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]

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.

Maillet I et al. (2000) European Worm Meeting "Identification of new genes involved in growth cone steering of the excretory canal"

Roelens IG, Maillet I, Bogaert T

[European Worm Meeting, 2000]

Cell migrations are an essential part of the development of any multicellular organism, but the underlying genetic and biochemical signalling pathways still are poorly understood. We use the excretory cell of the nematode Caenorhabditis elegans as a model to study both dorso-ventral and antero-posterior migrations. This cell sends out two lateral projections, which, after growing along the D-V axis, each split to form an anterior and a posterior branch (the excretory canals). We analysed the excretory canals in the strains containing unc-53(n152) , unc-71(e541)and unc-5(e53) as well as in all double and the triple mutant combinations. We observed a strong mutual enhancement of the outgrowth of ectopic ventral excretory canals caused by mutations in these genes. Figure 1: Percentages represent the amount of ectopic ventral canals seen in the different mutant backgrounds, lines stand for multiple mutant strains. We can conclude that these three genes are representative for different pathways playing a role in D-V migration. Based on these data, we designed a genetic screen for mutants with ventral excretory canals in the unc-53 background. Up to now, we isolated 8 mutants, of which 5 have been mapped. bg5 maps to the left arm of chromosome II, bg2, bg4and bg6 map to the right arm of chromosome III and bg10 maps to chromosome IV. Thereby they define at least three genes which we are currently analyzing.

Roelens IG et al. (1998) European Worm Meeting "Analysis of Growth cone steering of excretory canal"

Maillet I, Bogaert T, Roelens IG

[European Worm Meeting, 1998]

Migratory growth cones read guidance cues in both dorso-ventral and antero-posterior axes (e.g. the excretory canals, neuronal axons). The goal of this project is to disover how the many different signals are integrated and translated to the final direction a growth cone follows. The excretory cell is an excellent model to study both A-P and D-V migrations as it sends out 4 projections that make their trajectory along both axes. From the phenotype of unc-53 mutants, which stop posterior excretory canal outgrowth near the vulva, we know that unc-53 interferes with antero-posterior growth cone steering. unc-53 also plays a role in the integration of D-V signals, since mutants with ventral excretory canal outgrowth are strongly enhanced in an unc-53 background, whereas there is no enhancement of the A-P migration. To identify new components of the pathways involved in the integration of both types of signals, we designed a genetic screen using an unc-53 mutant. This provides us with a sensitized background to find animals with excretory cells that send out ventral projections. One mutation, bg2, has no canal phenotype by itself in the unc-53 background. From the other mutants we isolated, we choose the four with the highest penetrance, bg4 to bg7, and are currently analyzing those.

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.

Allen TS et al. (1995) International C. elegans Meeting "Analyses of Troponin T Mutations in C. elegans."

Myers CD, Goh P-Y, Bogaert T, Bucher EA, Allen TS

[International C. elegans Meeting, 1995]

To elaborate the mechanisms by which troponin T (TnT) functions in the assembly and contraction of developing and mature muscle, we are studying a TnT homolog in C. elegans that is encoded by the mup-2 gene locus. mup-2 is defined by a heat sensitive allele, designated e2346ts, and by an unconditional allele, up1, believed null. The e2346ts mutation creates a premature stop at codon 342, eliminating 64 residues. The up1 mutation creates a premature stop at codon 94, preventing expression of the regions homologous to the functional domains of vertebrate TnT. Both alleles cause muscle positioning defects (Mup) during embryogenesis and result in kinked, dead larvae. Because mutants for these alleles can show muscular contraction prior to death, the embryonic phenotype is distinct from the paralysed, arrested elongation at two-fold (Pat) phenotype shown by worms with mutations in genes identified tentatively as encoding troponin C and tropomyosin (Williams and Waterston. J. Cell Biol. 124:475, 1994), two proteins that interact with TnT. Our working hypothesis is that the putative tropomyosin and troponin C mutations prevent activation (disinhibition) of thin filaments and thus block muscular contraction. Conversely, the mutations in TnT impair inhibition (thin filaments are "unregulated") and thus allow aberrant force production, causing tearing of the dorsal muscle quadrants ventrally. mup-2 also has larval and adult functions. When e2346ts mutants are shifted from 15 C to 25 C after embryogenesis, the muscle quadrants remain properly positioned, but the worms tremble when placed in liquid solution. The trembling suggests dysfunctional contraction/relaxation cycles, in line with our working hypothesis. Additionally, the temperature-shifted hermaphrodites are sterile: oocytes accumulate in the oviduct, presumably because contraction of the oviductal myogenic sheath is defective. Our analysis also shows defects in migration of the gonadal sheath cells to the distal gonadal arms. A function of TnT in cell migration and in nonstriated tissue contraction is surprising, because vertebrate TnT is found exclusively in striated muscle; however, in situ hybridization studies support that mup-2 is expressed in these cells. Genetic interaction studies, site-directed mutagenesis, and physiological assays that we are developing will contribute towards defining the in vivo functions of TnT.

Sara Maxwell et al. (2002) European Worm Meeting "Functional analysis of the C. elegans T-box gene family"

Alison Woollard, Roger Pocock, Lizzie Clarke, Sara Maxwell

[European Worm Meeting, 2002]

T-box genes are a group of developmentally important transcription factors united by a common DNA binding domain. T-box genes are present in all metazoan species so far analysed but are absent from yeast. There are 20 T-box genes in C. elegans, more than twice the number found in Drosophila. Many of the C. elegans T-box genes are highly diverged from those found in other species while others have clear orthologues present throughout the metazoan kingdom. One highly conserved T-box gene is mab-9, a member of the tbx20 sub-family1. This was the first C. elegans T-box gene to be identified by mutation and is required for cell fate specification during hindgut and male tail development, and aspects of nervous system function. One other conserved T-box gene has recently been reported to be important for a particular muscle cell fate specification2. We have inactivated the remaining C. elegans T-box genes by RNAi and have found obvious phenotypes only in very few cases. These phenotypes include embryonic lethality, L1 lethality, and a Dpy phenotype with weakly penetrant male tail defects, and will be described in detail. The remaining T-box genes give no obvious phenotype by RNAi. Phylogenetic analysis reveals that several pairs of T-box genes are very similar to eachother and are therefore likely to be the result of recent duplications. This might suggest functional redundancy. Double RNAi experiments have revealed this to be the case with at least two of the T-box gene pairs (see also poster by Pocock et al). Study of the expression patterns of the whole T-box family may suggest other potential redundancy relationships which can be explored by RNAi. Comparison of the C. elegans T-box genes with the set of T-box genes now defined for C. briggsae is being used as a tool for defining potentially important regulatory regions present in orthologous genes.

Pulak RA et al. (1983) International C. elegans Meeting "the 1983 genetic map of Panagrellus redivivus."

Pulak RA, Bogaert T, Madrid A, Ager D, Samoiloff MR

[International C. elegans Meeting, 1983]