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[
Worm Breeder's Gazette,
2013]
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[
Science,
1985]
The biologists who investigate nature's deepest and longest-running mystery often use the term fate map to describe the startling transformations that lie in store for the fertilized egg. It is one of the more venerable terms in embryology, and one of the most appropriate, too, for destiny and geography indeed intersect within the magnificent speck of DNA and cytoplasm that is an egg on the edge of becoming a organism. In this one cell, the entire genetic bill of lading for an animal, be it fruit fly or human, is stored, waiting to unfold with miraculous precision. It is that process of life unfurling-of cells becoming brain or backbone, of genes selectively flashing on and herding cells toward their certain fates, of tissues aggregating and differentiating toward ever more specific tasks-that both confounds and as surely delights developmental biologists.
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[
Worm Breeder's Gazette,
1998]
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[
Development & Evolution Meeting,
2006]
In the beginning, rather little was known about what types of cells, tissues or organelles might lie within the nematode C. elegans. The very small size of the animal pushed us to begin exploring the anatomy using the electron microscope. Most tissue types and cell types in the worm emerged gradually after 1968, when Sydney Brenner, John White, and Nichol Thomson first began to study the animal in depth by serial thin sections. They were soon joined by a host of others in Cambridge and Pasadena, and eventually in many more labs. By 1980, most of this TEM work was completed, and the community moved on to biochemical, molecular and genomic studies, and relied more heavily on other modalities of microscopy. Exact characterization of all individual neurons and their synapses by TEM took still longer (1986), and a few cell types were still a mystery into the 21st century. Similarly, many familiar organelles and cell junctions were quickly identified by TEM in virtually all cells. Indeed the nematode is made up of simple cells that share many common features with higher animals. To a lesser extent, morphogenesis in the embryo and the larval stages has also been characterized by TEM.
Since 1980, TEM has also been used to explore the pathology of cells gone wrong in the worm: in developmental mutants, under toxic conditions, in aging, and during key events such as cell death. Even given the advent of GFP probes, FRET, confocal imaging, and laser ablations, we continue to lean on TEM techniques to answer certain questions. I will briefly survey some of the events, cells and organelles whose anatomy remains mysterious, and the techniques that show promise to reveal them at high resolution.
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[
Worm Breeder's Gazette,
1977]
Two young adult C. elegans have been serially sectioned and reconstructed from the tail tip forward through the anterior end of the pre-anal ganglion. Thirty-nine neurons can be identified in the tail, twelve cells in each lumbar ganglion, twelve cells in the pre- anal ganglion, and three cells in the dorso-rectal ganglion. Each cell in the tail can be reproducibly identified on the basis of a set of morphological features, including cell body position, fiber projections, fiber size, and cytoplasmic appearance. Eleven neurons in each lumbar ganglion are bilaterally homologous. Many lumbar cells have sensory dendrites in the tail. Two pairs of lumbar cells which lack sensory dendrites are prominent interneurons in the synaptic interactions of the tail. Virtually all synaptic contacts in the tail are found in the pre-anal ganglion. Most synapses involve lumbar fibers and fibers from cells whose cell bodies lie anterior to the reconstructed region. Pre-anal ganglion cells themselves are relatively minor participants in these synaptic interactions. A complete connectivity matrix has been constructed for both animals, involving about 150 synapses in each case. Certain ceIls make repeated contacts with one another (up to thirteen contacts) in both animals. Other instances of non-reproducible synapses are found, usually involving one contact in one animal and none in the other. No self-synapses are observed, but sensory cells frequently synapse onto their bilateral homologues. Homologously paired cells make similar sets of synaptic contacts. One class of reciprocal synapse formation is found. Eighty per cent of the contacts are dyadic, with one pre-synaptic cell and two post-synaptic ones. Ten per cent of the contacts are triadic; the remaining ten per cent are apparently conventional synapses with a single post-synaptic element. Each dyadic synapse generally involves three different types of neurons - none homologous to another - such that A- B/C. Each type of pre-synaptic neuron (A) contacts only a few preferred pairs of fibers (B, C). Most dyadic contacts are involved in multiple routes of information flow, such that A- B/C and, elsewhere B-C. The formation of dyadic synapses appears to follow strict rules which may reflect important factors in the development of the nervous system. Most synaptic Interactions can be included in a simple wiring diagram by which information flows from sensory cells through multiple routes to converge on a pair of interneurons which project forward into the ventral cord. Positional information is used to identify three pairs of interneurons which are important both in ventral cord synaptic patterns and in the synaptic interactions of the pre-anal ganglion.
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[
International C. elegans Meeting,
1997]
This method has been used to study the expression pattern of a protein at high resolution in identified cells. A post-embedding procedure was performed on LR Gold thin sections of lightly fixed animals, marking MH27 binding with a gold-linked anti-mouse IgG secondary antibody. Methods for fixation, embedding, sectioning and antibody procedures were modified from those of Selkirk et al. (1). We will discuss improvements to the procedure, particularly to label fragile tissues and embryos. Adherens junctions (AJs) were intensely labelled in intestine, pharynx, seam cells and hypodermis. In the intestine, a continuous narrow band of apical AJs link adjacent pairs of intestinal cells. Gap junctions are known to lie very near to the apical AJs in both intestine and hypodermis, but were not labelled by MH27. Smooth septate junctions were heavily labelled between epithelial cells in the spermatheca. Pleated septate junctions immediately adjacent in the same membranes showed no labelling. Negative staining with lanthanum was used to further characterize the septate junctions. The high antigenicity and ubiquitous nature of AJs in intestine and hypodermis along the length of the nematode make the MH27 antibody useful when testing immunochemical procedures in C. elegans. MH27 antibody was generously provided by Jim Waddle and Ross Francis (2). 1. M.E. Selkirk et al. (1990) Mol. & Biochem. Parasitol. 42: 31. 2. G.R.Francis & R.H.Waterston (1985) J. Cell Biol. 101: 1532.
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[
Development & Evolution Meeting,
2008]
The pages of WormAtlas are getting a fresh look and organization.These changes will start from the front page and then be implemented throughout the Handbook and many other portions of the website.Here we will explain the principles of the new organization, and show you how to find your favorite features.This is the first major revamping of WormAtlas since its launch in 2002.We hope you will find the site simpler to navigate and we expect it will be more intuitive for beginners.As much as possible, these changes should not disrupt any previous weblinks you have established to your favorite pages.Inside the WormAtlas website, there will be several major changes. First will be an improved adult hermaphrodite handbook; it will include several completely revised chapters and a new one covering the nervous system. Second will be the launch of a handbook for anatomy of the worm embryo.Third will be the addition of more data to Slidable Worm.Lastly, we will be adding many new Neuron pages for the male nervous system in order to highlight new synaptic patterns emerging from the Wired Worm project conducted together with Scott Emmons. The WormImage website is expanding steadily.It now presents much more mutant data, particularly for genes affecting the nervous system.As before, we are relying heavily on MRC datasets, but we will continue to add more from the Riddle and Hall lab files.We encourage more laboratories to share your own best archival TEM and SEM images for this purpose.We are very grateful to many labs that have already contributed ideas, advice and experimental results that are featured on these websites.This work is supported by NIH RR12596.
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[
Worm Breeder's Gazette,
1996]
Immunofluorescence studies using MH27 antibody have shown a wide variety of tissues to be labelled in patterns which define apical cell borders in C. elegans. Disappearance of MH27 binding during development often marks cell fusions in hypodermis and in the male tail (Podbilewicz and White, 1994; Fitch and Emmons, 1995). We have used an immunoEM method to learn the nature of membrane structures marked by MH27. A post-embedding technique was used to apply MH27 Ab to thin sectioned worms, followed by a gold-linked secondary Ab (for methods, see Selkirk et al., 1991; Hall, 1995). Electron microscopy reveals that MH27 binds only to a few types of cell junctions. By immunoEM, MH27 can be seen to bind to zonula adherens junctions in intestine, at hypodermal/seam cell borders, and at pharyngeal muscle/support cell borders. In adult pharyngeal muscle, small longitudinal stripes of adherens "junction" are also retained on the apical surface where pairs of muscle cells had fused to become syncytial. Thus MH27 binding can sometimes mark the vestiges of an old cell border, long after cell fusion. Spermathecal cells are held closely together by two types of "septate" junction, which together cover almost all of their lateral borders. Extensive, dark-staining apical junctions, which look vaguely adherens-like, are not labelled by MH27. In more basal regions, MH27 binds heavily to another class of extensive sinuous junctions, which lack any dramatic staining characteristics. Neither class of junction has osmiophilic septa visible in ordinary sections. The apical junctions are rather closely apposed and show periodic striping of the thick cytoplasmic density bordering the junctions. The basal, sinuous junctions show an even, widened extracellular space between adjacent plasma membranes, with faint periodic striations crossing the extracellular space between the cells. There is no cytoplasmic density associated with the sinuous junctions. Lanthanum infiltration has been used to negatively stain extracellular septa in the spermathecal junctions. The apical junctions have long, wavy septa; they may comprise a novel class of septate junction, characterized by the thick densely-staining coat on the cytoplasmic face of each cell. The sinuous junctions have not been well infiltrated with lanthanum yet. In one instance we did observe short septa at regular intervals, spanning the extracellular space, similar to those expected in "smooth septate junctions" as described in other invertebrate species. These two classes of septate junction may resist the stresses which would otherwise tear apart the spermatheca during the passage of an oocyte. Our current technique does not permit resolution of the exact locus of MH27 antigen within the adherens junction or the smooth septate junction. It could be cytoplasmic, extracellular, or within the plasma membrane. In glancing sections of both types of junction, it is clear the antigen is found evenly along the entire face of the cell-cell appositions, possibly at a fixed distance in relation to the plasma membrane. High resolution studies would require a more direct labelling method, such as attaching a small gold tag directly to a Fab fragment of the MH27 antibody. This would bring the gold tag into closer proximity to the true antigenic site within a thin section. We thank Jim Waddle and Ross Francis (Washington U.) for generously supplying MH27 antibody. Fitch and Emmons (1995) Dev. Biol. 170: 564-582. Hall (1995) In C. elegans: Modern Biological Analysis of an Organism. H.F. Epstein and D.C. Shakes (eds.). Academic Press, New York, pp. 395-436. Podbilewicz and White (1994) Dev. Biol. 161: 408-424. Selkirk et al. (1991) J. Biol. Chem. 266: 11002-11008.