Egg development in parthenogenetic nematodes: variations in meiosis and axis formation

In the well studied model nematode Caenorhabditis elegans entrance of the sperm induces an anterior-posterior polarity in the egg and determines the orientation of the primary embryonic axis. Subsequently, fusion of two haploid gamete nuclei results in a diploid zygote as a prerequisite for normal embryogenesis. Here we analyze the establishment of embryonic polarity and diploidy in the absence of sperm in three parthenogenetic nematode species from three different families, Diploscapter coronatus (Diploscapteridae), Acrobeloides nanus (Cephalobidae) and Plectus sp. (Plectidae). We find that they not only differ from C. elegans in these two aspects but also from each other, indicating variant solutions for the same developmental challenges and supporting the view that the parthenogenetic mode of reproduction has been acquired multiple times independently.     

Developmental potential of fused Caenorhabditis elegans oocytes: generation of giant and twin embryos

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.     

Functional morphology and evolutionary origin of the three-part pharynx in nematodes

The distinct morphological regions of the typical tripartite pharynx found in the nematode taxon Secernentea have distinctive functions. Besides the basic functions of sucking and pumping food against the pressure in the body cavity, the pharynx of Secernentea such as rhabditids serves two additional functions restricted to two pharyngeal subunits. The corpus traps bacteria behind the stoma and at its posterior end. The newly discovered pharyngeal pocket valve helps to trap particles behind the corpus in the rhabditid Poikilolaimus oxycercus and the cephalobid Acrobeles ciliatus (both Secernentea). The grinder of the terminal bulb serves for chewing trapped bacteria. The separated sites of trapping and chewing are connected by the isthmus that transports bacteria towards the grinder. It is likely that this complex feeding structure originated step by step from a two-part pharynx comprising a propharynx and the terminal bulb as in "Plectidae" (that probably include the closest relatives of the Secernentea within the "Adenophorea"). Analysis of video sequences of feeding rhabditids and plectids provided new data to reconstruct this transformation. Within the "Plectidae" two types of grinders occur. The first type or "parietinus type" has triangular chewing plates that can bulge medially and crush food particles. When they retract, new ingested particles are drawn into the grinder. The second type with more solid chewing plates called "butterfly valves" occurs in Ceratoplectus, Plectus parvus, and Wilsonema and can be homologized with the grinder in Secernentea ("Plectidae" is a paraphyletic taxon). Because butterfly valves cannot be retracted, the evolution of such valves required the evolution of an alternative mechanism to fill the grinder with bacteria. The differentiated closing pattern of the dilated pharynx lumen in Ceratoplectus, Plectus parvus, and Wilsonema can be interpreted as the first step in the development of a functional separation of trapping bacteria and of transporting them towards the grinder, which led to the morphologically discernible units of corpus and isthmus found in the Secernentea.     

Characteristics of the ovum cleavage and the importance of the preblastula phase in the embryogenesis of the Nematoda

The figure of tetrahedron is formed in certain species of Plectus and in Tobrilus gracilis at the stage of 4 blastomeres rather than a rhombus which is formed in most highly organized nematodes. The analysis of the Nematoda's embryogenesis allows to conclude that tetrahedron, rhombus as well as some other figures play the role of preblastula sustaining the most expedient disposition of the first blastomers for transition to the formation of the blastula. With the increasing organization of nematodas the tetrahedron preblastula turns into a rhombic, linear-rhombic and at last in aphelenchoid-tylenchoid one. The character of the distribution of structural elements of organs and tissues of the definitive animal in the cytoplasm of the egg of Plectus and Tobrilus confirms the rightness of the division of the class of nematodas into subclasses Enoplia and Chromadoria rather than subclasses Adenophorea and Secernentea.     

Embryonic gut differentiation in nematodes: endocytosis of macromolecules and its experimental inhibition

During embryogenesis of Caenorhabditis elegans cytoplasmic components are transferred from nongut cells into the developing gut primordium and an exo/endocytosis mechanism has been hypothesized (Bossinger and Schierenberg 1992). To test endocytotic activity of the gut primordium, we compared the uptake of different fluorochrome-conjugated marker molecules in two nematode species, C. elegans and Cephalobus spec., which differ in the pattern of early cleavage and cell-cell communication. We found no uptake of dextran (as a marker for pinocytosis) but rapid internalization of 30-fold larger transferrin molecules (as a marker for receptor-coupled endocytosis) into the differentiating gut primordium in both nematodes. The two studied species differ with respect to when this process starts. While the uptake of macromolecules in the fast developing C. elegans is first observed at a stage when essentially all cells of the hatching juvenile have been generated, in the slow developing Cephalobus endocytosis begins during the early proliferation phase when only two gut precursor cells are present. We found that the polysulfated hydrocarbon dye trypan blue and the cationic amphiphilic drug chlorpromazine both inhibit endocytosis into the gut primodium.     

Autophagy and apoptosis are redundantly required for C. elegans embryogenesis

Apoptosis, the main form of regulated (or programmed) cell death, allows the organism to tightly control cell numbers and tissue size, and to protect itself from potentially damaging cells. This type of cellular self-killing has long been assumed to be essential for early development. In the nematode Caenorhabditis elegans, however, the core apoptotic cell death pathway appears to be dispensable for embryogenesis when most developmental cell deaths take place: mutant nematodes defective for apoptosis develop into adulthood, with superficially normal morphology and behavior. Accumulating evidence indicates a similar situation in mammalian systems as well. For example, apoptosis-deficient mice can grow as healthy, fertile adults. These observations raise the possibility that alternative cell death mechanisms may compensate for the lack of apoptotic machinery in developing embryos. Interestingly, C. elegans embryogenesis can also occur without autophagy, an alternative form of cellular self-destruction (also called autophagic cell death). In an upcoming paper we report that simultaneous inactivation of the autophagic and apoptotic gene cascades in C. elegans arrests development at early stages, and the affected embryos exhibit severe morphological defects. Double-mutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. This novel function of autophagy genes in morphogenesis indicates a more fundamental role for cellular self-digestion in tissue patterning than previously thought.     

Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans.

The nematode Pristionchus pacificus (Diplogastridae) has been described as a satellite organism for a functional comparative approach to the model organism Caenorhabditis elegans because genetic, molecular, and cell-biological tools can be used in a similar way in both species. Here we show that P. pacificus has three juvenile stages, instead of the usual four found in other nematodes. Embryogenesis is lengthened and many developmental events that take place during the first juvenile stage in C. elegans occur during late embryogenesis in P. pacificus. Video imaging and transmission electron microscopy revealed no embryonic moult. The timing of later developmental events relative to the moults differs between P. pacificus and C. elegans. In addition, the post-embryonic blast-cell divisions display a specific change in timing between the two species, resulting in heterochrony between different cell lineages, such as vulval and gonadal lineages. Developmental events appear to come into register during the last larval stage. Thus, differences in developmental timing between P. pacificus and C. elegans represent a deep heterochronic change. We designate the three juvenile stages of P. pacificus as J1 to J3. Comparison with other species of the family Diplogastridae indicates that this pattern represents an apomorphic character for the monophylum Diplogastridae.     

Mechanisms of cell positioning during C. elegans gastrulation

Cell rearrangements are crucial during development. In this study, we use C. elegans gastrulation as a simple model to investigate the mechanisms of cell positioning. During C. elegans gastrulation, two endodermal precursor cells move from the ventral surface to the center of the embryo, leaving a gap between these ingressing cells and the eggshell. Six neighboring cells converge under the endodermal precursors, filling this gap. Using an in vitro system, we observed that these movements occurred consistently in the absence of the eggshell and the vitelline envelope. We found that movement of the neighbors towards each other is not dependent on chemotactic signaling between these cells. We further found that C. elegans gastrulation requires intact microfilaments, but not microtubules. The primary mechanism of microfilament-based motility does not appear to be through protrusive structures, such as lamellipodia or filopodia. Instead, our results suggest an alternative mechanism. We found that myosin activity is required for gastrulation, that the apical sides of the ingressing cells contract, and that the ingressing cells determine the direction of movement of their neighboring cells. Based on these results, we propose that ingression is driven by an actomyosin-based contraction of the apical side of the ingressing cells, which pulls neighboring cells underneath. We conclude that apical constriction can function to position blastomeres in early embryos, even before anchoring junctions form between cells.     

Occurrence, hosts, morphology, and molecular characterisation of Pasteuria bacteria parasitic in nematodes of the family Plectidae

Parasitic bacteria of the genus Pasteuria are reported for three Anaplectus and four identified and several unidentified Plectus species found in eight countries in various habitats. The pasteurias from plectids agree in essential morphological characters of sporangia and endospores as well as in developmental cycle with those of the Pasteuria species and strains described from tylenchid nematodes, but appear to be mainly distinguished from these by absence of a distinct perisporium in the spores and the endospores obviously not being cup- or saucer-shaped. The wide range of measurements and morphological peculiarities of sporangia and endospores suggest that probably several Pasteuria species have to be distinguished as parasites in Plectidae. From an infected juvenile of an unidentified plectid species the 16S rRNA gene sequence of Pasteuria sp. was obtained. Substantial sequence divergence from described Pasteuria species and its phylogenetic position on molecular trees indicate that this Pasteuria sp. could be considered as a new species. Preliminary results of the analysis of DNA phylogeny of Pasteuria spp. and their nematode hosts provide evidence for incongruence of their phylogenetic history and of host switching events during evolution of the bacterial parasites.     

Reproductive isolation in Caenorhabditis: terminal phenotypes of hybrid embryos

SUMMARY Several interspecific combinations of the "elegans" group of Caenorhabditis species are cross-fertile. Most F1 hybrids from these crosses arrest during embryogenesis. Developmental defects observed in hybrid embryos include defects in gastrulation initiation, defects in embryonic compaction, and defects in embryonic elongation. These reproductive barriers have arisen multiple times in the evolution of Caenorhabditis.     

Gastrulation in the nematode Caenorhabditis elegans

Gastrulation in Caenorhabditis elegans has been described by following the movements of individual nuclei in living embryos by Nomarski microscopy. Gastrulation starts in the 26-cell stage when the two gut precursors, Ea and Ep, move into the blastocoele. The migration of Ea and Ep does not depend on interactions with specific neighboring cells and appears to rely on the earlier fate specification of the E lineage. In particular, the long cell cycle length of Ea and Ep appears important for gastrulation. Later in embryogenesis, the precursors to the germline, muscle and pharynx join the E descendants in the interior. As in other organisms, the movement of gastrulation permit novel cell contacts that are important for the specification of certain cell fates.     

Developmental Determinants in Embryos of Caenorhabditis elegans

C. elegans is proving useful for the study of cell determination in early embryos. Breeding experiments with embryonic lethal mutants show that abnormal embryogenesis often results from defective gene function in the maternal parent, suggesting that much of the information for normal embryonic development is laid down during oogenesis. Analysis of a gut-specific differentiation marker in cleavage-arrested embryos has provided evidence that the potential for this differentiation behaves as a cell-autonomous internally segregating developmental determinant, which is present from the 2-cell stage onward and is partitioned into the gut precursor cell during early cleavage divisions. Visible prelocalized cytoplasmic granules that segregate with a particular cell lineage have heen observed in the embryonic germline precursor cells by fluorescent antibody staining. Whether these granules play a role in germline determina... [remainder of abstract missing in original]     

Unusual cleavage and gastrulation in a freshwater nematode: developmental and phylogenetic implications

Early embryogenesis in nematodes as seen in Caenorhabditis elegans and many other species of this phylum features several characteristic events. These include the visible presence of a germline from the very beginning generating different somatic lineages via asymmetric cleavages, the absence of a coeloblastula and a unique type of gastrulation with immigration of just two gut precursor cells. Here it is shown by using Nomarski optics that development of the freshwater nematode Tobrilus diversipapillatus differs from this pattern in two prominent aspects. (1) No asymmetric cleavages and no distinct cell lineages are generated; (2) in contrast to all other nematodes studied so far, a prominent coeloblastula is formed and gastrulation resembles the classical pattern found all over the animal kingdom. These developmental peculiarities are considered to be plesiomorphic and thus the order Triplonchida, to which Tobrilus belongs, may occupy a phylogenetic position at the base of the nematode phylum. The findings reported here allow us to reject a number of conceivable correlations between the type of gastrulation and other developmental parameters.Edited by D. TautzThis revised version was published online in December 2004 with corrections to typographical errors in the abstract.     

Free‐living nematodes as prey for higher trophic levels of forest soil food webs

Nematodes are the most abundant invertebrates in soils and are key prey in soil food webs. Uncovering their contribution to predator nutrition is essential for understanding the structure of soil food webs and the way energy channels through soil systems. Molecular gut content analysis of consumers of nematodes, such as soil microarthropods, using specific DNA markers is a novel approach for studying predator–prey interactions in soil. We designed new specific primer pairs (partial 18S rDNA) for individual soil‐living bacterial‐feeding nematode taxa (Acrobeloides buetschlii, Panagrellus redivivus, Plectus velox and Plectus minimus). Primer specificity was tested against more than 100 non‐target soil organisms. Further, we determined how long nematode DNA can be traced in the gut of predators. Potential predators were identified in laboratory experiments including nine soil mite (Oribatida, Gamasina and Uropodina) and ten springtail species (Collembola). Finally, the approach was tested under field conditions by analyzing five mite and three collembola species for feeding on the three target nematode species. The results proved the three primer sets to specifically amplify DNA of the respective nematode taxa. Detection time of nematode DNA in predators varied with time of prey exposure. Further, consumption of nematodes in the laboratory varied with microarthropod species. Our field study is the first definitive proof that free‐living nematodes are important prey for a wide range of soil microarthropods including those commonly regarded as detritivores. Overall, the results highlight the eminent role of nematodes as prey in soil food webs and for channelling bacterial carbon to higher trophic levels.     

Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development

Defects in the human XPV/POLH gene result in the variant form of the disease xeroderma pigmentosum (XP-V). The gene encodes DNA polymerase eta (Poleta), which catalyzes translesion synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers (CPDs) and other lesions. To further understand the roles of Poleta in multicellular organisms, we analyzed phenotypes caused by suppression of Caenorhabditis elegans POLH (Ce-POLH) by RNA interference (RNAi). F1 and F2 progeny from worms treated by Ce-POLH-specific RNAi grew normally, but F1 eggs laid by worms treated by RNAi against Ce-POLD, which encodes Poldelta did not hatch. These results suggest that Poldelta but not Poleta is essential for C. elegans embryogenesis. Poleta-targeted embryos UV-irradiated after egg laying were only moderately sensitive. In contrast, Poleta-targeted embryos UV-irradiated prior to egg laying exhibited severe sensitivity, indicating that Poleta contributes significantly to damage tolerance in C. elegans in early embryogenesis but only modestly at later stages. As early embryogenesis is characterized by high levels of DNA replication, Poleta may confer UV resistance in C. elegans, perhaps by catalyzing TLS in early embryogenesis.