Theresa Moser et al. (2004) East Coast Worm Meeting "Analysis of an UNC-13 protein expressed from an internal promoter"

Brooke Swalm, Monique Spencer, Theresa Moser, Rebecca Kohn, Lydia Sanchez, Bethany Stitt, Kristin Servent

[East Coast Worm Meeting, 2004]

Several protein products are expressed from unc-13 . The predominant product localizes to synapses (Kohn, 2000) and is important for vesicle priming (Richmond, 2001). We are interested in understanding the roles of the other proteins expressed from the gene and are examining their expression patterns. Previous cosmid rescue experiments indicated that an internal promoter is used for expression of an UNC-13 protein product. We constructed a fusion of the putative promoter with the GFP gene and injected it into worms. We also developed antibodies to recognize the protein product expressed from the internal promoter. These antibodies bind UNC-13 proteins on Western transfers. GFP expression and immunohistochemical studies will determine the localization patterns of the less abundant form of UNC-13.

Theresa Moser et al. (2005) International Worm Meeting "Analysis of different forms of UNC-13 proteins in the Caenorhabditis elegans nervous system"

Bethany Stitt, Lydia Sanchez, Chanelle Houston, Cristina Polinsky, Erika Darrah, Graciela Gallo, Monique Spencer, Nancy Fernandez, Dana Francis, Theresa Moser

[International Worm Meeting, 2005]

We are studying the expression and function of unc-13 protein products in the Caenorhabditis elegans nervous system. The predominant protein expressed from unc-13 interacts with syntaxin and affects SNARE complex formation. This form of UNC-13 localizes to presynaptic plasma membranes and is important in priming synaptic vesicles (Richmond, 2001). Expression and function of other UNC-13 protein products are not completely characterized. We are analyzing regions of unc-13 for internal promoters regulating expression of shorter UNC-13 protein products. We fused regions of unc-13 including possible promoters to GFP and injected constructs into C. elegans. Progeny were analyzed for GFP expression. A construct incorporating 2.5kb of a 7.5kb intron did not express GFP. A construct including a larger region of this intron and a construct including a different intron are being tested for promoter activity. To identify additional proteins that interact with UNC-13, we used a yeast two-hybrid screen and analyzed a suppressor mutant. The yeast two-hybrid screen identified three candidates for proteins interacting with the predominant form of UNC-13. Five candidates for proteins interacting with a short form of UNC-13 were found. Eight candidates for proteins that may interact with all forms of UNC-13 were identified. A mutation that partially suppresses paralysis in an unc-13 missense mutant was analyzed. Genetic crosses showed that the suppressor is recessive and unlinked to unc-13. The mutation also suppresses an unc-13 allele that does not express the predominant unc-13 protein product. We are mapping the suppressor gene using snip-SNP mapping.

Wolfgang Maier et al. (2006) European Worm Meeting "Identification of Genes Involved in the Neural Regulation of Lifespan"

Martin Regenass, Joy Alcedo, Wolfgang Maier, Monique Thomas

[European Worm Meeting, 2006]

Wolfgang Maier, Martin Regenass, Monique Thomas and Joy Alcedo. Aging is influenced by several factors, which include the environment of the animal. For example, mutations that impair sensory function extend the lifespan of C. elegans, which suggests that lifespan, of at least this animal, is regulated by the perception of environmental cues (1). Specific sensory neurons appear to regulate lifespan, at least in part, through the insulin/IGF-1 pathway (1, 2). Since several putative insulin-like ligands are also expressed in these neurons (3), it is possible that an environmental signal(s) sensed by these neurons regulates the production of these ligands. To elucidate further the molecular mechanisms of the sensory influence on lifespan, we are carrying out an RNA-mediated interference screen for enhancers and suppressors of the lifespan phenotype of sensory mutant animals. As we identify genes involved in the sensory influence on lifespan, we will study their expression patterns using GFP-fusion reporter constructs to determine in which cells they function. We also plan to measure the lifespans of strains in which the particular gene was knocked out or overexpressed. Furthermore, we will conduct pertinent studies on how the gene products function, such as testing if the functions of these genes require each other or involve the daf-2 insulin/IGF-1 pathway or other signaling pathways in the worm known to influence its lifespan. References: (1) Apfeld, J., and C. Kenyon. 1999. Nature (Lond.). 402: 804 809; (2) Alcedo, J., and C. Kenyon. 2004. Neuron 41: 45 55; (3) Pierce, S. B., et al. 2001. Genes Dev. 15: 672 - 686.

Monique van der Voet et al. (2006) European Worm Meeting "The Function of LIN-5 in Chromosome Segregation and Asymmetric Division"

Matilde Galli, Christian Berends, Monique van der Voet, Justin Peters, Sander Van Den Heuvel, Mike Boxem, Marjolein Wildwater, Adri Thomas, Inge The

[European Worm Meeting, 2006]

Monique van der Voet1, Marjolein Wildwater1, Mike Boxem, Justin Peters1, Christian Berends1, Matilde Galli1, Inge The1, Adri Thomas1 and Sander van den Heuvel. The position of the spindle apparatus determines the plane of cell division. During asymmetric division, proper positioning of the spindle is needed in the generation of daughter cells that differ in size, cytoplasmic determinants and developmental fate. We have focused our attention on the C. elegans gene lin-5 to identify novel regulatory mechanisms and molecules that control spindle-mediated functions.. Our previous studies demonstrated that lin-5 is generally required for chromosome segregation and spindle positioning during meiotic and mitotic M phase. lin-5 encodes a coiled-coil protein that localizes to the cell cortex as well as spindle asters, and recruits the G protein regulators GPR-1/GPR-2 to the same locations. Results from our lab as well as others support that LIN-5 and GPR act in concert with the GOA-1/GPA-16 G?i/o subunits of heterotrimeric G proteins in an evolutionarily conserved pathway for spindle positioning. Many aspects of this process remain poorly understood. In particular, it is unclear how polarity determinants at the cortex affect LIN-5/GPR/G?i/o function to create asymmetry in the pulling forces that act on the spindle asters. In addition, the downstream targets of the LIN-5/GPR/G?i/o pathway remain unknown. To better understand the molecular mechanisms of these processes, we continue to use a combined genetic and biochemical approach. We have identified novel candidate partners of LIN-5 through immunoprecipitation from embryonic lysates followed by mass spectrometry. In addition, we identified multiple residues of LIN-5 and GPR that are phosphorylated in vivo. We are currently testing the in vivo relevance of these interactions and modifications and hope to present our results at the meeting.

Reinke, Valerie et al. (2011) International Worm Meeting "The C. elegans transcriptome."

Leng, J, Gerstein, Mark, Miller, III, David M, Reinke, Valerie, Robilotto, Rebecca, Slack, Frank J, Spencer, W Clay, Kato, Masaomi, Strasbourger, Pnina, Wang, Guilin, Green, Phil, High, Amber, MacCoss, Michael, Hillier, LaDeana, Thompson, Owen, Ratsch, Gunnar, Zeller, Georg, Ewing, Brent, Waterston, Robert H, Merrihew, Gennifer, Henz, S

[International Worm Meeting, 2011]

As part of the modENCODE consortium, we are characterizing the C. elegans transcriptome using tiling arrays, RNA-seq, RT-PCR and mass spectrometry. Our earlier studies on whole animals of various stages and conditions and on specific cells and tissues led to a much improved set of protein coding genes covering greater than 95% of all genes including more than 12,413 trans-spliced leaders, 20,515 different trans-spliced transcript start sites, 28,199 polyA sites, 111,786 confirmed splice junctions, >7,000 inferred non-coding (nc) RNAs, and over 50 new miRNAs (1-5). More recently, we have (1) analyzed biological replicates with RNA-seq for different stages and conditions, validating the observed expression levels; (2) closed gaps in RNA-seq coverage of weakly expressed genes with RT-PCR; (3) characterized the RNA content of more finely staged embryos with RNA-seq; (4) tested methods that deplete rRNA to allow direct analysis by RNA-seq of ncRNAs and smaller samples, such as specific embryonic cells and tissues; (5) analyzed polyA+ RNA from selected stages of C. briggsae, C. remanei, C. brenneri and C. japonica; (6) analyzed miRNAs under additional stresses and conditions; and (7) characterized the proteins present in 12 size fractions from 16 different stages and conditions. All of the data are available through the modENCODE Data Coordinating Center and increasingly through WormBase. Our goal is to provide the community with a comprehensive description of the transcripts of the C. elegans genome, providing information about their specific utilization where possible. References 1. Hillier et al. Genome Research PMID: 19181841 2. Gerstein et al Science PMID: 21177976 3. Lu et al. Genome Reseaarch PMID: 21177971 4. Allen et al. Genome Research PMID: 21177958 5. Spencer et al. Genome Research PMID: 21177967.

Kiyokazu Morita et al. (2004) West Coast Worm Meeting "Identification and analysis of several genes involved in Pn.p cell generation and vulval development"

Lei Ding, Kiyokazu Morita, Sean Keenan, Keiko Hirono, Min Han, Andy Spencer

[West Coast Worm Meeting, 2004]

As reported previously in worm meetings, to identify additional genes that act in the late stage of vulval differentiation and that act in P cell generation, we have performed two screens for suppressors of the Muv phenotype of a lf allele of lin-31 , which acts downstream of the MAPK in the vulval induction pathway. After screening more than 100,000 haploid genomes, we isolated 10 suppressor mutations that have now been determined to be alleles of six genes. All alleles suppress the Muv phenotype ranging from 100% to less than 10%. We have used traditional and SNP mapping methods to locate these genes in the genome. Three of the genes have been cloned and studied extensively, and two others have been located to specific cosmid regions. Phenotype characterization and genetic interaction analysis suggest that none of these genes act directly downstream of the RTK/Ras-mediated cell signalling pathway for vulval induction. Instead, some of these may act in specifying the competence of vulval precursor cells for induction or morphogenesis. Positional cloning of a gene defined by two of the suppressors indicated that these are alleles of the let-21 gene (2) which encodes an exchange factor for Rho GTPases that is homologous to mouse ECT2 and Drosophila Pebble. One of the alleles, ku427 , appears to cause more P specific defects: Pn.p cells are frequently missing due to defects in migration as well as cytokinesis of P cells. This result is consistent with our previously published result that Rho family GTPases are involved in P cell migration and cytokinesis (3). While LET-21 appears to function both in cytokinesis and migration of P cells, UNC-73, an exchange factor that may act on both RHO and RAC small GTPases, seems to act only on migration. (1) Miller et al. (1993) Gene Dev. 7, 933-947. (2) Ohmachi et al 2000 MWM, Canevascini et al 2003 IWM (3) Spencer et al. (2001). Proc. Natl. Acad. Sci. USA 98, 13132-13137.

Stephen Von Stetina et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Gene Expression Profiling the Nervous System"

Stuart Kim, Joseph Watson, David Miller III, Stephen Von Stetina, Peter Roy, Kellen Olszewski, W. Clay Spencer

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

The anatomy and development of the C. elegans nervous system are defined with unprecedented precision; the lineal origin, morphology and connectivity of every neuron are known. Similarly, the genome is fully sequenced and most genes identified. The problem now is to wed neural development and architecture to a gene expression map of the nervous system. As a first step toward achieving this goal, we are using the mRNA-tagging method to profile gene expression in C. elegans neurons. In this method, a neuron-specific promoter is used to drive expression of epitope-tagged poly-A-binding protein (FLAG-PAB-1). mRNAs cross-linked to FLAG-PAB-1 are captured by co-immunoprecipitation with anti-FLAG antibody and applied to the C. elegans Affymetrix array. We have tested the feasibility of this approach by profiling the entire nervous system (Von Stetina, Watson et al., 2006). These microarray experiments identified ~1600 neural-enriched transcripts at the L2 stage in comparison to the average C. elegans cell. 90% of the genes in this dataset with expression patterns listed in WormBase are expressed in neurons in contrast to the overall frequency of 56% of all genes with known expression. Enrichment for neuronal genes was also confirmed by construction of 23 GFP reporter lines for previously uncharacterized genes in this dataset; 22 (~95%) exhibit neuronal expression. We have now extended the mRNA tagging method to subclasses of C. elegans neurons. For example, an mRNA tagging profile of VA class motor neurons detects ~400 enriched genes. The most highly elevated transcript in this list, unc-4, is expressed in VA-class motor neurons and specifies synaptic inputs to these cells (see Von Stetina, et al., Fox et al., this meeting). Paralleling the results of the pan-neural profiling experiments, ~90% of genes with known expression patterns in the VA list are found in neurons and ~90% of GFP reporters (16/18) are expressed in VAs. Other classes of C. elegans neurons that we are profiling with the mRNA tagging method include VB motor neurons (lineal sisters of the VAs), PVD nociceptors (Treinen, et al., IWM 2005), and the command interneurons (Spencer, et al., this meeting). In addition to defining genes with specialized roles in the development and function of specific neuron types, this analysis is also expected to reveal a core set of genes with common roles in all neurons.

Musset-Bilal F et al. (1996) East Coast Worm Meeting "Structural and functional similarities between mammalian and C. elegans SPARC proteins"

Musset-Bilal F, Schwarzbauer JE, Fitzgerald MC

[East Coast Worm Meeting, 1996]

The basement membranes of nematodes and vertebrates are composed of similar protein components including laminin, perlecan, type IV collagen, and SPARC. The sequence conservation of these proteins suggests that basement membrane structure and function are conserved. To determine the extent of the structural similarities between C. elegans and vertebrate SPARC proteins, we have raised polyclonal antibodies against a bacterial fusion protein containing one of the calcium binding domains of nematode SPARC. Immunoblots of whole nematode lysates and of NP40 soluble proteins showed that nematode SPARC is a 27 kD protein (1). When electrophoresed under nonreducing conditions, SPARC shows increased mobility demonstrating that it has compact, disulfidebonded structure. Similarly, treatment with N-glycosidase F, which removes complex carbohydrates, resulted in increased mobility. Therefore, SPARC is glycosylated, probably on the conserved asparagine residue in the cysteine-rich domain. Together these biochemical analyses show that nematode SPARC is structurally very similar to its vertebrate counterparts. In mammalian cell culture, excess SPARC protein has been shown to reverse cell adhesion and cause cell rounding. Overexpression of wild type SPARC in nematodes causes body deformities and paralysis which could result from defects in muscle cell adhesion (2). To determine the effects of SPARC on muscle cell structure, a transgenic line was generated that carries extra copies of the SPARC gene (ost-l) along with the rol-6(d) gene N2;Ex[ost-1;rol-6(d)]). L4 larvae from this line develop an Unc phenotype with a protruding vulva, paralysis, and sterility. These animals were stained with fluorescently labeled phalloidin to visualize actin filament organization. While wild type animals form distinct sex muscle cell structures that attach at the vulva, the N2;Ex[ost-l;rol-6(d)] animals show no actin organization in their sex muscle cells. We conclude that the vulval protrusion observed in the presence of excess SPARC results from improper cytoskeletal organization of the sex muscle cells. As actin filament structure is dependent on cell adhesion, these results support the idea that in these transgenic animals SPARC is disrupting muscle cell adhesion. 1. Schwarzbauer, J.E., F. MussetBilal, and C.S. Ryan (1994) The extracellular matrix associated protein SPARC/osteonectin in Caenorhabditis elegans. Meth. Enzymol. 245, 257-270. 2. Schwarzbauer, J.E. and C.S. Spencer (1993) The Caenorhabditis elegans homolog of the extracellular calcium binding protein SPARC/osteonectin affects nematode body morphology and mobility. Mol. Biol. Cell 4, 941- 952.

LS Kaltenbach et al. (1999) International C. elegans Meeting "tlp-1 encodes a variant TATA-binding protein that is essential for embryogenesis"

M Domeier, SE Mango, LS Kaltenbach

[International C. elegans Meeting, 1999]

One component of the basal transcription machinery is a 700kD complex called TFIID. TFIID is composed of TATA-binding protein (TBP; CeTBP in C. elegans 1 ) and several TBP-associated factors. The prevailing view is that this complex is expressed in all cells, at all stages, and that it plays an important role in promoter recognition and recruitment of additional components of the basal machinery to target genes. We have identified a variant of TBP (called tlp-1 for T BP- l ike p rotein) in a yeast two-hybrid screen designed to identify proteins that interact with conserved C-terminal sequences of the pharynx identity factor PHA-4 2 . TBP variants have also been found in flies, humans and rodents 3 . Given the ubiquitous and fundamental role of TBP during transcription, why do animals need a second TBP-like protein? Our data suggest that tlp-1 has an essential function during embryogenesis. First, we examined tlp-1 expression. We defined the tlp-1 gene structure and constructed a tlp-1::GFP chimera. This gene is expressed widely from early embryogenesis until adulthood. By contrast, CeTBP::GFP expression does not initiate until the 3-fold stage. Second, we used RNA interference to determine the loss-of-function phenotypes of tlp-1 and CeTBP . The phenotype of most tlp-1(RNAi) embryos mimics the effects of blocking RNA polymerase II 4 : expression of an early zygotic GFP reporter construct is extinguished, E and MS blastomeres fail to gastrulate normally, and embryos arrest at about the 100-cell stage with little overt differentiation. CeTBP(RNAi) embryos, on the other hand, progress through embryogenesis and arrest as fully differentiated L1 larvae. In summary, we propose that i) PHA-4 activates transcription by targeting the basal transcription machinery via the PHA-4 C-terminus and ii) variant TBP-like proteins play an essential role during embryogenesis to mediate RNA polymerase II-dependent transcription. 1. Lichsteiner and Tjian, 1993. 2. Thanks to Bob Barstead and Phillip James for yeast two-hybrid reagents. 3. Crowley et al., 1993; Hansen et al., 1997; Ohbayashi et al., 1999. 4. Edgar et al, 1994; Powell-Coffman et al., 1996. Thanks to Spencer Wright for technical assistance.

Warner, Adam D. et al. (2013) International Worm Meeting "Determining the time and tissue specific expression of genes during embryogenesis."

Huynh, Chau, Waterston, Robert H., Warner, Adam D.

[International Worm Meeting, 2013]

Embryonic development is a tightly controlled and regulated process in many species, with a complex network of transcription factors regulating gene activity. In C. elegans, the process is so tightly controlled that the lineage of cell divisions is invariant and has been fully mapped (Sulston et al, 1983). The set of genetic messages at an embryo's earliest stage is completely maternal, transitioning over time to being generated completely from its own DNA. Determining which genes become active at specific time points provides insight into when key regulatory events occur, and determining the total network of gene expression in each cell type over time will help map out developmental processes unique to each tissue. Previous studies have utilized a combination of FACS, SAGE, and microarrays (Meissner et al, 2009; Spencer et al, 2011) to gain insight into what genes are expressed in late and mixed stage embryonic tissues. More recently, RNA-Seq has been used for expression studies, producing gene expression data with advantages over SAGE and microarrays such as a broader range of expression levels, differentiation between gene isoforms, and information on every transcript expressed (reviewed in Wang et al, 2009). We have previously shown the ability to isolate time synchronized whole embryos at specific time points throughout embryonic development (see abstract from Max Boeck), and we aim to do the same with isolated tissue types by utilizing a collection of GFP and mCherry labeled C. elegans strains (Murray et al, 2012; Sarov et al, 2012) that label individual cells and tissues. Using FACS to isolate specific cell subsets at discrete time points and RNA-Seq for transcript identification and quantification, we will be able to create a map of embryonic gene expression not only specific to individual cells and tissues, but specific to those cell and tissue types over time. This will build upon and complement previous data by adding temporal information and added sensitivity. Clustering of genes with similar expression patterns to genes with known roles in development, combined with emerging ChIP-Seq data, will help identify novel genes involved in specific developmental processes.