Jansen G et al. (1996) European Worm Meeting "THE ROLE OF THE PIM RELATED KINASES, prk-1 AND prk-2, IN SIGNAL TRANSDUCTION."

Jansen G, Neels J, Plasterk RHA

[European Worm Meeting, 1996]

The pim proto-oncogenes encode protein kinases, that play a role in haematopoietic cell differentiation. Overproduction of the Pim proteins in mouse lymphocytes results in increased leukemia incidence. The signal transduction pathway involved in this developmental proces is largely unknown. Two pim gene homologues from C. elegans, pim related kinase 1 and 2 (prk-1 and prk-2) have been cloned and sequenced. Using both GFP and lacZ fusion constructs, the expression pattern of the prk genes has been elucidated. Both genes are predominantly expressed in the pharynx, in muscle and epithelial cells. Furthermore, prk-1 expression was found in body wall muscle and vulval muscles. prk-2 is also expressed in cells surrounding the anus. The role of the PRK proteins in signal transduction was investigated further by the generation of several different mutant animals. Tc1 insertions were identified in both prk-1 and prk-2, which are homozygous viable and show no obvious phenotype. Among the progeny of the prk-1 Tc1 insertion containing nematodes mutants have been isolated in which the Tc1 element and approximately 2 kb flanking genomic DNA was deleted. This deletion is probably a loss of function allele. However, also this mutation is homozygous viable and shows no obvious phenotype. The screening for deletion mutants of the prk-2 gene is currently in progress. Furthermore, transgenic animals have been generated that carry multiple copies of the wild type prk genes or a dominant negative version of the prk genes, regulated by their own promoter or the heat shock promoter. Also these mutant nematodes show no obvious phenotype. Mutant animals that exhibit an obvious phenotype will be used in suppressor studies to search for upstream and downstream factors in the Prk signalling pathway. An alternative mutant screen, which will be employed if no phenotypes can be discerned will be discussed.

Jansen G et al. (1997) International C. elegans Meeting "THE 19 G PROTEIN-ALPHA SUBUNITS OF C. ELEGANS"

Kato Y, Plasterk RHA, Thijssen KL, Jansen G, Hazendonk E, Werner P

[International C. elegans Meeting, 1997]

Heterotrimeric G proteins transduce signals from transmembrane receptors to intracellular second messengers. Thus far, two G-beta subunit genes (gpb-1 and 2), one G-gamma subunit gene (gpc-1) and one member of each of the mammalian classes of G-alpha genes (goa-1, gsa-1, egl-30 and gpa-12) have been identified in C. elegans. Furthermore the genome sequencing project revealed the existence of 15 additional C. elegans-specific G-alpha genes (gpa-1 to gpa-15 and odr-3). We studied the function of the C. elegans G-alpha genes by determining their expression patterns and by phenotypic analysis of mutant animals (null mutants, overexpression and dominant active mutants). The Go, Gs, Gq and G12 genes and one of the C. elegans specific genes, gpa-7, show an ubiquitous expression in many neurons and in other tissues such as body wall muscle. The other 14 G-alpha genes show a highly restricted expression pattern. Interestingly, they all show expression in a subset of the amphid neurons, suggesting they all act in sensory transduction. For example gpa-5 is specifically expressed in the AWA amphid neurons. We found a negative regulatory role of gpa-5 in trimethylthiazole odor perception (and less in diacetyl and pyrazine perception): loss-of-function mutants are more attracted to trimethylthiazole, gain-of-function mutants less. Expression of gpa-5 is regulated by odr-7, a member of the nuclear receptor superfamily, which also regulates expression of the diacetyl receptor, odr-10 (Sengupta et al., 1996, Cell 84, 899-909).

Jansen G et al. (1995) International C. elegans Meeting "The role of pim related kinases prk-1 and prk-2 in signal transduction."

Jansen G, Youngman S, Plasterk RHA

[International C. elegans Meeting, 1995]

The pim proto-oncogenes encode protein kinases that play a role in haematopoietic cell differentiation. pim-1 was originally identified as a common integration site for murine RNA-tumor viruses, resulting in leukemia due to overproduction of the Pim protein. Neither the targets of the Pim kinases nor the factors upstream of it in the signalling pathway are currently known. Recently, two pim homologues were cloned and sequenced from C. elegans: pim related kinase 1 and 2 (prk-1 and prk-2). Both genes are located on LGIII. In order to investigate the role of the prk genes in signal transduction, reverse genetics tools are applied. First, Tc1 insertions were identified in both prk-1 and prk-2. Two Tc1 insertions were found in prk-1, one in intron 7 and one in exon 3 in a region that encodes a highly conserved protein kinase domain, probably resulting in loss of function of prk-1. Both mutants are homozygous viable and show no obvious phenotype. For prk-2 one Tc1 insertion has been identified, located in intron 4. These Tc1 insertions are currently used to generate deletion mutants of both prk genes. Other approaches to determine the function of prk-1 and prk-2 in C. elegans involve the generation of transgenic nematodes harboring extra copies of the prk genes under the control of the endogenous or the heat shock promoter. Alternatively the effect of expression of prk constructs in which the kinase active site domain is mutated will be determined. By analogy with other kinases, one can expect that the mutant proteins can interact with the normal substrates and regulator proteins. They are therefore expected to titrate away factors normally interacting with endogenous Prk proteins, and thus interfere negatively with the signalling cascade.

Jansen G et al. (1998) European Worm Meeting "At least 15 G-alpha genes are involved in perception in C. elegans"

van Horst M, Jansen G, Werner P, Plasterk RHA, Thijssen KL, Hazendonk E

[European Worm Meeting, 1998]

Heterotrimeric G proteins are signal transduction molecules situated at the interface between extracellular signals received via receptors and intracellular second messenger systems. We set out to study the function of all G-protein genes in C. elegans. Thus far, two G-b subunit genes (gpb-1 and 2), one G-g gene (gpc-1) and 19 G-a genes have been identified in the genomic sequence. Besides one member of each of the mammalian G-a classes (goa-1, gsa-1, egl-30 and gpa-12) there are 15 new genes (gpa-1 to 11, 13 to 15 and odr-3). Based on the expression patterns of the G-a genes they can be divided into two groups. The Go, Gs, Gq and G12 genes and gpa-7 show an ubiquitous expression in many neurons and muscle cells. The other 14 new G-a genes show a highly restricted expression pattern. Interestingly, they all show expression in a subset of the sensory neurons, the amphids, suggesting they all act in perception. To test the involvement of the 15 new G-a genes in perception we generated both loss and gain of function mutant alleles, using chemical mutagenesis to generate deletions and overexpression by transgenesis of the wildtype gene, respectively. Thus far we isolated null alleles for 13 of the 15 new G-a genes and made gain of function mutants for 10 genes. All mutants that were generated are homozygous viable. Only gpa-7 mutants show obvious behavioural phenotypes: gpa-7 l.o.f. animals are egl-d, gpa-7 overexpression results in hyperactivity and an egl-c phenotype. For mutants of eight genes we found abnormalities in perception. Our preliminary results indicate that gpa-1 is involved in thermotaxis, gpa-2, 3 (Zwaal et al. 1997, Genetics 145, 715-727) and 10 in perception of the dauer pheromone, gpa-2, 3, 7, 10, 11 and odr-3 in chemotaxis towards water soluble compounds and that gpa-3, 5 and odr-3 (Roayaie et al. 1998, Neuron 20, 55-67) are involved in olfaction. Analysis of further defects in perception is ongoing.

G Jansen et al. (1999) International C. elegans Meeting "The complete family of G protein genes of Caenorhabditis elegans"

RH Plasterk, M van der Horst, E Hazendonk, KL Thijssen, G Jansen, P Werner

[International C. elegans Meeting, 1999]

One of the new possibilities in post-sequence genetics is the analysis of complete gene families at once. We studied the family of heterotrimeric G proteins. C. elegans has 20 G a , 2 G b and 2 G g genes. There is one clear homologue of each of the four mammalian classes of G a genes, Go/Gi, Gs, Gq, G12, and there are sixteen new a genes. While the conserved G a subunits are expressed in many neurons and muscle cells, GFP fusions indicate that 14 of the new G a genes are expressed almost exclusively in a small subset of the amphid cells, sensory neurons in the head of the nematode, or other putative sensory neurons. We isolated null alleles for all G a genes, using target selected gene inactivation. Furthermore gain-of-function alleles were generated by introducing the wild type genes as transgenes (XS). None of the amphid expressed genes are essential for viability. Based on the specific expression of many G a subunits in chemosensory neurons, all mutants were tested for chemotaxis to water soluble and volatile attractants and repellents. Only four loss-of-function alleles show chemotaxis defects. In contrast, nine gain-of-function alleles show altered behavior in the various chemotaxis assays. The lack of loss-of-function phenotypes can be explained by functional redundancy, as has been described for gpa-2 and odr-3 (Roayaie et al., Neuron 20: 55-67, 1998). Alternatively, the G a genes may be involved in the perception of compounds that have not yet been tested. Furthermore, they may have negative regulatory functions, as was found for gpa-5 and gpa-6 . We are further testing these hypotheses by combining several of loss-of-function alleles. Based on functional analysis the 20 G a genes can be divided into two groups: subunits that affect muscle activity (homologues of Gi/Go a , Gs a and Gq a ), and 14 new G a genes that are probably all involved in perception (Jansen et al., Nature Gen. In press).

Plasterk RHA et al. (2000) European Worm Meeting "Extensive modulation of G-protein mediated signaling in olfaction"

Plasterk RHA, Jansen G

[European Worm Meeting, 2000]

C. eleganscan detect at least 60 volatile attractants and discriminate among many of them using only two pairs of sensory neurons, AWA and AWC. This complex task is probably accomplished by the expression of multiple different chemosensory receptors per cell and the presence of 6 different heterotrimeric G-protein a subunits in the 2 pairs of olfactory neurons: gpa-2, 3, 5, 6, 13 and odr-3. However, loss-of-function of only odr-3 resulted in significantly altered odorant perception. What would be the function of the other 5 a subunits? Is there functional redundancy between the Ga proteins? Do they have negative regulatory functions? Or are they not involved in the perception of the odorants tested? To determine the interactions between these V Ga genes we generated animals that had lost the function of 2 to 5 Ga subunits and tested their behavior in odorant chemotaxis. Our results show that C. elegans uses ODR-3 as main stimulatory signaling route. 2 to 4 G a proteins modulate this signal. These a subunits may have stimulatory or inhibitory roles, depending on the odorant and its concentration. E.g. pyrazine detection uses both GPA-3 and ODR-3 as stimulatory signals, and GPA-2, GPA-5 and GPA-6 as redundant negative regulators. Our findings reveal extensive modulation of what seemed a simple G-protein mediated signal transduction cascade.

E Cuppen et al. (1999) International C. elegans Meeting "Heterotrimeric G proteins of C. elegans: cDNA sequence analysis and interaction studies"

G Jansen, RH Plasterk, E Cuppen

[International C. elegans Meeting, 1999]

Heterotrimeric G proteins are signal transduction molecules situated at the interface between extracellular signals received via G protein-coupled receptors (over 600 predicted in the C.elegans genome) and intracellular second messenger systems. We set out to study the function of all heterotrimeric G protein genes in C.elegans . Thus far, two G b subunit genes ( gpb-1 and 2 ), two G g genes ( gpc-1 and 2 ) and 20 G a genes have been identified in the genomic sequence (Jansen et al., Nature Gen. in press ). Besides one member ( gsa-1 , goa-1 , egl-30 and gpa-12 ) of each of the mammalian G a classes (Gs, Go/Gi, Gq, and G12) there are 16 new C.elegans specific genes ( gpa-1 to 11 , 13 to 16 and odr-3 ). We have cloned the cDNA's for all the G protein subunits. RT-PCR and sequence analysis gave no indications for alternative splicing, but we found that about 10% of the exons of this highly conserved protein family were predicted incorrectly by Genefinder. We will employ all cDNA's in yeast two- and three-hybrid interaction studies to test for interaction specificity between the different subunits. Furthermore, we will screen cDNA libraries for interacting proteins. The interaction specificity of novel proteins can easily be tested using mating assays, because all C.elegans heterotrimeric G protein subunits are available. Since we know that multiple G a subunits are expressed in particular cells (Jansen et al., Nature Gen. in press ), specificity in interactions mediated by these subunits may provide an efficient mechanism regulating signaling via G protein coupled receptors.

Werner P et al. (1997) International C. elegans Meeting "HETEROTRIMERIC G PROTEINS OF C. ELEGANS: SEQUENCE ANALYSIS AND EXPRESSION STUDIES"

Plasterk RHA, Jansen G, Thijssen KL, Kato Y, Werner P, Hazendonk E

[International C. elegans Meeting, 1997]

Heterotrimeric G proteins regulate cellular effectors in reaction to extracellular signals. Each mammalian class of G-alpha subunits (Gs, Go, Gq, G12) is represented in C. elegans by one homologue. Four additional C. elegans G-alpha subunits (gpa-1 to gpa-3 and odr- 3) have been reported previously. The C. elegans genome sequencing project revealed 11 more G alpha candidate genes. Their predicted protein sequences share only 30 to 60 % of the amino acid residues with each other. These novel genes gpa-4 to gpa-11 and gpa-13 to 15 reside on chromosomes I, II, IV, V and X, individually or in loose clusters. We visualized their expression patterns in transgenic lines that carry translational fusions to green fluorescent protein. All 11 candidates show distinct although partially overlapping expression patterns. A common feature seems to be transcriptional activity in neurons, especially in amphid neurons. For all subunits, with the exception of gpa-7, a rather restricted expression pattern was observed. Other gpa-genes produced additional non-neuronal staining, e.g. in the vulva, spermatheca, gut, and the excretory cell. To further characterizate this gene family, we are currently screening for a complete set of gpa-deletion mutants and we are investigating the influence of overexpression and constitutively active variants on transgenic worms. The present number of 19 apparently active C. elegans G-alpha genes is high compared to other species and suggests a significant potential of differential signal processing in individual nematode cells.

William B Wood et al. (2001) International C. elegans Meeting "Embryonic handedness choice in C. elegans involves a Galpha protein encoded by the spn-1 gene"

Barbara Robertson, William B Wood, Dominique Bergmann

[International C. elegans Meeting, 2001]

The apparent left/right (L/R) symmetry of the early C. elegans embryo is broken between the 4- and 6-cell stages of cleavage when the L/R-oriented spindles in the anterior blastomeres ABa and ABp skew in a clockwise direction (viewed dorsally). The resulting cleavage produces an asymmetric 6-cell embryo and establishes the handedness of all subsequent L/R asymmetries in development (Wood, Nature 349:536, 1991). Handedness choice is essentially invariant, so that animals with reversed situs are not seen in wild-type populations (<10 -4 ). The ts maternal-effect mutation spn-1(it143) causes misorientation of spindles in the second and third cleavages at 25deg, affecting ABa and ABp most severely. The result is 70% embryonic lethality, with 40% of the surviving progeny showing reversed situs (Bergmann & Wood, Int. C. elegans Meeting Abstr. 187, 1999). We mapped spn-1 to a region of LGI including the predicted G a protein gene gpa-16 (Jansen et al., Nature Genet 21:414-419, 1999). The spn-1 mutation failed to complement gpa-16(pk481) , a deletion mutation (kindly provided by G. Jansen), which in a gpa-16(pk481);dpy-20(e1282) strain also causes incompletely penetrant maternal-effect embryonic lethality. Sequencing of gpa-16 cDNA from spn-1(it43) revealed a single base change, causing the substitution G202D in a conserved region of the predicted G a protein product. Together with the recent demonstration that this protein is involved in control of a G b g complex regulating centrosomal migration and hence spindle orientation in early embryos (Gotta & Ahringer, Nature Cell Biol 3:297-300, 2001), our findings begin to explain how the spn-1(it143) mutation affects handedness choice and suggest that the initial symmetry-breaking event may act through heterotrimeric G protein signaling.

Damien M O'Halloran et al. (2005) International Worm Meeting "The molecular phylogeny and mode of evolution of a nematode specific clade of heterotrimeric G-protein subunit genes."

Ann M Burnell, James O McInerney, Damien M O'Halloran, David A Fitzpatrick, Grace McCormack

[International Worm Meeting, 2005]

In C. elegans, and most probably all animal olfactory systems, odorant molecules are detected by G-protein coupled receptors (GPCRs). Guanine nucleotide binding G-proteins relay signals from these GPCRs to intracellular second messenger pathways that trigger the opening of ion channels and local depolarization. The human genome contains 17 G genes which are divided into four subfamilies based on functional and sequence attributes. C. elegans contains 21 G genes, 14 of which are specifically expressed in the amphid sensory neurons1. In addition to having at least one member of each of the four mammalian G gene classes, our analysis shows that nematodes including C. elegans also possess at least one lineage-specific G gene expansion, homologues of which are not found in other organisms. We constructed a data set containing homologues of putative G genes from a variety of metazoans, protistans, fungi and plants. The final alignment contained 146 taxa and 751 aligned amino acid positions. Amino acids were recoded into the six categories corresponding to the PAM matrix. The recoded data was analysed using a Bayesian criterion with an appropriate model. The resultant phylogeny was compared to a second phylogenetic analysis based on a smaller number of exemplar G proteins. This analysis used maximum likelihood estimation to categorise sites into one of eight categories according to their rate of evolution. Overall, both analyses yielded near identical topologies and showed that the majority of nematode unique G genes are the result of a duplication of a putative ancestral Gi/o gene. We will present our phylogenetic analyses and an evaluation of the selective pressures acting on these nematode-unique G genes. A single C. elegans olfactory neuronal membrane contains multiple GPCRs and multiple heterotrimeric G proteins. These novel nematode specific G genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate and adapt to odor signals from the multiple distinct GPCRs expressed on their membranes2.1. Jansen, G. et al. (1999). Nature Genetics, 21, 414-419.2. Lans, H. et al. (2004. Genetics, 167, 1677-1687.