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[
International C. elegans Meeting,
1999]
Cell specification during embryogenesis of the model system C. elegans involves a combination of conditional and autonomous mechanisms. We have begun to study the development of other nematodes to investigate how well cell-specification mechanisms are preserved among closely related species. Here we report that the embryo of the soil nematode Acrobeloides nanus expresses a so far undescribed regulative potential. This allows - different to C. elegans - the development up to hatching and sometimes to fertile adults after elimination of early blastomeres, even if this means the loss of more than 50% of the embryos original volume. In our experiments eliminated cells are replaced in a sequential and hierarchical manner by specific neighbouring blastomeres. Thus, early somatic blastomeres in A. nanus are multipotent being capable to give rise to more than one somatic founder cell. Germline cells, however, cannot be replaced. A model is presented according to which in A. nanus cellular identities are assigned by a series of reciprocal inhibitory cell-cell interactions absent in C. elegans .
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[
Worm Breeder's Gazette,
1996]
The pattern of cell-cell communication in embryos of C.elegans has beeninvestigated by injecting tracer dyes into individual blastomeres (Bossinger and Schierenberg, 1992). It has been shown that in embryos from the 4-cell stage onward all blastomeres are well coupled for the diffusion of negatively charged Lucifer Yellow (LY; MW 457 Da) while uncharged Rhodamin-coupled dextran (MW 4.000 - 70.000 Da) remains restricted to the injected cell and its descendants. However, in Cephalobus spec. (another soil nematode), LY and Rhodamin-dextran (even at a molecular weight of 70.000 Da) diffuse along discrete pathways from cell to cell (Bossinger and Schierenberg, 1996). We extended our dye-coupling studies in C. elegans using negatively charged dextrans (LY-dextran and FITC-dextran) with molecular weights of 10.000 and 70.000 Da. Suprisingly, we found that in embryos between the 4-and the 24-cell stage all blastomeres are coupled for negatively charged dextrans whereas under all tested conditions uncharged Rhodamin-dextran (MW 10.000 Da) remains in the injected cell and its descendants. The diffusion of the negatively charged dyes appears to occur quickly and freely between all blastomeres. In contrast to Cephalobus we did not observe preferential pathways of dye spread from the injected cell, suggesting that all blastomeres are equally coupled. However at the 24-cell stage, when the primordial germline cell P4 has been generated, diffusion into D and P4 cells becomes retarded.The diffusion block lasts approximately 5 minutes until D joins the somatic compartment and P4 remains uncoupled for at least 20 min. A similar observation has been made for the much smaller LY in C. elegans indicating that it is a diffusion block and not a reduction in channel diameter. In summary, our findings suggest that in the developing C. elegans embryo all somatic blastomeres are coupled by communication channels much larger than conventional gap junctions. These channels seem to be permeable for negatively charged molecules up to a molecular weight of at least 70.000 Da but are impermeable to uncharged dextrans. Moreover, these channels seem to be equally present between all blastomeres of the somatic cells. Presently we can only speculate about the function of these communication pathways. Bossinger, O; Schierenberg, E. (1992) Dev. Biol. 151: 401-409 Bossinger, O; Schierenberg, E. (1996) Dev. Genes Evol. 206: 25-34
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[
Worm Breeder's Gazette,
1997]
Gut differentiation in C. elegans is easily detectable due to the autofluorescence and birefringence of rhabditin granules (Babu, 1974; Laufer et al., 1980). However, in experimental manipulated embryos both markers are sometimes hardly detectable. In other nematodes we found these markers to be completely absent. Alternative markers for gut differentiation such as visualization of gut esterase activity (L. Edgar, 1995; see below) requires experimental experience and antibody staining detects gut in late embryogenesis, only. Moreover, both methods are time consuming and only fixed material can be processed. Here, we present an easy and fast method to reliably detect gut differentiation of early and living embryos in a variety of nematode species. The method relies on the endocytotic activity of the gut primordium which can be visualized by the uptake of fluorescently-labelled transferrin (Bossinger et al., 1996). Embryos of appropriate stages are mounted in a drop of distilled water on a polylysine-coated slide. The water is replaced by culture medium supplemented with 0.1 mg/ml Texas Red-coupled transferrin (Molecular Probes, T- 2871). We advise either to use the Leibovitz L- 15 medium based recipe or EGM (both developed and described by L. Edgar: Blastomere culture and analysis in: Methods in Cell Biology 48: 303- 321,1995) or the medium used by C.A. Shelton and B. Bowerman (Development 122: 2043-2050, 1996). Embryos are covered with a coverslip sealed with vaseline on the edges to prevent desiccation. In order to allow the access of transferrin to the blastomeres we perforate the eggshell and the underlying vitelline membrane by short laser pulses. Depending on the laser used it will be necessary to stain eggshells to allow absorption of laser energy. We add 8mg/ml trypan blue (Sigma) to the culture medium and use a nitrogen-pumped dye- laser with Rhodamin 6G as laser dye. Medium containing trypan blue should be replaced by medium with transferrin after perforation of the eggshell, because trypan blue inhibits the uptake of this endocytotic marker. However, instead of laser perforation it is sufficient to squeeze embyros by applying pressure on the coverslip in order to disrupt the vitelline layer. Alternatively, embryos may be devitellinized as described by L. Edgar (1995; see above). Embryos are incubated for 5-30 min at room temperature. Finally, the excess of free transferrin-conjugate is removed in order to reduce background fluorescence by replacing it with transferrin-free medium. Endocytosis of transferrin is analyzed with epifluorescence at 515- 565 nm. Applying this method in the soil nematode Acrobeloides nanus we found that endocytotic activity is already detectable at the 2-E cell stage which is considerably earlier than in C. elegans, where uptake of transferrin starts with 16-E cells present. Nevertheless, in both nematodes the endocytosis is among the first markers of tissue- specific differentiation. In all nematodes species we tested so far, transferrin is specifically accumulated in the gut primordium. This method may also be usefull to identify genes involved in endocytosis.
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[
Worm Breeder's Gazette,
1996]
To better understand the evolution of cell-specification mechanisms we study development in other nematode species. One of them is Cephalobus spec., an apparently more primitive representative of rhabditid nematodes (Skiba and Schierenberg, 1992, Dev. Biol. 151:597). Cephalobus lacks autofluorescence and birefringence of the gut granules as found in C.elegans. In order to analyse development of the intestine we therefore take advantage of 1.) its tissue-specific endocytotic activity leading to accumulation of fluorescently labelled transferrin (Bossinger et al., 1996, Roux's Arch. Dev. Biol. 205: 494) and 2.) the mAb 1CB4 which specifically recognizes gut cells in C.elegans (Okamoto and Thomson, 1985, J. Neurosci. 5: 643) and Cephalobus. In the last WBG we reported that gut differentiation in Cephalobus does not depend on an induction by P2 but may depend on AB. Here we present additional data leading to a modified interpretation. When AB was extruded at the 2-cell-stage, 87% (35/40) of the P1-derived partial embryos displayed gut differentiation while 13% (5/40) did not. When AB was extruded at the 3-cell stage 45% (15/33) of the partial embryos showed gut differentiation while 55% (18/33) did not (or very faintly). From this we conclude that in Cephalobus gut differentiation can take place without inductions from either P2 or AB (descendants). In another set of experiments we extruded P1 from 2-cell stages. Much to our surprise, 62% (20/32) of the emerging AB-derived embryos developed strong gut differentiation although lineage analysis shows that normally the gut in Cephalobus comes exclusively from descendants of the E- cell as in C. elegans. To exclude a potential confusion of AB/P1 (despite their size differences), we ascertained the typical different early lineage patterns in the non-extruded and the extruded cell. We found that in 65% (17/26) of the embryos AB was still able to produce gut-like cells when EMS and P2 were extruded in late 3-cell stages. In 35% (6/17) of these we even observed an overexpression of the gut markers in the AB lineage with 40-60 cells being recognized by the transferrin assay and nicely outlined by the antibody. These cells were considerably smaller than the normal 20-24 gut cells in untreated Cephalobus embryos, suggesting one or two additional rounds of cell division. Our results indicate that in Cephalobus both of the first two blastomeres carry the potential to develop gut and that an inhibitory interaction between AB and EMS (or their descendants) is necessarty to restrict the gut fate to the E-lineage. They also show that the relatively few divisions that normally take place in the gut lineage are not a prerequisite for proper differentiation. They can be interpreted as an early example of two cells competing for a primary fate as observed later in equivalence groups. Preliminary observations suggest that in manipulated embryos after the 8-AB cell stage some AB descendants acquire a slower cell-cycle rhythm and give rise to descendants with gut characteristics. This pattern is reminiscent of early embryogenesis in Enoplus brevis, a marine nematode in which a visible soma/germline differentiation is absent. In addition, other nematode species have been described in which gut is derived from the AB blastomere (V.V. Malakhov, 1994, Nematodes, Smithsonian Institution Press).
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[
Worm Breeder's Gazette,
1996]
Gut induction is a well known example for cellular interaction in the early C. elegans embryo (Schierenberg, 1987; Goldstein, 1992). Contact between the germline cell P2 and the gut precursor cell EMS in the early 4-cell stage is necessary to specify the gut fate. As a consequence of the interaction, EMS divides into an anterior MS cell and a posterior E cell and the gut primordium is formed by the progeny of the latter.
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[
Dev Biol,
1999]
Cell specification during embryogenesis of the model system Caenorhabditis elegans involves a combination of inductive and autonomous mechanisms. We have begun to study the development of other nematodes to investigate how well cell-specification mechanisms are preserved among closely related species. Here we report that the embryo of the soil nematode Acrobeloides nanus expresses a so far undescribed regulative potential. When, for instance, the first somatic founder cell AB is eliminated it is replaced by its posterior neighbor EMS, which in turn is replaced by the C cell. This allows-different from C. elegans-the development of partial embryos up to hatching and sometimes to fertile adults. Thus, early somatic blastomeres in A. nanus are multipotent, each being capable of giving rise to more than one somatic founder cell. Lost germ-line cells, however, are not replaced. A model is presented, according to which in A. nanus cellular identities are assigned by specific reciprocal inhibitory cell-cell interactions absent in C. elegans. Differences and similarities in cell specification between the two species are discussed and related to different developmental strategies.
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[
International C. elegans Meeting,
1997]
Experimental analysis of C. elegans development has revealed that specification of cell fate requires a number of cell-cell interactions between adjacent early blastomeres. We compared the pattern of gut specification in C. elegans with that in Cephalobus spec., another representative of free-living rhabditid nematodes. Surprisingly, we found considerable differences. a) In C. elegans gut specification requires an inductive interaction in the 4-cell embryo between the germline cell P2 and its somatic sister EMS (gut precursor). In contrast, in Cephalobus gut specification does not require induction: The EMS blastomere produces gut cells autonomously. b) Cell isolation experiments show that in C. elegans EMS is the only cell which carries the potential to form gut. In Cephalobus, however, EMS and its somatic neighbour AB, both, are able to generate gut cells, if cultured in isolation. c) Since in normal Cephalobus development, as in C. elegans, gut derives exclusively from descendants of the EMS blastomere, AB and EMS seem to compete for gut fate. Thus, gut specification in Cephalobus does not depend on an inductional event as in C. elegans but instead requires inhibitory cell-cell interaction.
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[
International C. elegans Meeting,
1995]
Communication channels (CC; made of connexin, ductin and other proteins) and cytoplasmic bridges provide intercellu- lar pathways for the exchange of molecules. They appear to play an important role for normal development in various systems. We try to identify different CC on the ultrastruc- tural level and analyze their role in the coordination of physiological and developmental processes during embryoge- nesis of nematodes. As an extension of our recent descrip- tion of intercellular communication in C. elegans, using fluorescent marker dyes, we have begun to study other spe- cies of free-living nematodes. Embryogenesis of Cephalobus spec. was found to differ considerably from that in C. ele- gans. It was hypothesized that Cephalobus may be a more primitive representative of rhabditid nematodes. We can show that a different pattern of cleavage and spatial arrangement of cells in the Cephalobus embryo, - compared to C. elegans -, goes along with a modified pattern of cell-cell communi- cation. The main findings include: (1) In Cephalobus cell-cell communication is established in a stepwise fashion from anterior to posterior. (2) Already before the 30-cell stage, tissue-specific dye- coupling compartments are established. (3) In contrast to C. elegans high molecular weight dyes can diffuse between somatic cells in Cephalobus, probably due to midbodies, but never into the germline (4) The diffusion of high molecular weight markers into all somatic cells appears to take place along specific routes. (5) During early gastrulation communication via midbody-like pathways appears to change into a communication via gap junction-like channels.
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[
Dev Biol,
1998]
The classic view of a strictly cell-autonomous development in nematode embryos has been overturned in recent years with the demonstration of various inductive interactions during early development of Caenorhabditis elegans. To examine how conserved the pattern of embryonic cell specification is among nematodes, we have begun to study the pattern in other species after selective elimination of certain early blastomeres. Here we report considerable differences in specification of the gut lineage between C. elegans and Acrobeloides nanus, another free-living soil nematode belonging to the same order. In C. elegans none of the early blastomeres is by itself able to establish a gut lineage for which an inductive interaction between the somatic EMS cell and its germline sister P2 is required. In contrast, in A. nanus all blastomeres of the 3-cell stage carry the potential to generate gut cells. Our data suggest that repressive interactions take place among blastomeres to ensure that under normal conditions only one of them executes the gut fate. Thus, in related species of nematodes with a very conserved morphology, the assignment of cell fate during early embryogenesis appears to involve quite different strategies.
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[
Parasitol Res,
1987]
The midbody regions of female worms of six Onchocerca species (O. flexuosa, O. tarsicola, O. lienalis, O. gutturosa, O. armillata, O. gibsoni) were studied by transmission electron microscopy. The cuticular layering was rather similar in all species with the ridges built up by the cortical layers and the inner cuticular striations by the median or basal layers. Differences in the epicuticular morphology were considerable. O. flexuosa and O. lienalis had a thin epicuticle without protuberances, the epicuticle of O. armillata carried small knobs, and O. tarsicola, O. gutturosa, and O. gibsoni had a thick trilaminar epicuticle with long protuberances. Extreme hypertrophy of hypodermis and reductions of somatic musculature were observed in O. flexuosa and O. gibsoni. Less extended thickenings of the hypodermis were observed in the other species. No degenerative alterations were found in the muscle cells of O. gutturosa and O. lienalis. The intestinal lumen of most of the species was in a central position, but in O. tarsicola and O. gibsoni the lumen was reduced to small clefts between the intestinal cells. In these species, numerous electron-dense, concentric granules were observed in the cytoplasm of the intestinal cells. The proportions of the various organs differed considerably from species to species, e.g., the uteri contained the embryos filed one behind the other in O. tarsicola, whereas 50 or more embryos were found beside one another in cross-sections of the uterus of O. gibsoni. The comparative study showed that O. gibsoni and O. volvulus have many derived morphological characteristics in common and that in the other species more primitive stages of development of these morphological marks can be observed.