amounts of genomic and expression data provides an excellent opportunity to examine the relationship between gene expression, orthologous genes and known intergenic regulatory elements to search for novel functional regulatory elements in C. elegans on a genome-wide scale. For each gene in C. elegans, a list of putative coregulated genes consisting of coexpressed genes and orthologues from other nematode species is assembled. The coexpressed genes are obtained from available expression data, such as the cDNA microarray data used in Stuart et al (Science 302(5643): 249-55 (2003)), and locally available SAGE data (elegans.bcgsc.ca), while the orthologues are obtained from WormBase and from WABA comparisons of C. elegans genes to unannotated genomic sequence of other nematodes (until their orthologues with C. elegans are published). Potential regulatory elements will be generated by using existing motif discovery algorithms, in a pipeline previously developed for mammalian genomes (www.cisred.org), to search for over-represented motifs in the upstream regions of the genes in each set. Regulatory elements will be predicted for each C. elegans gene for which appropriate coexpressed and orthologous genes are available. Known regulatory elements in C. elegans will be used as a positive control, while the upstream regions of sets of genes that are not coregulated will be used as a negative control. Putative regulatory elements will be validated in the lab via collaborations. Results from validations will be used to improve the next round of predictions in an iterative process.

Rosoff ML et al. (1991) International C. elegans Meeting "MOLECULAR CHARACTERIZATION OF THE NEUROPEPTIDE GENE ENCODING FLRFamide."

Li C, Rosoff ML

[International C. elegans Meeting, 1991]

Single neurons can express multiple neurotransmitters and in addition, can switch their transmitter phenotype during development. We are interested in identifying what factors may be involved in determining transmitter specificity. Approximately thirty neurons in C. elegans stain with an antibody specific for the RFamide moiety of the neuropeptide FMRFamide [see WBG 9(3):110]. Screening of Stuart Kim's cDNA library with degenerate oligonucleotides against FMRFGK(R) yielded a 659 bp hybridizing clone whose putative translation product encodes eight potential neuropeptides. Seven of these neuropeptides share the same carboxy terminal -PNFLRFamide structure. Primer extension analysis has shown that this cDNA is not full length. Since screening of the MIT and MRC cosmid libraries yielded no hybridizing clones, we screened a lambda 1059 genomic library ( courtesy of Scott Emmons), and isolated thirteen hybridizing clones. By restriction mapping, the clones were separated into four overlapping classes. A hybridizing 3 kb EcoRI fragment was subcloned and sequenced from two separate isolates. The clones contain approximately 300bp upstream of the start of the cDNA, the entire cDNA, and a poly adenylation signal. Constructs are being made to analyze the promoter region using the beta-galactosidase gene as a reporter in microinjection studies. Concurrently, we have begun the purification of FMRFamide-like peptides from worm extracts. Radioimmunoassays show the presence of FMRFamidelike peptides, and further characterization of the extracts by reverse phase HPLC reveals at least five peaks of FMRFamide-like immunoreactivity.

Kara Thoemke et al. (2001) International C. elegans Meeting "Identification and characterization of putative male-specific sexual regulators and TRA-1 target genes"

Woelsung Yi, Jennifer Ross, Kara Thoemke, David Zarkower, Marc Sohrmann

[International C. elegans Meeting, 2001]

C. elegans is highly sexually dimorphic, with about 30% of the somatic cells in the adult hermaphrodite and 40% of the somatic cells in the adult male being sexually specialized. In C. elegans , all aspects of somatic sexual development are controlled by TRA-1A, a zinc finger transcription factor. Sexual dimorphism in C. elegans affects all tissue types and is achieved through diverse mechanisms including sex-specific cell divisions and migrations, sex-specific cell death, generation of sex-specific blast cells and sex-specific changes in gene expression. TRA-1A probably directly regulates multiple genes involved in restricted aspects of sexual differentiation, and an important goal is to identify these genes. We have identified a large number of male-enriched genes in C. elegans using microarray analysis with help from Stuart Kim and the Stanford Microarray Database curators. We have compared N2 XX hermaphrodites and tra-2(ts) XX pseudomales from the L2 through the L4 stages to generate a developmental profile of sex-specific gene expression. Many of these genes likely function as male-specific sexual regulators. We have also identified putative TRA-1A binding sites in the C. elegans genome using a hidden Markov model (HMM) computer algorithm and weight matrix searches. Combining these two approaches, we seek to identify and characterize male-specific TRA-1A target genes in an effort to understand the mechanisms by which C. elegans achieves sexual dimorphism. We are currently conducting functional analysis of these putative male sexual regulators using RNAi and GFP reporter analysis.

Estevez AOZ et al. (1995) International C. elegans Meeting "BONES AND WORMS AND daf-8"

Albert PS, Estevez AOZ, Estevez M, Riddle DL

[International C. elegans Meeting, 1995]

A bone morphogenetic protein (BMP) signal transduction pathway may be involved in regulating the ability of C. elegans to respond to environmental cues which alter its developmental fate. The multifunctional role of the BMP family of ligands has been well described in many organisms (dorsal-ventral patterning in flies, mesodermal cell fate in frogs, whisker and limb bud formation in mice, etc.), but little is known about the molecular components involved in regulating or transducing their signal. A signal transduction pathway regulating dauer larva formation includes two genes, daf-1 and daf-4, which encode receptor serine-threonine kinases similar to ones shown to bind members of the transforming growth factor (TGF)-B superfamily. When expressed in monkey COS cells, the daf-4 receptor binds BMP-2 and BMP-4 (Estevez, M., et al., Nature 365:644 649). Sequences encoding two potential natural ligands for these receptors have been cloned in our lab, daf-7, and a BMP-4 homolog. By indentifying the products of other daf genes, such as daf-8, the daf pathway provides an ideal model for understanding BMP regulated signalling mechanisms. For molecular analysis of the daf-8 gene, a novel Tc1 containing genomic fragment was isolated from one of three transposon induced mutants. Two overlapping cosmids hybridize to a 600 bp region flanking this novel Tc1 insertion site. Analysis of open reading frames in the first 1.6 kb of genomic sequence has not revealed any significant similarities in the databases, but the transposon insertion is within a region that encodes a ribosomal S6 kinase (rsk) homolog (Stuart Kim, pers. comm.).

Steven RM et al. (1991) International C. elegans Meeting "THE AXON GUIDANCE GENE UNC-73."

Culotti JG, Ruiz A, Steven RM, Mancillas JR

[International C. elegans Meeting, 1991]

A partial characterization of the Unc-73 phenotype has been carried out in a number of laboratories (Hedgecock et al., Development 100, 1987; Desai et al, Nature 336, 1988; unpublished results). Mutations in unc-73 generally affect the longitudinal positioning of certain axons and the longitudinal migrations of certain cells in C. elegans. These axons or cell migrations are either misplaced in a dorsal or ventral direction or their path is shortened and they do not reach their final destinations. Experiments are now underway to determine the null phenotype and the focus of action of unc-73. We previously reported the identification and cloning of two independent Tcl insertions in the unc-73 gene and the fact that cosmid rescue of the Unc-73 phenotype was obtained using DNA spanning the region of these two insertion sites (WBG 11:4). The sequencing of 6.8kb of genomic DNA in the region of these Tcl insertions reveals that both Tcls are inserted into putative exons and they are 4.6kb apart. Within this sequenced region ten putative exons comprising a total of 3.2kb have been found. No significant similarity of the protein encoded by these exons has been found to any of the proteins in the NBRF or Swiss-Prot databases. Fragments of exon-containing genomic DNA flanking the Tcl insertion sites were used to probe Stuart Kim's cDNA library. Two overlapping clones of 3.5kb and 3.0kb were isolated. Preliminary sequence data indicates that one of the cDNAs has a polyA tail and confirms a number of the exons predicted from the genomic sequence. We will use the RACE PCR procedure to obtain the 5' region of the unc-73 gene. Poster

CD Link et al. (1999) International C. elegans Meeting "Genetic analysis of beta amyloid toxicity in transgenic C. elegans"

CJ Johnson, A Fluet, CD Link, A Smith

[International C. elegans Meeting, 1999]

The beta amyloid peptide (Abeta) forms deposits in the brains of Alzheimer's disease patients and is believed to be central to the pathology of this disease. We have generated transgenic C. elegans animals expressing relatively high levels of human Abeta in hopes of modeling some aspects of this disease. Animals with muscle-specific expression of Abeta have amyloid deposits and show a distinctive, temperature-dependent progressive paralysis. Our recent studies suggest that this phenotype is a result of specific Abeta toxicity, and not due to non-specific effects of foreign protein expression or toxic properties of the transgenic array itself (e.g., titration of muscle-specific transcription factors). Specifically, we find that transgenic lines expressing equivalent or higher levels of the putative non-toxic met35cys or single chain dimer Abeta variants do not show the progressive paralysis. In addition, RNA inhibition of transgenic (wild-type) Abeta expression also completely blocks the paralysis phenotype. We have therefore undertaken initial screens to identify mutations that block Abeta toxicity, in order to identify genes involved in this process. From our initial small-scale screens, we have identified four mutations that significantly reduce the paralysis phenotype in transgenic animals. One extragenic mutant, dv54 , dramatically reduces transgene expression, and though intriguing, is presumably not relevant to Abeta toxicity. However, the other three mutants, dv53,55 , and 56 , express Abeta at parental levels and have amyloid deposits, yet still show suppression of the paralysis phenotype. These mutants are being further characterized. In collaboration with Stuart Kim, we are complementing this genetic analysis by examining gene expression profiles in amyloid and control animals using microarray hybridization.