Worby, Carolyn et al. (2015) International Worm Meeting "Fam20C function in C. elegans."

Desai, Arshad, Dixon, Jack, Oegema, Karen, Worby, Carolyn, Gerson-Gurwitz, Adena

[International Worm Meeting, 2015]

A new family of atypical secreted kinases in human was recently identified, an archetypal member of which is Fam20C. Fam20C phosphorylates S-x-E motifs in caseins and several secreted proteins implicated in bone mineralization. In humans, mutations in Fam20C cause an osteosclerotic bone dysplasia known as Raine syndrome. In addition to biomineralization, phosphorylation by FAM20C is likely to be important for other functions, since many secreted hormones, proteases and other factors have been shown to be phosphorylated on S-x-E motifs.Intriguingly, the C. elegans genome encodes a FAM20C ortholog, which contains a signal-peptide for secretion and phosphorylates proteins within S-x-E motifs in vitro. Since mineralization does not occur in C. elegans, we are hoping to identify new roles for FAM20C in this system.In collaboration with the Oegema and Desai laboratories at UCSD, we are addressing the significance of FAM20C kinase activity in C. elegans. Preliminary work reveals that FAM20C is expressed and localized in the spermatheca and that its deletion leads to germline phenotypes and ~40% embryonic lethality. We are currently investigating potential substrates of FAM20C in C. elegans to determine the molecular basis for the observed phenotypes.

Petersen, Carola et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "New insights into the natural ecology of C. elegans"

Petersen, Carola, Schulenburg, Hinrich

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Only little is known about the ecology of the model nematode Caenorhabditis elegans - in stark contrast to the enormous database on the nematode's biology under laboratory conditions. T o enhance our understanding of the worm's natural requirements and preferences, we sampled Caenorhabditis strains continuously across 1.5 years from rotting apples and compost heaps in three North German locations. C. elegans and the related nematode species C. remanei were isolated throughout summer and autumn from the same site, but rarely the same substrate sample, possibly suggesting niche separation. The occurrence of C. elegans itself was related to environmental humidity and rain, rather than temperature as previously demonstrated for other natural populations. Genetic differentiation was analysed for populations from two locations using microsatellite markers. Significant genetic structuring was detected between locations and, within single locations, across long time periods (i.e., a 10-year time span), but not throughout the 1.5 years sampling period. Our analysis also revealed significant phenotypic variation in the nematode's interaction with naturally coexisting bacteria and pathogens. Our study emphasizes the importance of field studies of C. elegans in order to fully appreciate the nematode's life history characteristics.

William B Raich et al. (2001) International C. elegans Meeting "A genetic dissection of the minibrain kinase gene family and its role in Down syndrome"

Ronald H A Plasterk, Celine Moorman, Clay O Lacefield, Oliver Hobert, Eric R Kandel, William B Raich

[International C. elegans Meeting, 2001]

Increased gene dosage of minibrain kinase (MnbK) has been implicated in the cognitive defects of Down syndrome, the most common cause of mental retardation. MnbK belongs to the DYRK subfamily of protein kinases, which are capable of phosphorylating both serine/threonines and tyrosines. Despite their potential importance, little is known about the normal function of these genes, and it is unclear how increased expression of MnbK produces cognitive defects. Towards these ends, we are investigating the C. elegans members of the DYRK kinase subfamily. Two members of the DYRK kinase subfamily were identified in the database, which we call mbk-1 and mbk-2 ( m ini b rain k inase), as well as a related kinase called hpk-1 ( h omeodomain interacting p rotein k inase). Based on sequence similarity of the kinase domains and overall gene structure, mbk-1 is the DYRK1A/MnbK homolog, and mbk-2 is equally similar to mammalian DYRK2 and DYRK3. To determine the expression and subcellular localization of these genes, we generated translational fusions with GFP. mbk-1::gfp is ubiquitously expressed, and the gene product primarily localizes to the nucleus. mbk-2 is alternately spliced into 3 forms; all 3 forms are cytoplasmic and are expressed in overlapping patterns that include subsets of neurons in the head, ventral nerve cord, and tail. hpk-1::gfp localizes to nuclear puncta and is expressed strongly in embryos and L1 larvae, and weakly in older larvae and adults. In an effort to model the consequences of increased MnbK gene dosage in Down syndrome, MBK-1 was overexpressed using its endogenous promoter. In addition, we overexpressed putative "kinase dead" and "kinase active" mutants of MBK-1. We found that overexpression of mbk-1(gf) produces strong defects in AWC-mediated olfaction. AWC sensed odors fail to be sensed; odors sensed by both AWA and AWC elicit only a partial response, while AWA-sensed odors elicit an intact response. The defects are dosage sensitive, since heterozygotes show partial odortaxis defects. Using a heat shock promoter, we determined that the defects result from acute MBK-1 activity, since a single heat shock one hour prior to the assay was sufficient to produce the defect, while heat shocking 24 hours prior to the assay had little effect. The odortaxis defect is at least partially cell autonomous, since mbk-1 overexpression from the gcy-10 promoter was sufficient to produce weak defects. Additional phenotypes for mbk-1(gf) include synergistic enhancement of partial loss-of-function mutations in let-60 and mpk-1 during vulval development, as well as a strong dauer constitutive phenotype with daf-7 at 15deg C. To determine the normal function of the minibrain genes, we have identified deletion mutants in mbk-1 and hpk-1 , and are screening for an mbk-2 deletion. Both mbk-1 and hpk-1 deletion mutants are viable, and are currently being outcrossed prior to characterization.

Asim A Beg et al. (2002) West Coast Worm Meeting "A GABA-gated non-selective cation channel in C. elegans."

Asim A Beg, Erik M Jorgensen

[West Coast Worm Meeting, 2002]

-aminobutyric acid A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in both vertebrates and invertebrates. GABA binding to these receptors causes the opening of an anion-selective channel that predominantly conducts chloride ions, thereby hyperpolarizing cells. However, there are numerous mechanisms by which GABA can serve as an excitatory neurotransmitter. To date, the molecular identity of an excitatory ionotropic GABA receptor has remained elusive.

Sarah Straud Anselmo et al. (2002) West Coast Worm Meeting "Developing a screen for HERG blocking drugs using the C. elegans pharynx"

Sarah Straud Anselmo, Leon Avery

[West Coast Worm Meeting, 2002]

The HERG (human ether-a-go-go related gene) potassium channel is expressed in the human heart and is responsible for proper repolarization of cardiac muscle at the end of the action potential. Compromising HERG function gives rise to Long QT Syndrome, a disorder in which one is predisposed to ventricular fibrillation and sudden death. Many drugs, including Seldane (an anti-histamine) and Propulsid (an acid reflux inhibitor), block HERG as an unwanted side effect and consequently have been taken off of the market.

Melissa M Harrison et al. (2004) East Coast Worm Meeting "Analysis of synMuv Protein Complexes in vivo and Characterization of the Class B synMuv Gene lin-61"

Melissa M Harrison, Bob Horvitz, Xiaowei Lu

[East Coast Worm Meeting, 2004]

Vulval induction in C. elegans is negatively regulated by at least three redundant functions provided by the synthetic Multivulva (synMuv) class A, B, and C genes. Loss-of-function mutations in members of any single class do not result in a Multivulva (Muv) phenotype, but animals with mutations in any two synMuv classes are Muv. The molecularly characterized synMuv class A genes encode novel proteins. Many of the class B and class C synMuv genes, including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, let-418 Mi-2, trr-1 TRRAP, mys-1 HAT, and epc- 1 E(Pc), have homologs in other species that are involved in chromatin remodeling and transcriptional modulation. Some of the proteins that act in the synMuv B pathway have been shown to physically interact in vitro (1,2), in yeast two-hybrid assays (3), or in vivo based on co-immunoprecipitation experiments (4). These results along with homology to proteins in other species suggest that many synMuv proteins may function together in transcriptional regulatory complexes. We are using co-immunoprecipitation experiments to further explore in vivo physical interactions among the synMuv proteins. Such studies may allow us to better understand the functions of novel synMuv proteins, to analyze the effects of various synMuv mutations on physical interactions, and to identify potential sub-complexes among the synMuv proteins. We have focused initially on the gene products of the class B synMuv genes lin-37 and lin-61 as well as the class A synMuv gene lin-56, since null mutants in these genes are viable and can serve as negative controls for the immunoprecipitation experiments. LIN-37 is a small hydrophobic protein with no known homologs outside of nematodes. LIN-61 contains four MBT ( m alignant b rain t umor) repeats, which are loosely defined sequences of approximately 100 amino acids found in a number of nuclear proteins including the Drosophila Polycomb group protein Sex Comb on Midleg. LIN-56 is a novel acidic protein with no canonical motifs. We have surveyed the interactions of these proteins with a number of class A and B synMuv proteins by immunoprecipitation followed by western blots and shown that a subset of class B proteins co-immunoprecipitate from embryo extract. We are developing reagents to perform immunoprecipitation studies of other synMuv proteins. We also hope to identify new proteins involved in vulval development on the basis of their interaction with the synMuv proteins by co-immunoprecipitations followed by mass spectrometry. We are also characterizing the class B synMuv gene lin-61 in detail as mutations in this gene cause a number of pleiotropic defects that differ from those of most other class B synMuv mutants. lin-61 loss-of-function alleles cause GFP transgene misexpression (see abstract by Schwartz, Wendell, and Horvitz), and Pothof et al. have shown that RNAi of lin-61 results in an increased mutation rate (5). We have made polyclonal antibodies that recognize LIN-61 and have shown that it is a ubiquitously expressed nuclear protein that is localized to condensed chromosomes in the germline. We have also shown that this localization remains unchanged in a large number of synMuv mutant backgrounds. (1) Ceol and Horvitz. Mol. Cell 7 : 461-473, 2001. (2) Lu and Horvitz. Cell 95 : 981-991, 1998. (3) Walhout et al. Science 287 : 116-122, 2000. (4) Unihavaithaya et al. Cell 111 : 991-1002, 2002. (5) Pothof et al. Genes Dev. 17 :443-448, 2003.

Schulze, Jens et al. (2011) International Worm Meeting "Plectus - a stepping stone in embryonic cell lineage evolution of nematodes."

Schulze, Jens, Schierenberg, Einhard, Uenk, Jana

[International Worm Meeting, 2011]

The outstanding feature of Caenorhabditis elegans is its invariant cell lineage generating blastomeres of fixed identity. In contrast, the early embryo of the basal nematode Enoplus brevis shows only symmetric early cell divisions and the only identifiable lineage generates the gut (Voronov and Panchin, 1998; Development 15:143-150). How an organism with invariant development like C. elegans can evolve from an ancestor with an indeterminate mode remains a central question. Our lineage analysis of Plectus sambesii offers an important piece of the puzzle. Unexpectedly, cell arrangements in the AB lineage of this species are rather variable but merge into six different stereotyped patterns which all are compatible with normal development. However, one of the six patterns is prevalent and shows the same early cell arrangement as found in C elegans. This observation can be interpreted as an evolutionary trend from an indeterminate to an invariant pattern of cells. Furthermore, our cell lineage analysis of hypodermal and pharyngeal cells reveals that AB cell fates in all six stereotyped patterns are determined in a position-dependent rather than a lineage-dependent manner resulting in a single distinct right-handed juvenile pattern. This differs fundamentally from the lineage-dependent C. elegans mechanism where a left-handed situs inversus can be generated experimentally by switching positions of AB cells in the early embryo (Wood, 1991; Nature 349:536-38).

Marc Hammarlund et al. (2002) West Coast Worm Meeting "Mapping a new class of Ric mutants using an improved SNP mapping technique"

Tracey Harrach, Erik M Jorgensen, Marc Hammarlund

[West Coast Worm Meeting, 2002]

In an effort to identify additional molecules that function in synaptic transmission we have conducted several large screens for Aldicarb resistance. Aldicarb is an inhibitor of acetylcholinesterase and mutants that secrete less neurotransmitter are resistant to this drug. The mutants obtained from these screens fall into two classes: those that are uncoordinated, and those that are not uncoordinated.

Laskova, Valeriya et al. (2013) International Worm Meeting "Mapping the entire connectome of C. elegans L1 larvae."

Zhen, Mei, Kasthuri, Bobby, Shalek, Richard, Lichtman, Jeff, Samuel, Aravi, Pfister, Hanspeter, Laskova, Valeriya, Kaynig-Fittkau, Verena, Wen, Quan, Berger, Daniel, Guan, Asuka

[International Worm Meeting, 2013]

To investigate the poorly understood mechanisms of development and function of the nervous system, connections between neurons must be deciphered first. The adult nervous system of C. elegans was mapped to near completion by Dr. John White and his colleagues in 1970s. However, neuronal wiring in C. elegans larvae differs from that of an adult, since it undergoes multiple rounds of neuronal birth, apoptosis and rewiring during development. We utilize an Automatic Tape-Collecting Ultramicrotome (ATUM) to section an entire L1 stage animal into thousands cross-sections, followed by the automated imaging with a scanning electron microscope (SEM) to map an entire neuronal wiring diagram with synaptic resolution. The acquired wiring diagram will guide and be validated by calcium imaging to map the functional connection of the juvenile motor circuit.

Cynthia Wagner et al. (2006) Development & Evolution Meeting "The Role of gak-1 in Meiosis"

Gabrielle Howard, Cynthia Wagner, Rachel Webster, Judith Yanowitz, Dana Hubbard

[Development & Evolution Meeting, 2006]

We performed an RNAi feeding screen to identify chromatin associated gene products, that when mutant, result in an increase in recombination. We screened greater than 300 genes and identified 20 genes that give between a two-fold and four-fold enhancement of recombination. We are in the process of characterizing one of these genes, gak-1. gak-1 has previously been reported to function in meiotic chromosome segregation, including an increase in X-chromosome nondisjunction, which results in a him phenotype. We will present our cytological analysis on gak-1 mutants as well as detailed information on recombination effects in gak-1 mutants.