Bogaerts A et al. (2008) European Worm Meeting "Differential analysis of immune-induced proteins in Caenorhabditis elegans."

Schoofs L, Temmerman L, Lindemans M, Verleyen P, Husson SJ, Bogaerts A

[European Worm Meeting, 2008]

The striking similarities between vertebrates and invertebrates in their. innate defences as well as the amenability of Caenorhabditis elegans for. genetic analysis, have made this free-living ground nematode a popular. model system in the study of bacterial pathogenesis. Although genetic. studies have brought new insights, showing the inducibility and pathogen-. specificity of the immune response, there is still much to be discovered. about the exact mechanisms underlying resistance to infection. Our group. has tried to uncover a few of the remaining questions by approaching. immunity from a different angle. We report the application of differential. gel electrophoresis (DIGE), combined with mass spectrometry to search for. proteins that are differentially expressed in the worm after infection with. the gram-negative bacteria Aeromonas hydrophila. A total of 62 proteins. were identified. Interestingly, the most strongly induced protein was. galactose binding lectin (lec-1) which is believed to play a role as a. pathogen recognition molecule. Another interesting protein induced after. infection was DAF-16, confirming its role in the insulin-like receptor. immune pathway. In addition a number of unknown proteins, not yet. associated with the immune response, were isolated and identified.. Determining the exact function (recognition/effector) of all these proteins. in the immune system of C. elegans, remains a very exciting challenge which. will shed more light on the complexity of innate immunity in general.

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.

Bogaert T et al. (1989) International C. elegans Meeting "genes required for body wall and sex muscle attachment."

Bogaert T, Goh P-Y

[International C. elegans Meeting, 1989]

Bogaert T et al. (1989) International C. elegans Meeting "characterization of e2346, a mutant affecting body wall muscle development (attachment)."

Bogaert T, Goh P-Y

[International C. elegans Meeting, 1989]

Temmerman L et al. (2009) European Worm Neurobiology Meeting "Unravelling neuropeptide function by a combined transcriptome and proteome study."

Meelkop E, Temmerman L, Franssens L, Schoofs L, Boerjan B, Cardoen D, Bogaerts A, Janssen T

[European Worm Neurobiology Meeting, 2009]

Innumerate organisms rely on internal biological clocks in order to prepare themselves for periodic environmental changes, thereby using an impressive set of molecules. The best studied and perhaps most appealing molecular timekeeper is the circadian clock, which keeps track of the day-night rhythm. In invertebrates, the neuropeptide Pigment Dispersing Factor (PDF) is the key molecule that links the circadian clock to a fit output. Deletion of the insect pdf gene results in animals with aberrant rhythmic behaviour. Despite the discovery of daily rhythms in C. elegans behaviour, its molecular basis remains elusive, and might even be fundamentally different from the currently known timekeeping mechanisms. Our lab has identified two pdf genes in C. elegans, which could be coupled to the regulation of locomotor activity. So far, it could not be shown that C. elegans pdf influences the circadian activity rhythm. It is therefore likely that unravelling the PDF-system in C. elegans will provide insight into a more primitive or basic function of this neuropeptide. To assess the molecular effects of the pdf-1 gene, we opted for a combined transcriptome and proteome study in which a pdf-1 null mutant was compared to wild type worms. After RNA isolation, whole genome transcript levels were investigated by means of microarray analysis and validated by real-time PCR. Protein isolates were labelled and separated by 2D-DIGE. Differentially expressed proteins were excised from the gels, trypsin digested and identified by Peptide Mass Fingerprint after MALDI-TOF mass spectrometry. Using the discriminating power of these techniques, we were able to identify a surprisingly limited set of differentially transcribed or expressed genes; thereby providing a more complete insight into the C. elegans PDF-system.

Roelens IG et al. (2000) European Worm Meeting "High density C. elegans screening: an automated phenotype analysis platform, applied to the unc-53 (Steerin) pathway"

Maillet I, Roelens IG, Geysen J, Ver Donck K, Van Osta P, Bogaert T, Bols L

[European Worm Meeting, 2000]

Green fluorescent protein (GFP) technology enables cellular resolution in the living animal. Using a microscope with sensitive cameras for low level fluorescence detection, automated image acquisition and image analysis, one can observe, analyze and derive objectively calculated parameters from composite digital images: a novel technology termed high density nematode screening. We developed this technology platform for microtiterplate in vivo nematode phenotype drug screening, using a transformed C. elegansstrain, expressing GFP in the vulval myoblasts under the control of the ceh-25 promoter. The vulval myoblasts were previously shown to be dependent on steerin (unc-53) for their correct positioning in larval development (Bogaert, unpublished). The screening is run on an inverted fluorescence microscope, equipped with an automated (XYZ) stage and an intensified camera. SCIL-image software (University of Amsterdam) running on Silicon Graphics unix was used to control the stage. The entire plate was scanned using automated focusing (patent pending), seamless image grabbing and generation of composite images. Worm images are picked semi-automated, and subsequently analyzed automatically for quantification of the wild type or compound-induced phenotype. Using this assay, a lead compound previously shown active in agar plate cultures was confirmed and novel compounds discovered as in vivo inhibitors of the steerin signaling pathway. The activity of this compound has been confirmed using mouse neuroblastoma cells overexpressing nematode steerin. Computer simulations using the crystal structure of GRB-2 C-terminal SH3-domain suggest that this compound may be a diproline mimetic capable of inhibiting SH3-binding to proline-rich helices.

K Ver Donck et al. (1999) International C. elegans Meeting "'High density C. elegans screening': a drug discovery platform applied to the steerin (unc-53) pathway"

P Van Osta, K Ver Donck, L Bols, I Roelens, T Bogaert, J Geysen, I Mailliet

[International C. elegans Meeting, 1999]

Green fluorescent protein (GFP) technology enables cellular resolution in the living animal. When combined with light sensitive cameras and with up-to-date automated stage driving, image acquisition and image analysis, microscopists can observe, analyse and derive objectively calculated parameters from composite digital images: a novel technology termed 'high density nematode screening'. We developed this technology platform for 96-well plate in vivo nematode phenotype drug screening, using a transformed C. elegans strain, expressing GFP in the vulval myoblasts under the control of the ceh-25 promoter. The vulval myoblasts were previously shown to be dependent on steerin (unc-53) for their correct positioning in larval develpment (Bogaert, unpublished). The screening was run on an inverted fluorescence microscope, equipped with an automated (XYZ) stage and an intensified camera. SCIL-image software (University of Amsterdam) on Silicon Graphics unix was used to write programs for stage driving, automated focussing (patent pending), seamless image grabbing, generation of composite images and semi-automated image picking for visual analysis of the wild type or compound-induced phenotype. Using this screening assay, a lead compound previously shown active in agar plate cultures was confirmed and novel compounds discovered as in vivo inhibitors of the steerin signaling pathway. The activity of this compound has been confirmed using mouse neuroblastoma cells overexpressing nematode steerin. Computer simulations using the crystal structure of GRB-2 C-terminal SH3-domain suggest that this compound may be a diproline mimetic capable of inhibiting SH3-binding to proline-rich helices.

Goh P-Y et al. (1991) International C. elegans Meeting "PHENOTYPIC AND GENETIC CHARACTERIZATION OF THE MUP-1 GENE AND MOLECULAR CHARACTERIZATION OF MUP-2(e2346)."

Goh P-Y, Bogaert T

[International C. elegans Meeting, 1991]

Mup mutants are larval lethals with permanent bends and kinks in the body due to ectopic body wall musde attachments and/or peeled off musde quadrants. We isolated 10 alleles of the mup-l gene, including putative null alleles. The body wall musdes of bean to comma mup-l embryos have a wild type myofilament pattern but may extend ectopic processes. Later in embryogenesis some body wall muscdes detach from the hypodermis. Genetic analysis suggests that mup-l has both a maternal and zygotic component and is not required for postembryonic muscle growth and attachment. Hypomorphic mup-l alleles are suppressed by several dpy mutations and unc-52, suggesting the mup-l product may be a component of or interact with the cell surface / extracellular matrix. (see Goh & Bogaert, Development, in press 1991) So far one only one temperature sensitive mup-2 allele has been isolated. Genetic characterization suggests it is a strong hypomorph. The temperature sensitive period (tsp) for the body wall muscle phenotype of mup-2(e2436ts) is in the last three quarters of the threefold stage and ends at hatching. Mup2(e2346ts) is also required for oviduct function, probably to squeeze oocytes from the oviduct into the spermatheca. mup-2(e2346ts) was mapped relative to RFLP's in wild type strains and narrowed down to a 8.6 kb region by transformation rescue. We sequenced a 1.5 kb cDNA that maps within this fragment. The ORF in this cDNA shows 38 % identity over 316 amino acids with Drosophila troponin-T. Troponin and tropomyosin comprise an actin filament associated complex that regulates actomyosin interactions in response to free calcium. We still lack the amino-terminus of the putative mup- 2 protein. We do not know yet whether mup2 encodes C. elegans troponin- T or a related molecule.

N Pujol et al. (1999) International C. elegans Meeting "Axonal pathfinding defects in CePhox2/ceh-17 mutants"

JF Brunet, J Ewbank, N Pujol, R Terranova

[International C. elegans Meeting, 1999]

We are studying the C. elegans homologue of two vertebrate prd-like homeobox genes, Phox2a and Phox2b, involved in neuronal-subtype specification (1,2,3,4). The predicted amino acid sequence of CEH-17 is 88% identical to murine Phox2 proteins within the homeodomain, a level of similarity often associated with functional conservation between homeobox genes of vertebrates and invertebrates. An antibody raised against the C-terminus of CEH-17 reveals an expression restricted to 5 neurons located in the head ganglia: ALA and 4 ventral neurons tentatively identified as SMB. Interestingly, all those neurons send posteriorly directed axons along the lateral and sublateral cords. The expression is strongest from the 3-fold stage to L1 and decreases thereafter to a faint level in adults. In addition, different GFP fusions show a variable expression in other neurons not substantiated by immunohistochemistry and whose significance is therefore unclear. We have isolated a deletion allele in ceh-17 . The 1.3 kb deletion extends from 0.6 kb upstream of the transcription start site to the first helix of the homeodomain and is thus expected to create a null allele. Indeed, immunostaining of the mutant reveals no CEH-17 expression. The hermaphrodite and male homozygous mutants are viable and fertile. Crosses between the mutant strain and a strain in which GFP is expressed under the control of a unc-53 promoter (see abstract by Pujol and Bogaert) allowed ALA axonal pathfinding to be assessed in the mutant. In the wild type, the two ALA axons migrate on the lateral cords as far as the tail, beginning at the 3 fold stage until early L1 stage (i.e. a period corresponding to the strongest expression of CEH-17). In the ceh-17 mutant, the axons of 80% of the scored ALA neurons stop at the level of the vulva. In 5% of the cases, the axon stop at the vulva but then returns half way along the same lateral cord. Therefore, CePhox2/ceh-17 is required for the posteriorly directed growth of ALA axons in the second half of their trajectory, from the gonad to the tail. We are now ectopically expressing CEH-17 and assessing the functional conservation of CEH-17 and murine Phox2. (1) Morin et al. 97 Neuron 18 411-423 (2) Pattyn et al. Nature 99 in press (3) Ewbank et al. 98 WBG 15,3:17 (4) Pujol et al. 99 WBG 15,5:29

Ralph Menzel et al. (2003) International Worm Meeting "Xenobiotically induced cytochrome P450 genes in Caenorhabditis elegans: GFP-fusions and embryonic lethality by RNAi."

Ralph Menzel, Ruth Boose, Sarah Forster

[International Worm Meeting, 2003]

Cytochromes P450 (CYP) are a super family of heme containing and NADPH dependent monooxygenases which catalyze the oxidative metabolism of many exogenous and endogenous compounds. While the investigation of CYP induction pathways in mammals has made considerable progress, very little is known about P450s from invertebrates, such as the nematode Caenorhabditis elegans. C. elegans possesses, however, a remarkably high number of 80 different cytochrome P450 genes. The functions of the encoded proteins are almost unknown. In a previous study it could be demonstrated that the five different CYP forms 35A1-A5 are strongly inducible by PAHs, PCBs and different drugs [1]. In order to study this xenobiotically induced gene expression different transgenic C. elegans lines expressing GFP under control of the CYP35A1-5 promoter regions were constructed. With this tool it was possible to follow the GFP production in vivo depending on the applied inducer. Induction with -naphthoflavone resulted in all cases in an expression of the green fluorescent protein exclusively in the worm intestine, identifying this compartment as the likely tissue of P450 35A localization. For the CYP35A2 and A5 forms promoter-deletion experiments were performed revealing a 400 bp and a 250 bp fragment, respectively, to be sufficient to induce GFP production. While the 35A2 promoter caused GFP production in all developmental stages, the 35A5 based gene expression was mainly detectable in eggs and in L1/L2 larvae. In particular in case of the 35A2 promoter region a striking correspondence to mammalian xenobiotic response elements (XRE) has been found. To obtain more information about the biological functions of the five different CYP35A forms dsRNA producing E. coli strains for RNAi by feeding experiments were produced. Surprisingly, embryonic lethality was observed as the loss of function phenotype for all CYP35A forms with exception of 35A2. This might indicate that at least one or more CYP35A forms play an essential role in the embryogenesis of C. elegans. Using semi-quantitative RT-PCRs the cross-reactivity level of the individual RNAi constructs was determined. Future efforts will be focused to characterize the blocked embryonic development more in detail.[1] Menzel R., Bogaert T. and Achazi A. (2001) A systematical gene expression screen of the Caenorhabditis elegans cytochrome P450 genes revealed CYP35 as strong xenobiotic inducible. Arch. Biochem. Biophys. 395, 158-168.