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]

N Pujol et al. (1999) International C. elegans Meeting "unc-53 expression pattern and phenotype"

N Pujol, T Bogaert

[International C. elegans Meeting, 1999]

We have characterised the expression of unc-53 using a GFP reporter strategy. The unc-53 gene spans over 30 kb, contains very large introns in its 5' region, and has several SL1-spliced transcripts. We identified two different promoter regions, each associated with a distinct SL1. Each promoter element leads to GFP expression in a subset of pioneer neurons, sex muscles, sensory socket cells, distal tip cells and the excretory cell. The expression begins at the early comma stage and is present during process outgrowth. We found an expression in a majority of the cells known to be affected in unc-53 mutants (e.g. DA and AS motoneurons, vulval, diagonal and spicule retractor muscles, the excretory cell) suggesting that the observed expression patterns accurately reflect the in vivo expression pattern of the gene. During the morphogenesis of the sex muscles, we observed that in wild type, while the sex myoblasts undergo their three divisions, they migrate longitudinally, sending longitudinally processes along the edge between the seam and muscles to reach the hypodermis. Those processes could serve to sense migration cues. In unc-53 mutants, these structures don't form and the A/P migration is dramatically reduced. Moreover, while in unc-53 mutants the mature vulval muscles have an organized and attached myofilament cytoskeleton, they make a morphologically different adhesive structure with the hypodermis, characterized by a rounded appearance. We used the defined promoters fused to the cDNA of unc-53 to construct minigenes directing the expression of unc-53 in specific cells. In unc-53 mutants, the axons of the paired neurons ALN, which normally run along the entire animal on the sub-lateral cords, stop before the vulval region and turn to join the dorsal cord. By expressing UNC-53 in those neurons in the mutant, we were able to rescue the mutant phenotype, suggesting that unc-53 acts cell autonomously in those cells. Moreover, when we over-expressed UNC-53 in the mec cells, which are normally bipolar, these neurons send ectopic filopodia-like outgrowths in different directions. Together with our previously reported work, these data suggest that expression levels or activation of the UNC-53 protein must be highly regulated to set up the normal direction and levels of outgrowth.

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.

IG Roelens et al. (1999) International C. elegans Meeting "New mutations involved in growth cone steering of the excretory cell"

IG Roelens, I Maillet, R Feichtinger, T Bogaert

[International C. elegans Meeting, 1999]

During the development of an organism, migratory growth cones encounter guidance cues for both dorso-ventral and antero-posterior axes (e.g. the excretory canals, neuronal axons). The excretory cell is an excellent model to study both D-V and A-P migrations. This cell sends out two lateral projections, which, after growing along the D-V axis, split to form an anterior and a posterior branch (the excretory canals). The phenotype of these canals in unc-53 , unc-5 and unc-71 mutants shows that they are very sensitive to changes in the signaling pathways responsible for directional migration. Based on the results of prior genetic and molecular research, we propose the following model for the migration of the excretory canals. 1. together with unc-5 and unc-71 , unc-53 is important for the dorso-ventral migration of the lateral branches of the excretory canals (Buesa et al., EWM 1996) 2. for the migration from the lateral branching point to the vulva the excretory canal integrates a gonad-dependent signal as well as a signal from the unc-53 pathway (Chen et al.,1997, Dev Biol 182, 88-100) 3. unc-53 is needed for the migration of the excretory canals from the vulva into the tail To identify new components of the pathways involved in the integration of dorso-ventral signals, we designed an F2 screen in an unc-53 mutant. This provides us with a sensitized background to find animals with excretory cells that send out ectopic ventral projections. Up to date, we isolated seven mutants. Three mutants ( bg2 , bg4 and bg6 ) map on the right arm of chromosome III. We are performing complementation analysis with these mutants. We are also performing PCR-based mapping of two more mutants, one of which lies on the left arm of chromosome II( bg5 ). 1 as research assistant at the Fund for Scientific Research-Flanders

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.

Hisako Takeshita et al. (2005) International Worm Meeting "Direction of cell polarity is determined by Wnt in C.elegans"

Hisako Takeshita, Hitoshi Sawa, Kota Mizumoto

[International Worm Meeting, 2005]

Asymmetric cell division is a fundamental process that produces cellular diversity during development. In C. elegans, asymmetric divisions of many cells are regulated by Wnt signaling. In particular, asymmetry of the T cell division is regulated by LIN-44/Wnt and the LIN-17/Frizzled receptor. LIN-44/Wnt is expressed in hypodermal cells at the tip of the tail, posterior to the T cell. In lin-44 mutants, the polarity of the T cell division is frequently reversed, while in lin-17 mutants, the division is symmetric. It has been proposed that LIN-44 activates the LIN-17 receptor on the posterior side of the T cell to polarize it. To determine whether LIN-44/Wnt acts as an asymmetric cue that polarizes the T cell, we ectopically expressed LIN-44 in cells anterior to the T cell using the egl-5 promoter (egl-5::LIN-44) to find that polarity reversal phenotype of lin-44 was strongly enhanced. Moreover lin-17 was required for egl-5::LIN-44 to reverse the T-cell polarity. These results suggest that LIN-44 polarize the T cell through the LIN-17 receptor. To further confirm that LIN-44 directly acts on LIN-17 in the T cell, we analyzed the localization of LIN-17::GFP fusion proteins expressed by the seam cell specific promoter (SCM::LIN-17::GFP). This construct rescued defects of lin-17 mutants in the T cell division, indicating that lin-17 functions cell-autonomously in the T cell. We found that LIN-17 localization was localized in the posterior cortex of the T cell in wild type. In lin-44 mutants, the LIN-17 localization was uniform on the cortex. Furthermore, egl-5::LIN-44 reversed the LIN-17 localization to the anterior cortex. These results strongly indicate that LIN-44 directly acts on LIN-17 in the T cell to orient the polarity.

Hryshkevich, Uladzislau et al. (2011) International Worm Meeting "Core promoter T-blocks correlate with gene expression levels in C. elegans."

Yanai, Itai, Hryshkevich, Uladzislau, Hashimshony, Tamar

[International Worm Meeting, 2011]

Core promoters mediate transcription initiation by the integration of diverse regulatory signals encoded in the proximal promoter and enhancers. It has been suggested that genes under simple regulation may have low-complexity permissive promoters. For these genes, the core promoter may serve as the principal regulatory element; however the mechanism by which this occurs is unclear. We report here a periodic poly-thymine motif, we term T-blocks, enriched in occurrences within core promoter forward strands in C. elegans. An increasing number of Tblocks on either strand is associated with increasing nucleosome eviction. Strikingly, only forward strand T-blocks are correlated with expression levels, whereby genes with ³6 T-blocks have five fold higher expression levels than genes with 3 T-blocks. We further demonstrate that differences in T-block numbers between strains predictably affect expression levels of orthologs. Highly expressed genes and genes in operons tend to have a large number of T-blocks, as well as the previously characterized SL1 motif involved in trans-splicing. The presence of T-blocks thus correlates with low nucleosome occupancy and the precision of a trans-splicing motif, suggesting its role at both the DNA and RNA levels. Collectively our results suggest that core promoters may tune gene expression levels through the occurrences of T-blocks, independently of the spatio-temporal regulation mediated by the proximal promoter.

TM Morse et al. (1999) International C. elegans Meeting "Time-series analysis of pirouettes in C. elegans chemotaxis."

TM Morse, JT Pierce, SR Lockery, ML Moravec, TC Ferree, SE Owens, AB Bates

[International C. elegans Meeting, 1999]

Chemotaxis in C. elegans involves a series of abrupt turns (pirouettes) triggered by movement down a gradient of chemical attractant * . Analysis of the time series of concentration change experienced by a worm, together with its pirouette record, suggests a three-stage chemotaxis mechanism in which chemical concentration (C(t)) is differentiated (dC(t)/dt), smoothed (q(t)), and converted into pirouette probability by a nonlinear function (P(q[t])). To test the plausibility of this mechanism, we constructed a computer model in which the smoothing filter and the nonlinearity were estimated from the time series of dC(t)/dt and the pirouette records of real worms. Pirouettes were modeled by sampling randomly, via a Poisson process with probability P(q[t]), from the distribution of direction changes associated with pirouettes in real worms. The average chemotaxis index (time-average of normalized C(t)) of model worms (0.47 +/- 0.05 SD, n = 2000) closely matched the average chemotaxis index of real worms (0.45 +/- 0.04 SD, n = 45), indicating that the three-stage mechanism is quantitatively sufficient to account for C. elegans chemotaxis. To determine the form of the smoothing filter and nonlinearity directly, we have devised a new behavioral assay that subjects unrestrained worms to negative-going impulses in dC/dt as they swim across a sharp border between high and low concentrations of attractant. Preliminary results show that immediately after a border crossing, large impulses make P(t)~= 1, while small impulses make 0 t) >< C. elegans . * Pierce, J.T., and Lockery, S.R., J. Neurosci. (Submitted)