Korswagen HC et al. (1996) Worm Breeder's Gazette "Tc1 Sequence Tagged Sites, An Update"

Smits MT, Korswagen HC, Plasterk RHA, Durbin RM

[Worm Breeder's Gazette, 1996]

In an effort to establish a high density Tc1 STS map as an extension of the mapping strategy developed by Williams et al. (1), we shotgun sequenced Tc1 insertion sites present in the high Tc1 copy number strains RW7000, CB4000 and KR1787 (2). To map these Tc1 insertion sites, flanking sequences were compared to the genomic sequence. In principle, all Tc1 insertion sites will fall in place when the complete genomic sequence of C. elegans becomes available. So far, we mapped 151 polymorphic Tc1 insertion sites in 40 Mbp of genomic sequence; a density of about one insertion in every 265 kb. Information on these mapped Tc1 insertion sites is available through ACeDB. These Tc1 insertions can be used as polymorphic markers, but can also be used for deletion mutatgenesis when a Tc1 insertion is located in or close to a gene of interest. (1) Williams, B. D. (1995) in Caenorhabditis elegans: Modern biological analysis of an organism, eds. Epstein, H. F. & Shakes, D. C. (Academic Press, San Diego), pp. 81-96. (2) Korswagen et al. (1995) Worm Breeder's Gazette 13(5): 90. Mapped polymorphic Tc1 insertion sites: (Indicated are approximate position on the genetic map, the cosmid the Tc1 is inserted in and the position of that Tc1 insertion in the cosmid sequence. Strain designations are A. RW7000, B. CB4000 and C. KR1787)

Korswagen HC et al. (1996) European Worm Meeting "A constitutive activating mutation in gsa-1 results in neural degeneration"

Park J-H, Plasterk RHA, Ohshima YM, Korswagen HC

[European Worm Meeting, 1996]

Heterotrimeric G proteins relay information from transmembrane receptors to various intracellular effector systems. We cloned a C. elegans homolog of Gsalpha, the G protein alpha-subunit gene that couples to adenylate cyclase. gsa- 1 encodes a 375 amino acid protein that is 66% identical to rat Gsalpha. A gsa-1 GFP reporter construct is expressed throughout the nervous system and in muscle cells, including body wall muscles, the pharyngeal muscle cells and the muscle cells of the vulva. gsa-1 is an essential gene; loss of function of gsa-1 results in larval lethality. Animals homozygous for the deletion allele gsa-1(pk75) arrest in the first stage of larval development. We analyzed the effects of a dominant activating mutation of gsa-1. To generate a constitutively active mutation in gsa-1, we mutated a conserved glutamine to a leucine (Q208L) in the gsa-1 sequence. This mutation destroys the GTPase activity of the G protein and locks it in the active state. Introduction of this dominant active gsa-1QL construct in transgenic animals results in lethality of the F1 transformants. Animals show vacuoles at the positions of neurons in the ventral nerve cord, the nerve ring and neurons in the tail. In addition, there is a strong hypercontraction of body wall muscle cells. Identical phenotypes are produced by expression of gsa-1QL under heat-shock promotor control. The phenotype of the vacuolated neurons is similar to the neural degeneration observed in the degenerin ion channel mutants, suggesting that gsa- 1QL activates ion channels and that an ion imbalance leads to the observed vacuolated neurons. Mutations in the degenerins are in homologs of amiloride sensitive sodium channels. These dominant channel mutations are suppressed by loss of function mutations in mec-6(e1342). We tested if mec-6 could also suppress the neural degeneration of gsa-1QL. mec- 6;gsa-1QL double mutants still show the gsa-1QL induced neural degeneration. Therefore, gsa-1QL may not act via degenerin type ion channels. Suppressor screens and pharmacological studies to identify the gsa-1QL activated ion channels are in progress.

Korswagen HC et al. (1995) International C. elegans Meeting "Reverse genetics of C.elegans: current approaches and future directions"

Durbin RM, Plasterk RHA, Smits MT, Korswagen HC, van der Linden KM

[International C. elegans Meeting, 1995]

Transposon mediated deletion mutagenesis is now a method of choice for reverse genetics. First a Tc1 insertion allele of the gene of interest is isolated. In case this insertion is not a loss-of-function allele, the Tc1 insertion can be used to select deletion derivatives. As reported previously, we constructed a library of Tc1 insertion mutants. We have screened this library on a routine basis for laboratories all over the world and have obtained now over seventy Tc1 insertion alleles of researcher's pet genes. In line with the genome sequencing project we have started sequencing Tc1 insertions in high copy number strains. Flanking sequences of these insertions are mapped by comparison with the genome sequence; all insertions will fall in place once the entire genome sequence is known. These polymorphic Tc1 insertions serve two functions: Insertions in or close to genes can be used to generate knock-outs of those genes. Second, the set of Tc1 insertions forms a dense grid of "PCR visible" genetic markers. We amplified and sequenced Tc1 flanks of the high copy number strains RW7000, CB4000 and KR1787 and obtained a set of at least 500 distinct Tc1 insertions. We mapped 35 of these insertions by alligning with the 10 Mbp of genome sequence. Strategies to use these insertions in mapping new mutant genes will be discussed.

Korswagen HC et al. (2000) Nature "Distinct beta-catenins mediate adhesion and signalling functions in C. elegans."

Herman MA, Clevers HC, Korswagen HC

[Nature, 2000]

In flies and vertebrates, Armadillo/beta-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signalling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative beta-catenin homologues have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homologue POP-1 mediate Wnt signalling by a mechanism that has challenged current views of the Wnt pathway. Here we show that BAR-1 is the only beta-catenin homologue that interacts directly with POP-1. BAR-1 mediates Wnt signalling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only beta-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. We conclude that a canonical Wnt pathway exists in C. elegans. Furthermore, our analysis shows that the functions of C. elegans beta-catenins in adhesion and in signalling are performed by separate proteins.

Korswagen HC et al. (1996) Worm Breeder's Gazette "An activating mutation in the G protein gene gsa-1 results in neural degeneration"

Ohshima YM, Korswagen HC, Park J-H, Plasterk RHA

[Worm Breeder's Gazette, 1996]

Heterotrimeric G proteins relay information from transmembrane receptors to various intracellular effector systems. We cloned a C. elegans homolog of Gsalpha, the G protein alpha-subunit gene that couples to adenylate cyclase. gsa- 1 encodes a 375 amino acid protein that is 66% identical to rat Gsalpha. A gsa-1 GFP reporter construct is expressed throughout the nervous system and in muscle cells, including body wall muscles, the pharyngeal muscle cells and the muscle cells of the vulva. gsa-1 is an essential gene; loss of function of gsa-1 results in larval lethality. Animals homozygous for the deletion allele gsa-1(pk75) arrest in the first stage of larval development. We analyzed the effects of a dominant activating mutation of gsa-1. To generate a constitutively active mutation in gsa-1, we mutated a conserved glutamine to a leucine (Q208L) in the gsa-1 sequence. This mutation destroys the GTPase activity of the G protein and locks it in the active state. Introduction of this dominant active gsa-1QL construct in transgenic animals results in lethality of the F1 transformants. Animals show vacuoles at the positions of neurons in the ventral nerve cord, the nerve ring and neurons in the tail. In addition, there is a strong hypercontraction of body wall muscle cells. Identical phenotypes are produced by expression of gsa-1QL under heat-shock promotor control. The phenotype of the vacuolated neurons is similar to the neural degeneration observed in the degenerin ion channel mutants, suggesting that gsa- 1QL activates ion channels and that an ion imbalance leads to the observed vacuolated neurons. The dominant degenerin channel mutations are suppressed by loss of function mutations in mec-6(e1342). We tested if mec-6 could also suppress the neural degeneration of gsa-1QL. mec- 6;gsa-1QL double mutants still show the gsa-1QL induced neural degeneration. Therefore, gsa-1QL may not act via degenerin type ion channels.

Korswagen HC et al. (1997) Genes Dev "An activating mutation in a Caenorhabditis elegans Gs protein induces neural degeneration."

Ohshima YM, Korswagen HC, Park J-H, Plasterk RHA

[Genes Dev, 1997]

Heterotrimeric guanine nucleotide-binding proteins (G proteins) act as signal-transducing molecules that connect serpentine-transmembrane receptors to a variety of intracellular effectors. We characterized a Caenorhabditis elegans G(s) gene, gsa-1, which encodes a G(s) alpha-subunit (G alpha(s)) that is expressed throughout the nervous system and in muscle cells. gsa-1 is an essential gene; a loss-of-function mutation in gsa-1 results in lethality at the first stage of larval development. Partial (mosaic) loss of G alpha(s) expression or overexpression of the protein results in reciprocal defects in movement and egg-laying, suggesting a role for G alpha(s) in the regulation of these behaviors. Expression of a constitutively active form of G alpha(s) from an inducible promotor results in hypercontraction of body-wall muscle cells and vacuolization and degeneration of neurons within hours of induction. Neurons that are susceptible to the degeneration induced by activated G alpha(s) are predominantly motoneurons located within the ventral nerve cord. Phenotypic analysis shows that the induced neural degeneration is not the result of programmed cell death but is probably caused by the activation of ion channels. A genetic suppressor of activated G alpha(s) was isolated that identifies a putative downstream target of G(s) signaling.

Korswagen HC (2002) Bioessays "Canonical and non-canonical Wnt signaling pathways in Caenorhabditis elegans: variations on a common signaling theme."

Korswagen HC

[Bioessays, 2002]

Wnt glycoproteins are signaling molecules that control a wide range of developmental processes in organisms ranging from the simple metazoan Hydra to vertebrates. Wnt signaling also plays a key role in the development of the nematode C. elegans, and is involved in cell fate specification and determination of cell polarity and cell migration. Surprisingly, the first genetic studies of Wnt signaling in C. elegans revealed major differences with the established (canonical) Wnt signaling pathways of Drosophila and vertebrates. Thus, the Wnt-dependent induction of endoderm in the early embryo and the specification of several asymmetric cell divisions during larval development are mediated by as yet novel Wnt signaling pathways that repress, rather than activate the TCF/LEF-1 transcription factor POP-1. Recently, however, it has been shown that, in addition to these divergent Wnt pathways, C. elegans also has a canonical Wnt pathway that converts POP-1 into an activator and controls the expression of several homeobox genes. Interestingly, these different Wnt pathways use distinct beta-catenins to control POP-1 function: the endoderm induction pathway requires the beta-catenin WRM-1 and parallel input from a mitogen-activated kinase (MAPK) pathway to downregulate POP-1, whereas the canonical Wnt pathway employs the beta-catenin BAR-1 to activate Wnt target gene expression.

Korswagen HC et al. (1995) International C. elegans Meeting "PHYSICAL MAPPING OF Tc1 SEQUENCE-TAGGED SITES IN THREE Tc1-POLYMORPHIC STRAINS"

Plasterk RHA, Shmookler Reis RJ, Korswagen HC, Thaden JJ

[International C. elegans Meeting, 1995]

Williams et al. [(1992) Genetics 131, 609] have described a genetic mapping system based on polymorphic sequence-tagged sites (STS). It is an alternative to classical two- or three-factor crosses, and can also be used to map polygenic traits [Ebert et al. (1993) Genetics 135,1003; Ayyudevara et al. poster]. One weakness has been that fewer than 80 C. elegans polymorphisms had been sequenced and mapped. This has changed. By shotgun sequencing, Korswagen et al. [WBG 13(5),90] have generated over 800 sequences adjoining Tc1 insertions in strains RW7000, KR1787, and CB4000. At this writing, 35 Tc1 alleles map to sequenced genome regions. This number will slowly increase as sequencing proceeds. We are mapping Tc1 alleles by YAC grid annealing to pooled DNA probes. Pooling is via the "matrix addressing" strategy [Zwaal et al. (1993) PNAS 90, 7431]; DNAs are arranged in 3-D arrays and pooled along each plane. An allele thus contributes to three orthogonal probe pools. A YAC or adjacent YACs labeled by only those three pools will in principle map the allele. In preliminary work, we identified and set aside alleles that align with repetitive DNA elements or anneal to total genomic DNA. We are evaluating probe mixtures of increasing complexity, to optimize 3-D array size.

Korswagen HC et al. (1997) International C. elegans Meeting "GENETIC ANALYSIS OF ACTIVATED Gs-INDUCED NEURAL DEGENERATION IN C. ELEGANS"

Plasterk RHA, Park J, Ohshima YM, Korswagen HC

[International C. elegans Meeting, 1997]

Heterotrimeric guanine nucleotide binding proteins (G proteins) act as signal transducing molecules that connect serpentine-transmembrane receptors to a variety of intracellular effectors. In humans, a Gs protein is causally involved in several endocrine tumors. We characterized a C. elegans Gs gene. gsa-1 encodes a Gs a-subunit (Gas) which is expressed throughout the nervous system and in muscle cells. gsa-1 is an essential gene; a loss-of-function mutation in gsa-1 results in lethality at the first stage of larval development. Partial (mosaic) loss of Gas expression or overexpression of the protein results in reciprocal defects in movement and egg-laying, suggesting a role for Gas in the regulation of these behaviors. Expression of a constitutively active form of Gas from an inducible promotor results in hypercontraction of body-wall muscle cells, and vacuolization and subsequent degeneration of neurons within hours of induction. Neurons that are susceptible to the activated Gas-induced degeneration are predominantly motorneurons located within the ventral nerve cord. Epistatic analysis shows that the induced neural degeneration is not the result of programmed cell death. The induced swelling and lysis of neurons is probably the result of direct or indirect activation of ion channels. However, loss-of-function mutations in ion channel genes such as the degenerin mec-6 and Ca2+ channels such as unc-2, unc-36 and egl-19 did not suppress the activated Gas-induced degeneration. Several genetic suppressors of activated Gas that identify putative targets in Gas signaling were isolated in a screen for revertants of the neural degeneration phenotype. One of these suppressors, sgs-1, maps on chromosome III and is located 1-2 map units to the right of unc-32.

Korswagen HC et al. (1995) Worm Breeder's Gazette "Shotgun Sequence Analysis of Transposon Tc1 Alleles In High Copy Number Strains."

Durbin RM, Korswagen HC, Plasterk RHA, Smits MT

[Worm Breeder's Gazette, 1995]

Shotgun sequence analysis of transposon Tc1 alleles in high copy number strains