-
[
Worm Breeder's Gazette,
1992]
One question we are interested in, is, whether a fixed polarity is already present in the unfertilized egg of the nematode as described for other systems. In order to investigate this question we have developed a technique to fuse oocytes with a laser microbeam. Generally, the most distal oocytes (positioned closest to the spermatheka) are suited best for such a manipulation. Giant oocytes generated this way can be fertilized. So far, we have succeeded to obtain giant embryos derived from a fusion of 2 or of 3 oocytes. Not all fused oocytes become fertilized or initiate cleavage. So far, in 37 cases giant zygotes derived from 2 oocytes were found to start cleavage. All of them expressed a normal early cleavage pattern with the generation of 6 lineages. The vast majority of such embryos arrested as monsters, most of them with signs of tissue-specific differentiation However, 20% (8/37) developed into normal-looking giant juveniles [See Figure 1, B1 -B5].Most of these (6/8) reached the adult stage with essentially normal properties but having approximately twice the volume of a regular worm They reproduced with a low (15% of normal) brood size (reduced number of eggs produced; frequent embryonic arrest) with the fraction of males much higher than normal (up to 30%). During subsequent generations the number of males returned to the normal percentage (ca 0.2%). These results show that the uncleaved egg can regulate to form a (nearly) normal animal from 2 oocytes They indicate that no fixed polarity and prelocalization of cytoplasmic determinants are present in the oocyte. The high number of males and low fecundity appears to be due to triploidy and aneuploidy as observed by others (see Bob Herman, worm book). After fusion of 3 oocytes (C1) we observed 3 different pathways of development (C-E). In two thirds of the cases (23/33) the giant oocyte was fragmented into a smaller anucleate and a larger triploid part. The anucleate fragment could be fertilized and develop as an apparently haploid embryo with a normal early pattern to eventually arrest during late embryogenesis (C2-C5), often quite normal morphogenesis in the head region (10/23) and terminal cell differentiation (muscle twitching, gut birefringence). The triploid part usually lysed during the passage through the spermatheka In no case it started cleavage. Alternatively, such a super-giant oocyte could be fertilized in total and initiate normal early cleavage with the complete series of unequal germline divisions (5/33). So far, in all observed cases of this type embryos arrested before the beginning of gastrulation (< 30 cells) and often lysed, apparently due to a weak eggshell ( D2 -D5).Despite the arrest, these findings show that even a zygote derived from 3 oocytes can regulate to behave like a normal egg. Some eggs derived from 3 oocytes behaved quite differently (E2-E5). Pronuclei (a single one at one pole, two at the other pole) approached each other from the poles and both formed a cleavage spindle along the anterior-posterior axis of the egg resulting in 2 small cells at the ends and a large cell in the center (5/33). With the next division the large central cell formed a tetrapolar spindle and generated 4 blastomeres of similar size and future AB-behavior. The two small cells at the poles performed a series of germline-like, unequal cleavages like a P, cell Our interpretation that this represents a development of twins fused at their anterior (AB) ends is supported by the generation of 2 distinct E-cell groups with gut-specific birefringence (ES). So far, we consider 2 explanations for this unexpected finding: a the maternal centrosome has not been inactivated in these eggs and forms a second cleavage spindle together with the centrosome introduced by the sperm. However, so far no case has been reported that cleavage can be initiated in unfertilized eggs of C.e. or after removal of the sperm centrosome (Schierenberg and Wood; Dev. Biol 107, 337, 1985). Additionally, it is difficult to understand why a posterior pole is established under these conditions where normally the anterior pole would be. Therefore, we favor explanation b.: Because of the large egg size the assumed polyspermy-block initiated by the entrance of a sperm at the pole entering the spermatheka first is not completed (fast enough?) allowing the entrance of a second sperm at the other pole. If this assumption is true, it could mean that it is the entrance of a sperm which generates a posterior pole. Presently, we try to test whether a second sperm has been introduced in this type of egg If anyone has a smart idea (e.g. in-vivo marking of sperm) please contact us (E-mai} address: ESchier@Biolan Uni-Koeln De; FAX: 049-221-470-3109). Experiments to fuse oocytes of giant nematodes to generate even larger worms and fuse their oocytes etc. are under consideration. Donations of giant petri dishes are welcome.
-
[
International C. elegans Meeting,
1993]
-
[
Dev Genes Evol,
2002]
With their first cleavage blastomeres in Caenorhabditis elegans are fixed to very different developmental programs going along with differential segregation of maternal gene products. To investigate whether indications for a prelocalization of cytoplasmic components can already be found in unfertilized egg cells, we fused mature C. elegans oocytes with the help of a laser microbeam. Fertilization of two fused oocytes resulting in triploid zygotes showed an essentially normal early cleavage pattern with the establishment of five somatic cell lineages and a germline and also a normal spatial arrangement of blastomeres. A considerable fraction of such embryos hatched and developed into fertile giant nematodes. The numbers of cell nuclei in freshly hatched and adult giant animals were found to be essentially the same as in untreated controls. When three fused oocytes were fertilized, two alternative patterns of early embryogenesis were observed. Half of the embryos followed the normal cleavage mode. The other half, however, developed in a twin-like fashion with all cells present in two copies, apparently due to fertilization by two sperm. In such embryos, two areas of gastrulation were established, resulting in the generation of two separate gut primordia. In summary, our results suggest that (1) in contrast to the uncleaved zygote in the mature oocyte of C. elegans no cytoplasmic regionalization exists, (2) the invariable cell numbers typical for the C. elegans embryo are not controlled via cell size, and (3) the entry of a second sperm can induce a cascade of events in the egg leading to the formation of two complete embryo anlagen.
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[
J Neurosci,
2003]
Thermotactic behavior in Caenorhabditis elegans is sensitive to both a worm's ambient temperature (T-amb) and its memory of the temperature of its cultivation (T-cult). The AFD neuron is part of a neural circuit that underlies thermotactic behavior. By monitoring the fluorescence of pH-sensitive green fluorescent protein localized to synaptic vesicles, we measured the rate of the synaptic release of AFD in worms cultivated at temperatures between 15 and 25degreesC, and subjected to fixed, ambient temperatures in the same range. We found that the rate of AFD synaptic release is high if either T-amb > T-cult or T-amb > T-cult, but AFD synaptic release is low if T-amb congruent to T-cult. This suggests that AFD encodes a direct comparison between T-amb and T-cult.
-
[
Trends Mol Med,
2007]
Transforming growth factor beta1 (TGFbeta1), an important pleiotropic, immunoregulatory cytokine, uses distinct signaling mechanisms in lymphocytes to affect T-cell homeostasis, regulatory T (T(reg))-cell and effector-cell function and tumorigenesis. Defects in TGFbeta1 expression or its signaling in T cells correlate with the onset of several autoimmune diseases. TGFbeta1 prevents abnormal T-cell activation through the modulation of Ca(2+)-calcineurin signaling in a Caenorhabditis elegans Sma and Drosophila Mad proteins (SMAD)3 and SMAD4-independent manner; however, in T(reg) cells, its effects are mediated, at least in part, through SMAD signaling. TGFbeta1 also acts as a pro-inflammatory cytokine and induces interleukin (IL)-17-producing pathogenic T-helper cells (T(h) IL-17 cells) synergistically during an inflammatory response in which IL-6 is produced. Here, we will review TGFbeta1 and its signaling in T cells with an emphasis on the regulatory arm of immune tolerance.
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[
Genomics,
1995]
Recently, a novel family of genes with a region of homology to the mouse T locus, which is known to play a crucial, and conserved, role in vertebrate development, has been discovered. The region of homology has been named the T-box. The T-box domain of the prototypical T locus product is associated with sequence-specific DNA binding activity. In this report, we have characterized four members of the T-box gene family from the nematode Caenorhabditis elegans. All lie in close proximity to each other in the middle of chromosome III. Homology analysis among all completely sequenced T-box products indicates a larger size for the conserved T-box domain (166 to 203 residues) than previously reported. Phylogenetic analysis suggests that one C. elegans T-box gene may be a direct ortholog of the mouse Tbx2 and Drosophila omb genes. The accumulated data demonstrate the ancient nature of the T-box gene family and suggest the existence of at least three separate T-box-containing genes in a common early metazoan ancestor to nematodes and vertebrates.
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[
Glycobiology,
2006]
The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-Gal:GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122)(2). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by PCR using a C. elegans cDNA library as the template, contains 1,170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type-II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has 7 Cys residues in the lumenal domain including 6 conserved Cys residues in all of the orthologs. The Ce-T-synthase has 4 potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search and it contains 8 exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein constructs show that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc, and might require invertebrate-specific factors for formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and opens new avenues to explore O-glycan function in this model organism.
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[
Int J Syst Evol Microbiol,
2007]
A yellow-pigmented, Gram-positive, aerobic, non-motile, non-spore-forming, irregular rod-shaped bacterium (strain TAN 31504(T)) was isolated from the bacteriophagous nematode Caenorhabditis elegans. Based on 16S rRNA gene sequence similarity, DNA G+C content of 69.5 mol%, 2,4-diaminobutyric acid in the cell-wall peptidoglycan, major menaquinone MK-11, abundance of anteiso- and iso-fatty acids, polar lipids diphosphatidylglycerol and phosphatidylglycerol and a number of shared biochemical characteristics, strain TAN 31504(T) was placed in the genus Leucobacter. DNA-DNA hybridization comparisons demonstrated a 91 % DNA-DNA relatedness between strain TAN 31504(T) and Leucobacter chromiireducens LMG 22506(T) indicating that these two strains belong to the same species, when the recommended threshold value of 70 % DNA-DNA relatedness for the definition of a bacterial species by the ad hoc committee on reconciliation of approaches to bacterial systematics is considered. Based on distinct differences in morphology, physiology, chemotaxonomic markers and various biochemical characteristics, it is proposed to split the species L. chromiireducens into two novel subspecies, Leucobacter chromiireducens subsp. chromiireducens subsp. nov. (type strain L-1(T)=CIP 108389(T)=LMG 22506(T)) and Leucobacter chromiireducens subsp. solipictus subsp. nov. (type strain TAN 31504(T)=DSM 18340(T)=ATCC BAA-1336(T)).
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[
Genome,
1997]
The T-box gene family consists of members that share a unique DNA binding domain. The best characterized T-box gene, Brachyury or T, encodes a transcription factor that plays an important role in early vertebrate development. Seven other recently described mouse T-box genes are also expressed during development. In the nematode Caenorhabditis elegans, four T-box genes have been characterized to date. In this study, we describe three new C. elegans T-box genes, named
Ce-tbx-11,
Ce-tbx-12, and
Ce-tbx-17.
Ce-tbx-11 and
Ce-tbx-17 were uncovered through the sequencing efforts of the C. elegans Genome Project.
Ce-tbx-12 was uncovered through degenerate PCR analysis of C. elegans genomic DNA.
Ce-tbx-11 and
Ce-tbx-17 are located in close proximity to the four other previously described T-box genes in the central region of chromosome III. In contrast,
Ce-tbx-12 maps alone to chromosome II. Phylogenetic analysis of all known T-box domain sequences provides evidence of an ancient origin for this gene family.
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[
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.