Auswahl der wissenschaftlichen Literatur zum Thema „Embryons non-C. elegans“

Gastrulation in C. elegans embryos involves formation of a blastocoel and the ingression of surface cells into the blastocoel. Mutations in the par-3 gene cause abnormal separations between embryonic cells, suggesting that the PAR-3 protein has a role in blastocoel formation. In normal development, PAR proteins localize to either the apical or basal surfaces of cells prior to blastocoel formation; we demonstrate that this localization is determined by cell contacts. Cells that ingress into the blastocoel undergo an apical flattening associated with an apical concentration of non-muscle myosin. We provide evidence that ingression times are determined by genes that control cell fate, though interactions with neighboring cells can prevent ingression.

Vertebrates produce multiple chondroitin sulfate proteoglycans that play important roles in development and tissue mechanics. In the nematode Caenorhabditis elegans, the chondroitin chains lack sulfate but nevertheless play essential roles in embryonic development and vulval morphogenesis. However, assignment of these functions to specific proteoglycans has been limited by the lack of identified core proteins. We used a combination of biochemical purification, Western blotting, and mass spectrometry to identify nine C. elegans chondroitin proteoglycan core proteins, none of which have homologues in vertebrates or other invertebrates such as Drosophila melanogaster or Hydra vulgaris. CPG-1/CEJ-1 and CPG-2 are expressed during embryonic development and bind chitin, suggesting a structural role in the egg. RNA interference (RNAi) depletion of individual CPGs had no effect on embryonic viability, but simultaneous depletion of CPG-1/CEJ-1 and CPG-2 resulted in multinucleated single-cell embryos. This embryonic lethality phenocopies RNAi depletion of the SQV-5 chondroitin synthase, suggesting that chondroitin chains on these two proteoglycans are required for cytokinesis.

The endoderm of higher organisms is extensively patterned along the anterior/posterior axis. Although the endoderm (gut or E lineage) of the nematode Caenorhabditis elegans appears to be a simple uniform tube, cells in the anterior gut show several molecular and anatomical differences from cells in the posterior gut. In particular, the gut esterase ges-1 gene, which is normally expressed in all cells of the endoderm, is expressed only in the anterior-most gut cells when certain sequences in the ges-1 promoter are deleted. Using such a deleted ges-1 transgene as a biochemical marker of differentiation, we have investigated the basis of anterior-posterior gut patterning in C. elegans. Although homeotic genes are involved in endoderm patterning in other organisms, we show that anterior gut markers are expressed normally in C. elegans embryos lacking genes of the homeotic cluster. Although signalling from the mesoderm is involved in endoderm patterning in other organisms, we show that ablation of all non-gut blastomeres from the C. elegans embryo does not affect anterior gut marker expression; furthermore, ectopic guts produced by genetic transformation express anterior gut markers generally in the expected location and in the expected number of cells. We conclude that anterior gut fate requires no specific cell-cell contact but rather is produced autonomously within the E lineage. Cytochalasin D blocking experiments fully support this conclusion. Finally, the HMG protein POP-1, a downstream component of the Wnt signalling pathway, has recently been shown to be important in many anterior/posterior fate decisions during C. elegans embryogenesis (Lin, R., Hill, R. J. and Priess, J. R. (1998) Cell 92, 229–239). When RNA-mediated interference is used to eliminate pop-1 function from the embryo, gut is still produced but anterior gut marker expression is abolished. We suggest that the C. elegans endoderm is patterned by elements of the Wnt/pop-1 signalling pathway acting autonomously within the E lineage.

Co-factor homeodomain proteins such as Drosophila Homothorax (Hth)and Extradenticle (Exd) and their respective vertebrate homologs, the Meis/Prep and Pbx proteins, can increase the DNA-binding specificity of Hox protein transcription factors and appear to be required for many of their developmental functions. We show that the unc-62 gene encodes theC. elegans ortholog of Hth, and that maternal-effect unc-62mutations can cause severe posterior disorganization during embryogenesis (Nob phenotype), superficially similar to that seen in embryos lacking function of either the two posterior-group Hox genes nob-1 and php-3 or the caudal homolog pal-1. Other zygotically actingunc-62 alleles cause earlier embryonic arrest or incompletely penetrant larval lethality with variable morphogenetic defects among the survivors, suggesting that unc-62 functions are required at several stages of development. The differential accumulation of four unc-62transcripts is consistent with multiple functions. The C. elegans exdhomologs ceh-20 and ceh-40 interact genetically withunc-62 and may have overlapping roles in embryogenesis: neither CEH-20 nor CEH-40 appears to be required when the other is present, but loss of both functions causes incompletely penetrant embryonic lethality in the presence of unc-62(+) and complete embryonic lethality in the presence of an unc-62 hypomorphic allele.

AbstractTo determine whether embryogenesis of Caenorhabditis elegans is typical for nematodes in general, we started to analyse in comparison several aspects of development in various nematode species. The differences we observed can be subdivided into two classes, those visible in the intact embryo and those requiring experimental interference. Particularly obvious differences of both types were revealed between C. elegans (Rhabditidae) and Acrobeloides nanus (Cephalobidae). Not only does the spatial and temporal pattern of early events differ but also that of intercellular communication and cell specification. Our data suggest that some developmental variations are characteristic for certain nematode groups and therefore may be useful as phylogenetic markers. In contrast, we detected little evidence so far for environmental influence on early developmental processes. Pour déterminer dans quelle mesure l’embryogenèse de Caenorhabditis elegans est une caractéristique générale des nématodes, nous avons commencé l’analyse de plusieurs aspects du développement chez différentes espèces de nématodes. Les différences observées peuvent être divisées en deux catégories: celles observables chez l’embryon intact et celles nécessitant une intervention expérimentale. En particulier, des différences nettes entre les deux catégories ont été mises en évidence chez C. elegans (Rhabditidae) et Acrobeloides nanus (Cephalobidae). Diffèrent non seulement le schéma spatio-temporel des évènements précoces, mais également la communication intercellulaire et la différenciation cellulaire. Nos données suggèrent que certaines variations du développement sont caractéristiques de certains groupes de nématodes et pourraient donc être utiles comme marqueurs phylogénétiques. A contrario, une influence de l’environnement sur les processus précoces du développement n’a pas, jusqu’à présent, été détectée.

AbstractThe resistance of the nematode Caenorhabditis elegans towards the highly potent toxin ricin has been studied. Incubation of C. elegans in ricin did not affect life span or progeny production. However, micro-injection of the ricin A-chain into the distal, syncitial gonad caused degeneration and sterility in test specimens, confirming that C. elegans ribosomes are sensitive. Using transmission electron microscopy, it was observed that ricin is effectively internalised into the intestinal cells. When pre-labelled with gold, the toxin reached only the lysosomes. When native toxin was used, the toxin was either routed to the lysosomes or underwent transcytosis to the pseudocoelomatic cavity and incorporation into embryos. None of the ricin reached either the trans Golgi network or the Golgi apparatus, considered essential for toxicity. The observed oral non-toxicity is therefore due to alternate sorting of the toxin, a mechanism not previously observed. The data indicate that, although ricin can opportunistically bind to, and be internalised by, cell surface receptors, these receptors are not sufficient to elicit toxicity.

Telomeres are specialized protein–DNA structures that protect chromosome ends. In budding yeast, telomeres form clusters at the nuclear periphery. By imaging telomeres in embryos of the metazoan Caenorhabditis elegans, we found that telomeres clustered only in strains that had activated an alternative telomere maintenance pathway (ALT). Moreover, as in yeast, the unclustered telomeres in wild-type embryos were located near the nuclear envelope (NE). This bias for perinuclear localization increased during embryogenesis and persisted in differentiated cells. Telomere position in early embryos required the NE protein SUN-1, the single-strand binding protein POT-1, and the small ubiquitin-like modifier (SUMO) ligase GEI-17. However, in postmitotic larval cells, none of these factors individually were required for telomere anchoring, which suggests that additional mechanisms anchor in late development. Importantly, targeted POT-1 was sufficient to anchor chromatin to the NE in a SUN-1–dependent manner, arguing that its effect at telomeres is direct. This high-resolution description of telomere position within C. elegans extends our understanding of telomere organization in eukaryotes.

The C. elegans gene lin-26, which encodes a presumptive zinc-finger transcription factor, is required for hypodermal cells to acquire their proper fates. Here we show that lin-26 is expressed not only in all hypodermal cells but also in all glial-like cells. During asymmetric cell divisions that generate a neuronal cell and a non-neuronal cell, LIN-26 protein is symmetrically segregated and then lost from the neuronal cell. Expression in glial-like cells (socket and sheath cells) is biologically important, as some of these neuronal support cells die or seem sometimes to be transformed to neuron-like cells in embryos homozygous for strong loss-of-function mutations. In addition, most of these glial-like cells are structurally and functionally defective in animals carrying the weak loss-of-function mutation lin-26(n156). lin-26 mutant phenotypes and expression patterns together suggest that lin-26 is required to specify and/or maintain the fates not only of hypodermal cells but also of all other non-neuronal ectodermal cells in C. elegans. We speculate that lin-26 acts by repressing the expression of neuronal-specific genes in non-neuronal cells.

La division cellulaire asymétrique est un processus essentiel du développement. Ce processus ainsi que sa régulation ont fait l’objet de nombreuses études chez l’embryon de Caenorhabditis elegans. La division asymétrique de l'embryon unicellulaire est un processus conservé à travers les espèces de nématodes, cependant les caractéristiques cellulaires menant à la division sont étonnamment variables. Au cours de mon doctorat, j'ai voulu étudier ces différences en utilisant deux embryons non-C. elegans : Diploscapter pachys et Pristionchus pacificus. D. pachys est le parent parthénogénétique le plus proche de C. elegans. La polarité étant induite par le sperme chez C. elegans, on ne peut expliquer ce qui brise la symétrie chez D. pachys. Mes résultats montrent que le noyau occupe le plus souvent l’hémisphère de D. pachys qui deviendra le pole postérieur. Dans les embryons où il est astreint à un pôle par centrifugation, le noyau fini par revenir à son pôle préférentiel. Même si l’embryon est polarisé, l’agitation corticale et le cytosquelette d’actine semblent identiques aux deux pôles. D’autre part, la position du fuseau méiotique est corrélée avec la future cellule postérieure. Dans certains ovocytes, on observe des structures de microtubules émanant du fuseau méiotique combiné à un faible enrichissement en actine au future pôle postérieur. Finalement, mon principal projet de thèse montre que la polarité de D. pachys est atteinte durant la méiose, au cours de laquelle le fuseau méiotique pourrait jouer un rôle par un mécanisme présent mais inhibé chez C. elegans. Chez P. pacificus, la transgénèse biolistique a été récemment utilisée avec succès. Toutefois, par manque d’un marqueur de sélection fiable, il était illusoire de poursuivre cette approche. En conclusion, les résultats de ma thèse contribuent à une meilleure compréhension de l’embryogénèse hors C. elegans. Ils soulignent l’importance de ces espèces dans l’optique d’études comparatives
Asymmetric cell division is an essential process of development. The process and its regulation have been studied extensively in the Caenorhabditis elegans embryo. Asymmetric division of the single-cell embryo is a conserved process in nematode species, however, the cellular features leading up to division are surprisingly variable. During my PhD, I aimed to study these differences by using two non-C. elegans embryos: Diploscapter pachys and Pristionchus pacificus. D. pachys is the closest parthenogenetic relative to C. elegans. Since the polarity cue in C. elegans is brought by the sperm, how polarity is triggered in D. pachys remains unknown. My results show that the nucleus inhabits principally the hemisphere of the D. pachys embryo that will become the posterior pole. Moreover, in embryos where the nucleus is forced to one pole by centrifugation, it returns to its preferred pole. Although the embryo is polarized, cortical ruffling and actin cytoskeleton at both poles appear identical. Interestingly, the location of the meiotic spindle also correlates with the future posterior cell. In some oocytes, a slight actin enrichment along with unusual microtubule structures emanating from the meiotic spindle are observed at the future posterior pole. Overall, my main PhD project shows that polarity of the D. pachys embryo is attained during meiosis wherein the meiotic spindle could potentially be playing a role by a mechanism that may be present but suppressed in C. elegans. For P. pacificus, biolistic transgenesis has been shown recently successful. However, due to a lack of a stringent selection marker, the continuation of this project was unfeasible during my PhD. Altogether, the results of my PhD add to the understanding of non-C. elegans early embryogenesis and emphasizes on the importance of using these species for comparative studies

La morphogenèse de l'embryon de C. elegans est caractérisée par un allongement quadruple, qui se produit sans aucune division ou intercalation cellulaire. Ce processus de changement de forme cellulaire se produit en deux étapes distinctes, dont la seconde est déclenchée par un apport mécanique des muscles. Les deux étapes nécessitent de l'actomyosine dans l'épiderme. Ce travail est une investigation de la deuxième étape, notamment l'interaction entre les muscles et l'épiderme, et le rôle précis des deux myosines non musculaires NMY-1 et NMY-2. Cette paire de moteurs moléculaires est essentielle pour l'allongement, leur inhibition à l'aide de mutants sensibles à la température ayant provoqué un arrêt immédiat, malgré l'apport mécanique continu des muscles. De plus, après l'arrêt, les myosines peuvent retrouver leur état fonctionnel et l'allongement peut reprendre. Il a également été démontré ici que des moteurs de myosine inactifs forment des agrégats dans l'épiderme. Il est probable que cette paire de myosines soit chargée de tirer les câbles d'actine circonférentiels de l'épiderme l'un vers l'autre, ce qui fournit la force nécessaire à l'allongement
The morphogenesis in the C. elegans embryo is characterized by a four-fold elongation, which occurs without any cell division or intercalation. This process of cell-shape change occurs in two distinct stages, the second of which is triggered by an initial mechanical input from the muscles. Both stages require actomyosin in the epidermis. This work is an investigation of the second stage, especially the interplay between the muscles and the epidermis, and the precise role of the two non-muscle myosins NMY-1 and NMY-2. This pair of molecular motors is essential for the late stage elongation, their inhibition using temperature sensitive mutants having been shown to cause immediate arrest, despite continued mechanical input from the muscles. Furthermore, after arrest, the myosins can return to their functional state and elongation is able to resume. Inactive myosin motors also have been shown here to form aggregates in the epidermis. It is likely that this myosin pair is responsible for pulling circumferential actin cables in the epidermis towards one another, this providing the force necessary for elongation

Abstract This chapter is designed to help you to analyse the phenotype of your favourite worm or embryo by looking through a microscope. The worm is perfectly suited to analysis by light microscopy. The dimensions of the embryo (about 55 µm) or the adult (1 mm) are just in the right range, such that the whole embryo or significant parts of the body of larvae and adults can be viewed with the highest numerical aperture and therefore with the best achievable resolution. Thus, subcellular details can be seen without losing sight of the whole animal. Although a wealth of very helpful, molecular markers of cell fate are available (e.g. antibodies, transgenic lines with green fluorescent protein (GFP) or [3 galactosidase labelled proteins, or specific probes for RNA in situ), differentiated cells show very characteristic features when viewed directly through a light microscope fitted with Nomarski (DIC) optics, so that determining which tissue or organ a cell belongs to (e.g. pharynx, gut, hypodermis, nervous system, or body wall muscle) is quite easy. The use of 4D microscopy, which allows the development of whole embryos to be documented, adds a new level to the analysis of cells since the ‘behaviour ‘ of cells can also be followed. It is an interesting question whether the expression of a single molecular marker really defines the fate of a cell unambiguously. However, if a cell looks like, migrates, and intercalates like a hypodermal cell it is very likely a hypodermal cell. Many investigators have now become interested in C. elegans because the function of their favourite molecule can be readily analysed in this nematode. It is therefore the general aim of this chapter to introduce molecular biologists to the basics of microscopy. To determine the function of the gene, at least its biological function as opposed to its ‘chemical ‘ function, one has to look through a microscope. Although this is mainly a book about methods, concepts are also very important methods and therefore a short conceptual discussion about what is required to determine the function of the gene, that is characterization of the ‘phenotype ‘ of the organism lacking a gene function, is included in the section discussing 4D microscopy.

Abstract At the supramolecular level, at least, most animals start out “relatively simply”—a haploid egg is fertilized by a haploid sperm, resulting in a single diploid cell. While the rich heritage of that animal’s lineage is contained within this cell’s genetic template, the fertilized cell itself is simple in structure. From these humble beginnings arise the enormously complex adult forms containing several hundreds of cells of numerous types in some metazoans (e.g., C. elegans) to the hundreds of trillions of cells in large endothermic vertebrates. More impressive than sheer proliferation of cell number during development, however, is the increase in organismal complexity that occurs as the fertilized cell repeatedly divides to form differentiated cell types that move on to form tissues, then organs, and finally organ systems. Indeed, the combined wonders and travails of this developmental journey would seem to be reflected in the recurring theme for book titles on the subject—From Gene to Animal (De Pomerai 1985), From Egg to Embryo (Slack 1991), and From Conception to Birth (Tsiaras and Werth 2002). As is evident from the proliferation of not only scholarly works, but also coffee table and even children’s literature, there is clear and longstanding interest in the developmental journey of animals— where it starts, where it finishes, and the steps in between—as well as an appreciation for the increases in complexity that occur along this journey.