Tinney, Mathew et al. (2013) International Worm Meeting "C. briggsae genomic fosmid library."

Tinney, Mathew, Sarov, Mihail, Dahl, Andreas, Schloissnig, Siegfried, Loester, Elisabeth, Ernst, Susanne

[International Worm Meeting, 2013]

We have previously developed techniques for protein function exploration in vivo, using BAC and fosmid genomic DNA clones as transgenes and have applied them on a genome wide scale in C. elegans. Extending this work to other nematode species would provide a platform for addressing a wide range of evo-devo problems. As a first step towards this goal we generated a fosmid gDNA library for C. briggsae. We have developed a new deep sequencing based approach for clone mapping that significantly reduces the cost and effort required to map a large number of clones compared to the traditional BAC/fosmid end sequencing. The method can be easily extended to more nematode species. While for C. briggsae the genome sequence is available, we are currently working on an approach that combines de novo assembly and clone mapping and can be used for unsequenced genomes. The physical scaffolding provided by the clones and the reduced complexity compared to direct gDNA sequencing greatly simplifies the assembly process. We have started to use the resource to compare orthologous groups of gene expression regulatory proteins (transcription factors and RNA binding proteins) in C. elegans and C. briggsae.

Kaempfer, Philipp et al. (2015) International Worm Meeting "Improved C. briggsae genome assembly."

Dahl, Andreas, Schloissnig, Siegfried, Tinney, Mathew, Kaempfer, Philipp, Sarov, Mihail, Winkler, Sylke, Merret, Stephanie

[International Worm Meeting, 2015]

C. briggsae is and essential sister model to C. elegans for comparative genomics and evo-devo studies. Since the original genome was published in 2003 the genome assembly has been improved several times but it is still incomplete. We attempted to improve the quality of the C. briggsae genome assembly using the Pacific Bioscience sequencing platform and the more conventional approach of Illumina paired-end sequencing of total genomic DNA and an arrayed genomic fosmid library. We find that the PacBio data is sufficient for a very high quality assembly even in highly repetitive regions.The C. briggsae sequencing was our proof of principle experiment before we move on to other currently unsequenced or partially sequenced genomes that we need in order to study the evolutionary divergence of developmental programs. The meeting will be a good opportunity to compare our agenda with other similar initiatives to avoid duplication of effort and to optimise the use our resources.

Redemann, Stefanie et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Codon adaptation-based control of protein expression levels in C. elegans and its application to GPR-1 allows control of microtubule-based pulling force"

Redemann, Stefanie, Ernst, Susanne, Ayloo, Swathi, Bringmann, Henrik, Schloissnig, Siegfried, Pozniakowski, Andrej, Hyman, Anthony A

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The variation of the expression level of a protein could provide a powerful tool to study protein function. However, there is no method that allows the precise control of protein levels under a native promotor in eukaryotes. We developed a method, which enables us to fine tune the protein expression levels in C. elegans by using synthetic genes with adapted codons. By modifying the codon usage of a gene, the Codon adaptation index (CAI) can be changed and the level of protein expression can be controlled. We used this method to regulate the expression of the G-protein regulator GPR-1/2, which is involved in force generation during spindle positioning in the first asymmetric cell division in C. elegans. By gradually increasing the amount of GPR-1/2, we found that the amount of force acting on the spindle in C. elegans embryos is directly related to the amount of the G protein regulator GPR1/2 in the cell. In C. elegans GPR-1/2 is found in a complex, the force-generating complex,which is thought to consist of at least three proteins: GPR-1/2, LIN-5 and a G-alpha protein. Since increasing the amount of GPR1/2 is sufficient to increase the force, this suggests that the other proteins are there in excess and that GPR-1/2 is the limiting component. The modification of the CAI is a good example of how the ability to over-express proteins is essential for identifying components that are limiting as opposed to permissive for force generation. This method provides the first way to control the level of protein expression levels in C. elegans, and the first method for overexpression of proteins in the C. elegans germline. With this method the protein levels of a protein of interest can be varied, while maintaining all the wild type genetic regulation and the wild type protein sequence.

Sarov, Mihail S. et al. (2013) International Worm Meeting "A genome-scale resource for in vivo tag-based protein function exploration in C. elegans.."

Janosch, Stephan, Sarov, Mihail S., Murray, John I., Schloissnig, Siegfried, Ernst, Susanne, Pozniakovski, Andrei, Kapulkin, Wadim, Loester, Elisabeth, Hyman, Anthony A.

[International Worm Meeting, 2013]

Our understanding of biological systems at all levels of organization depends on our ability to probe the molecular function of their building blocks in vivo. To enable systematic protein function interrogation in a multicellular context, we have built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The resource covers 14,637 protein coding genes, or 73% of the proteome. The function of the tagged proteins can be interrogated with various standard, tag-based techniques - in vivo imaging, AP-MS, ChIP, PAR-CLIP. The resource not only simplifies the work required to characterize individual genes but opens up the way towards systematic function discovery on a proteome-wide scale. The resource is now available to the community and hundreds of researchers have already started to generate lines at a rate far exceeding what could be achieved by any single lab. We are providing web based tools that enable the exploration of the resource, simplify sample tracking throughout the transgenesis pipeline and the storage and collaborative annotation of imaging data. We will present the results of the project so far and our ongoing efforts to improve the available resources and to extend them to other nematode species as a tool to explore the evolution of developmental regulation.

Ambrose R Kidd III et al. (2004) Mid-west Worm Meeting "SYS-1, an atypical beta-catenin, acts with POP-1/TCF to control cell fates in C. elegans"

Judith Kimble, Jennifer A Miskowski, Ambrose R Kidd III

[Mid-west Worm Meeting, 2004]

POP-1/TCF controls numerous asymmetric cell divisions during C. elegans development. Our focus has been on an asymmetric division that generates the proximal-distal axis of the gonad. Both POP-1 and other classical Wnt/MAPK signaling components promote the distal fate in daughters of somatic gonadal progenitor cells (1). Mutations in the sys-1 (for sy mmetrical s isters) gene affect Z1/Z4 asymmetry in a similar way and interact genetically with pop-1 (1,2). We now have five sys-1 mutations. Two sys-1 deletions are embryonic lethal, which appears to be the null phenotype. Three other sys-1 mutations lead to sterility with defects in Z1/Z4 asymmetry; their molecular lesions include a missense mutation, an intronic change, and a silent mutation. The sys-1 promoter drives GFP in many cells throughout development. Given its genetic interaction with pop-1, its effect on Z1/Z4 asymmetry, its broad expression pattern and embryonic lethal null phenotype, we suggest that SYS-1 may control many asymmetric divisions during C. elegans development. A major clue to the mechanism by which SYS-1 controls fates comes from its molecular identification as a divergent b -catenin. SYS-1 contains at least three ARM repeats, a motif typical of b -catenin/Armadillo, and rescues a bar-1 null mutant when driven by a bar-1/ b -catenin promoter. Furthermore, by two-hybrid assay, SYS-1 binds POP-1 in a region that includes the b -catenin binding domain. Finally, a lack of SYS-1 does not affect POP-1 nuclear asymmetry (3). We suggest that SYS-1 works with POP-1 to control cell fates. Our favorite model is that SYS-1 acts with POP-1 as a transcriptional coactivator to control target genes. Predictions from this model are being tested and will be presented. --------------------- 1. Siegfried and Kimble (2002) Development 129, 443-453. 2. Miskowski et al. (2001) Developmental Biology 230, 61-73. 3. Siegfried et al. (2004) Genetics 166, 171-186.

Sarov, Mihail S. et al. (2011) International Worm Meeting "The TransgeneOme of C. elegans: a platform for in vivo analysis of protein function."

Zinke, Andrea, Tinney, Matthew, Hasse, Susanne, Pozniakovski, Andrei, Waterston, Robert, Rupprecht, Michaela, Janette, Judith, Angerman, Karolin, Murray, John, Schanze, Kristin, Kim, Stuart, Hyman, Anthony, Vinis, Elizabeth, Stewart, Francis, Ernst, Susanne, Reinke, Valerie, Schloissnig, Siegfried, Snyder, Michael, Teichgraber, Tina, Sarov, Mihail S.

[International Worm Meeting, 2011]

We have developed a genome wide tag based platform for rapid in vivo function analysis of any C. elegans protein of interest. We used high-throughput recombineering to systematically engineer a "synthetic genome" collection of large genomic fragments caring fluorescent and affinity tagged alleles for each protein coding gene in the C. elegans genome. Upon genome integration these large transgenes behave like additional alleles expressing a tagged version of the protein of interest, which can be localized, biochemically purified or quantified using generic, tag based assays. Because all cis acting elements are typically included in a construct of this size the endogenous regulation mediated by regulatory proteins and RNAs at the level of transcription, splicing, message turnover and translation are maintained and can reveal fine-grained expression pattern dynamics that are not possible with the traditional cDNA based transgenes or promoter reporter constructs. The resource was successfully utilized in the modENCODE project to describe transcription factor localization at both molecular level (by ChIP-seq) and cellular level (by in vivo fluorescent microscopy). The availability of the TransgeneOme resource opens the possibility to uncover the function of thousands of previously unstudied proteins, and would eventually allow us to build a systems wide atlas of protein localizations and interactions within the context of a multicellular model organism. Distribution of the effort throughout the community can significantly cut down the time required to achieve this goal. The use of a standard tag based techniques would make independently obtained data more comparable and easily reproducible. Finally, many of the transgenes would prove useful as reporters for loss of function studies, which will dramatically expand the accessible phenotypic space for both classic and reverse genetic approaches.

Green, Rebecca A. et al. (2011) International Worm Meeting "A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a ComplexTissue."

Schedl, Tim, Audhya, Anjon, Niessen, Sherry, Desai, Arshad, Swathi, Arur, Laband, Kimberley, Piano, Fabio, Mayers, Jonathan, Green, Rebecca A., Wang, Shaohe, Fridolfsson, Heidi, Gunsalus, Kristin, Schulman, Monty, Oegema, Karen, Kao, Huey-Ling, Starr, Daniel, Schloissnig, Siegfried, Hyman, Anthony

[International Worm Meeting, 2011]

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach- profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for 106 uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes- pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Bogaert T et al. (1991) International C. elegans Meeting "GENETIC AND MOLECULAR CHARACTERIZATION OF UNC-53."

Goh P-Y, Bogaert T

[International C. elegans Meeting, 1991]

In a search for genes required for sex musde pattern formation we identified an allele of unc-53 with specific defects in the hermaphrodite sex muscles: the 4 vm2 sex muscles have a misplaced or no dorsal attachment. The vml sex muscles have misguided dorsal attachments in that they are not sufflciently extended away from the vulva in the a/p axis. Ed Hedgecock and Siegfried Hekimi studied the neuronal phenotypes of some unc53 alleles. unc-53 mutants have also excretory canal extension defects (noticed by Ed Hedgecock) and seam cell differentiation defects (our observation). unc-53 mutations also cause extra left/right, dorsal/ventral and forward/backward turns of the distal tip of the migrating hermaphrodite gonad. We isolated new alleles in various pre-complementation screens which could yield complete loss of function alleles for the locus independent of the loss of function phenotype of the new alleles. One strong allele produces some dead embryos at 25 C, suggesting that the null phenotype of the gene may be embryonic lethal. The range of defects in unc-53 indicates that it is likely to be a widely distributed molecule. The phenotypic analysis of 8 alleles suggests that unc-53 may be a complex locus in which for example defects in extension of anterior vs. posterior vml sex muscles can be genetically separated. The unc-53 gene was mapped relative to RFLP's between wt strains and cloned by transformation rescue. A 3.8 kb cDNA spans most of the 16 kb region that rescues and detects a +/-150 bp internal deletion in one unc-53 allele and a different DNA rearrangement (possibly a partial duplication) in a second allele. The sequence of this cDNA will be presented and discussed.

Michael Chesney et al. (2006) Development & Evolution Meeting "Control of DTC Fate by CEH-22 Homeodomain Transcription Factor: What Are the Target Genes?"

Judith Kimble, Ngan Lam, Michael Chesney

[Development & Evolution Meeting, 2006]

The distal tip cell (DTC) is a key regulatory cell in the somatic gonad that controls germline proliferation and provides a stem cell niche. We are interested in the molecular specification of the DTC fate. In the early somatic gonad, each of two progenitor cells, called Z1 and Z4, divides asymmetrically to produce one distal daughter and one proximal daughter. Each distal daughter, in turn, produces one DTC; the proximal daughter does not. Previous studies showed that the Z1/Z4 asymmetric division is controlled by Wnt/MAPK signaling, and that the ceh-22 gene is a direct target of the POP-1/TCF and SYS-1/β-catenin Wnt signaling transcription factors. In the absence of Wnt/MAPK signaling or in the absence of CEH-22, the distal daughter fails to generate a DTC; by contrast, heat shock driven SYS-1/ β-catenin or CEH-22 generates extra DTCs (Kidd et al. 2005; Lam et al., 2006; Siegfried et al., 2004). CEH-22 is a member of the NK-2 family of homeodomain transcription factors and is best known for its role in pharyngeal development (Okkema and Fire, 1994; Okkema et al., 1997). CEH-22 responsive elements promoting pharyngeal expression have been identified in the promoters of myo-2 and ceh-22 (Okkema et al., 1994; Kuchenthal et al., 2001). We want to identify downstream targets of CEH-22 that specify the DTC fate. To this end, we are testing candidate genes that function in the DTC to ask if they are direct targets. The promoter sequences of candidate CEH-22 targets have been scanned for CEH-22 binding sites. We have also purified recombinant GST-CEH-22 protein for use in in vitro DNA binding assays with candidate promoter elements. This analysis will be coupled with the characterization of candidate promoters using GFP-reporter transgenes.

Trey Kidd et al. (2001) International C. elegans Meeting "The sys genes and early gonadogenesis"

Peggy Kroll-Conner, Judith Kimble, Kellee Siegfried, Trey Kidd

[International C. elegans Meeting, 2001]

The process of organogenesis involves cell proliferation, differentiation and morphogenesis. We have chosen the gonad as a model system to study the coordination of these events. The somatic structures of the gonad develop from two cells (Z1 and Z4) in the gonad primordium. In the wild type, Z1 and Z4 give rise to non-equivalent sister cells during L1. In hermaphrodites, Z1.a and Z4.p give rise to a distal tip cell (DTC), while Z1.p and Z4.a give rise to an AC/VU precursor. In males, Z1.a and Z4.p give rise to DTCs, while Z1.p and Z4.a give rise to the linker cell (LC). In addition to these regulatory cells, somatic blast cells are also generated, which become rearranged into a somatic primordium (SP) that prefigures the development of later somatic structures. We have isolated a collection of mutants that are defective in early gonadogenesis and are therefore sterile. Our recent focus has been on one group of these mutants that have similar early gonadal defects in hermaphrodites: loss of DTCs and the inability to form an SP. By mapping and complementation tests, we find that these mutations define six genes, designated sys-1 through sys-6 ( sys for sy mmetrical s isters ). Previous work demonstrated that the first divisions of Z1 and Z4 appear to generate equivalent cells in sys-1 hermaphrodites -- Z1.a and Z4.p appear to adopt the fates of their sisters(1). This phenotype is similar to that described for the frizzled homologue lin-17 (2) . Furthermore, the two sys-2 mutations are actually alleles of pop-1, consistent with the idea that the Wnt signaling pathway controls the polarity of this first division (see Siegfried abstract). Here we report our progress on characterizing sys-3 - sys-6. The sys-2 - sys-6 genes and lin-17 not only have similar phenotypes, but they also interact genetically with sys-1. Thus, for example, sys-1/ +; sys-4/ + double heterozygotes display Sys gonadal phenotypes even though both mutations are normally recessive. These genetic interactions suggest that the sys genes and lin-17 all function in a common pathway to control early gonadal development. However, with the exception of sys-2 , the other sys genes do not map to any known Wnt pathway components. Therefore, the sys genes may identify regulators of Wnt signaling that have previously not been identified. 1. Miskowski et al. (2001) Developmental Biology 230, 61-73. 2. Sternberg and Horvitz (1988) Developmental Biology 130, 67-73.