Zonula adherens formation in Caenorhabditis elegans requires dlg-1, the homologue of the Drosophila gene discs large.

The correct assembly of junction components, such as E-cadherin and beta-catenin, into the zonula adherens is fundamental for the function of epithelia, both in flies and in vertebrates. In C. elegans, however, the cadherin-catenin system is not essential for general adhesion, raising the question as to the genetic basis controlling junction morphogenesis in nematodes. Here we show that dlg-1, the C. elegans homologue of the Drosophila tumour-suppressor gene discs-large, plays a crucial role in epithelial development. DLG-1 is restricted to adherens junctions of all embryonic epithelia, which contrasts with the localisation of the Drosophila and vertebrate homologues in septate and tight junctions, respectively. Proper localisation of DLG-1 requires the basolateral LET-413 protein, but is independent of the cadherin-catenin system. Embryos in which dlg-1 activity was eliminated by RNA-mediated interference fail to form a continuous belt of junction-associated antigens and arrest development. Loss of dlg-1 activity differentially affects localisation of proteins normally enriched apically to the zonula adherens. While the distribution of an atypical protein kinase C (PKC-3) and other cytoplasmic proteins (PAR-3, PAR-6) is not affected in dlg-1 (RNAi) embryos, the transmembrane protein encoded by crb-1, the C. elegans homologue of Drosophila crumbs, is no longer concentrated in this domain. In contrast to Drosophila, however, crb-1 and a second crb-like gene are not essential for epithelial development in C. elegans. Together the data indicate that several aspects of the spatial organisation of epithelial cells and its genetic control differ between flies, worms, and vertebrates, while others are conserved. The molecular nature of DLG-1 makes it a likely candidate to participate in the organisation of a protein scaffold that controls the assembly of junction components into the zonula adherens.

A nematode kinesin required for cleavage furrow advancement.

Dividing cells need to coordinate the separation of chromosomes with the formation of a cleavage plane. There is evidence that microtubule bundles in the interzone region of the anaphase spindle somehow control both the location and the assembly of the cleavage furrow [1-3]. A microtubule motor that concentrates in the interzone, MKLP1, has previously been implicated in the assembly of both the metaphase spindle and the cleavage furrow [4-6]. To gain insight into mechanisms that might underlie interdependence of the spindle and the cleavage furrow, we used RNA-mediated interference (RNAi) to study the effects of eliminating MKLP1 from Caenorhabditis elegans embryos. Surprisingly, in MKLP1(RNAi) embryos, spindle formation appears normal until late anaphase. Microtubule bundles form in the spindle interzone and the cleavage furrow assembles; anaphase and cleavage furrow ingression initially appear normal. The interzone bundles do not gather into a stable midbody, however, and furrow contraction always fails before complete closure. This sequence of relatively normal mitosis and a late failure of cytokinesis continues for many cell cycles. These and additional results suggest that the interzone microtubule bundles need MKLP1 to encourage the advance and stable closure of the cleavage furrow.

The expression of the C. elegans labial-like Hox gene ceh-13 during early embryogenesis relies on cell fate and on anteroposterior cell polarity.

Clusters of homeobox-containing HOM-C/hox genes determine the morphology of animal body plans and body parts and are thought to mediate positional information. Here, we describe the onset of embryonic expression of ceh-13, the Caenorhabditis elegans orthologue of the Drosophila labial gene, which is the earliest gene of the C. elegans Hox gene cluster to be activated in C. elegans development. At the beginning of gastrulation, ceh-13 is asymmetrically expressed in posterior daughters of anteroposterior divisions, first in the posterior daughter of the intestinal precursor cell E and then in all posterior daughters of the AB descendants ABxxx. In this paper, we present evidence that supports position-independent activation of ceh-13 during early C. elegans embryogenesis, which integrates cell fate determinants and cell polarity cues. Our findings imply that mechanisms other than cell-extrinsic anteroposterior positional signals play an important role in the activation and regulation of the C. elegans Hox gene ceh-13.

Early embryonic induction in C. elegans can be inhibited with polysulfated hydrocarbon dyes.

During embryogenesis of Caenorhabditis elegans cellular interactions are necessary to determine the fate of blastomeres. In one of these interactions, taking place in the 4-cell stage, the germline cell P2 induces longitudinal orientation of the cleavage spindle in the neighboring EMS cell, its asymmetric division, and the establishment of a gut lineage. Application of several polysulfated hydrocarbon dyes (e.g., trypan blue, TB) in the 1- to 4-cell stages inhibits induction of the gut precursor cell. However, dye application from the late 4-cell stage onward does not interfere with gut induction, supporting the earlier finding of a short time window for this interaction. We also tested the effect of TB on the induction of pharyngeal muscle cells by the MS blastomere, which appears to involve a surface receptor-ligand interaction. We found that this process is inhibited as well. These and additional data indicate that polysulfated hydrocarbon dyes are suitable tools to generally interfere with cell-cell interactions in the nematode embryo.

The use of fluorescent marker dyes for studying intercellular communication in nematode embryos.

As more and more cases of necessary cell-cell interactions are revealed, the classical view of mosaic development in nematodes has to be replaced by a more dynamic picture showing different types of intercellular communication. To investigate the pattern and function of communication pathways between cells, we have developed different techniques to shunt fluorescent marker dyes into embryos and hatched animals and study their distribution in vivo. During embryogenesis we find that for a long time all somatic cells form a single dye-coupling compartment while transfer into the germline is restricted already at an early stage. Considerable variations between species with respect to the size of communication channels and the time during which these are functional are observed and can be correlated to differences in the developmental program. A different kind of intracellular communication can be visualized with the help of fluorescent dyes: a transfer of yolk proteins in two phases of the life cycle, in the adult hermaphrodite from the gut into the maturing germ cells, and in the embryo from non-gut cells into the gut primordium. Cell-cell interactions in the nematode embryo can be inhibited with polysulfated hydrocarbon dyes (e.g. Trypan Blue) which bind strongly to the plasma membrane. In summary our data indicate that fluorescent marker dyes can be helpful tools to identify and understand the role of intercellular communication and transfer processes in nematode development.

Cell-cell communication in nematode embryos: differences between Cephalobus spec. and Caenorhabditis elegans.

During early nematode embryogenesis a series of asymmetric cleavages in the germ line generates several somatic founder cells and a primordial germ cell. We have found previously that the two soil nematodes Cephalobus spec. and Caenorhabditis elegans express considerable differences in the order of events and spatial arrangement of cells during early embryogenesis. With the help of microinjected fluorescent marker dyes, we show here that these dissimilarities partner major differences in the pattern of intercellular communication. Whilst in C. elegans all early blastomeres become dye-coupled simultaneously, in Cephalobus communication is established progressively in the sequence in which cells are born. In addition, in Cephalobus but not C. elegans, sequential lucifer yellow accumulation indicates stepwise changes in the state of early blastomeres: if injected into the uncleaved zygote, for example, the dye becomes equally distributed to all cells at first but rapidly accumulates in a single blastomere in the 4-cell stage. We speculate that such a redistribution mechanism may be involved in the differential segregation of cytoplasmic components to individual blastomeres. The most dramatic difference between the two species was found with respect to the transfer of high molecular weight molecules. In contrast to C. elegans, in Cephalobus not only small lucifer dyes but also high molecular weight dextrans can diffuse along specific pathways between early somatic cells indicating the presence of large communication channels. However, a transfer of dextran into or out of germ line cells never takes place. The origin of these channels as midbodies of previous mitoses and their potential role for normal development is discussed. Tissue-specific dye-coupling compartments in the slow developing Cephalobus are established in the same order but at a considerably earlier developmental stage than in C. elegans suggesting that this process may depend more on parameters like available time for transcription rather than the number of cell cycles passed through.

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.

Cell-cell communication in the embryo of Caenorhabditis elegans.

We have investigated the pattern of cell-cell communication in embryos of the free-living soil nematode Caenorhabditis elegans. For this, we have established a method for microinjection of tracer dyes into individual blastomeres. After iontophoresis of fluorescent dyes of different molecular weights (Lucifer yellow, LY, M(r) 457; rhodamine-labeled dextran, RD, M(r) 4000), we can visualize intercellular communication pathways. The dye-spread of LY, indicating communication via gap junctions, becomes first visible in the late 2-cell stage. From the 4-cell stage onward all cells appear to be well coupled by communication channels, which allow the free diffusion of LY. In contrast, RD remains restricted to the injected cell and its descendants. After the primordial germcell P4 has been generated in the 24-cell stage, dye-spread of LY into this cell and its somatic sister D is delayed. However, the restricted dye-coupling of D is only temporary. After a brief period it joins the somatic compartment. With the beginning of the morphogenesis phase the two existing germline cells (the daughters of P4) are completely uncoupled from the soma, while the latter still forms a single dye-coupling compartment. Only during the second half of embryogenesis different separate somatic communication compartments are established. We followed the pattern of intercellular communication in the alimentary tract and found a progressive restriction into smaller dye-coupling units. Our data are compared to those found in other systems and discussed with respect to cellular determination and differentiation.