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WormBook,
2006]
In Drosophila and vertebrates, Hedgehog (Hh) signalling is mediated by a cascade of genes, which play essential roles in cell proliferation and survival, and in patterning of the embryo, limb buds and organs. In C. elegans, this pathway has undergone considerable evolutionary divergence; genes encoding homologues of key pathway members, including Hh, Smoothened, Cos2, Fused and Suppressor of Fused, are absent. Surprisingly, over sixty proteins (i.e. WRT, GRD, GRL, and QUA), encoded by a set of genes collectively referred to as the Hh-related genes, and two co-orthologs ( PTC-1 ,-3) of fly Patched, a Hh receptor, are present in C. elegans. Several of the Hh-related proteins are bipartite and all can potentially generate peptides with signalling activity, although none of these peptides shares obvious sequence similarity with Hh. In addition, the ptc -related ( ptr ) genes, which are present in a single copy in Drosophila and vertebrates and encode proteins closely related to Patched, have undergone an expansion in number in nematodes. A number of functions, including roles in molting, have been attributed to the C. elegans Hh-related, PTC and PTR proteins; most of these functions involve processes that are associated with the trafficking of proteins, sterols or sterol-modified proteins. Genes encoding other components of the Hh signalling pathway are also found in C. elegans, but their functions remain to be elucidated.
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Ann N Y Acad Sci,
1992]
The freeze-fracture technique offers a unique view of intramembrane particles (IMPs), which derive from large membrane-associated molecules such as gap junctions, receptors, and ion channels. We are particularly interested in the gap junction (gj) and its role in intercellular communication. The anatomy of the soil nematode, C. elegans, has been studied extensively in serial thin sections and gjs have been noted in many cell types. Although gjs vary in frequency and extent, their appearance in sectioned material is rather uniform. The freeze-fracture technique can be used to identify and differentiate gjs according to IMP size, packing density, and preferred fracture face. For instance, in the planarian, Dugesia, this technique revealed three classes of gjs occurring in different tissues. The nematode usually fractures lengthwise; the fracture plane preferentially travels along membranes, splitting the unit membrane into two opposing halves (the P- and E-faces). Many tissues are recognizable: hypodermis, muscle, neurons, nerve cords, intestine, and so forth...
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Methods Cell Biol,
2010]
We describe a method for automatically finding the location and conformations of microtubules in tomograms of high-pressure frozen, freeze substituted cells. Our approach uses two steps: a preprocessing step that finds locations in the tomograms that are likely to lie inside microtubules and a tracking step that uses the preprocessed data to identify the trajectories of individual microtubules. We test this method on a reconstruction of a Caenorhabditis elegans mitotic spindle and we compare our results with those obtained by a human expert who manually segmented the same data. At present, the method could be used to assist the analysis of large-scale tomography reconstructions. With further improvements, it may be possible to reliably segment cellular tomograms without human intervention.
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Methods Mol Biol,
2006]
High-pressure freezing (HPF) is capable of converting liquid water, to a depth of approx 0.6 mm, into amorphous ice nearly instantaneously. At midbody, an adult Caenorhabditis elegans hermaphrodite approaches its widest girth of approx 0.1 mm. In theory, an entire living adult animal can be physically immobilized instantly in amorphous ice by HPF, thus, providing a unique opportunity to examine cellular architecture with exquisite spatial preservation. The following chapter will discuss, in detail, procedures for freezing C. elegans under high pressure, for embedding frozen samples in resin after a freeze-substitution step, and for the postembedding immunogold labeling of proteins contained within thin sections of embedded animals. These protocols enable high-resolution analysis of both morphological features and molecular domains within most tissues of C. elegans.
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Int J Biochem Cell Biol,
2003]
Kallmann's syndrome (KS) is a genetic condition characterised by hypogonadotrophic hypogonadism (HH) and anosmia; although these are the defining features of the condition, additional neurological and non-neurological sequel may also occur depending on the specific mode of inheritance. KS affects about 1 in 8000 males and 1 in 40,000 females, with most presentations being of the 'sporadic' type. Of the inherited forms, hitherto, only the gene responsible for the X-linked form (X-KS), namely KAL-1, has been identified and the encoded protein, anosmin-1, consists primarily of a whey acidic protein (WAP) and fibronectin-like type III (FnIII) domains which appear to mediate distinctly different protein functions. The WAP/FnIII combination is conserved in anosmins across species and recent studies in rodents and in Caenorhabditis elegans demonstrate that anosmin functions in both axonal targeting and branching. Screening for loci that modify these phenotypes in C. elegans has identified heparan-6-O-sulpbotransferase as a key interactor mediating anosmin-1 function. Furthermore, over-expression and loss of function of the C elegans Kal-1 gene disrupt epidermal morphogenesis, resulting in ventral enclosure and male tail formation defects. These findings provide novel insights into the molecular pathogenesis of X-KS.
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Comp Biochem Physiol C Toxicol Pharmacol,
2002]
Oxygen is essential for most life forms, but it is also inherently toxic due to its biotransformation into reactive oxygen species (ROS). In fact, the development of many animal and plant pathological conditions, as well as natural aging, is associated with excessive ROS production and/or decreased antioxidant capacity. However, a number of animal species are able to tolerate, under natural conditions, situations posing a large potential for oxidative stress. Situations range from anoxia in fish, frogs and turtles, to severe hypoxia in organs of freeze-tolerant snakes, frogs and insect larvae, or diving seals and turtles, and mild hypoxia in organs of dehydrated frogs and toads or estivating snails. All situations are reminiscent of ischemia/reperfusion events that are highly damaging to most mammals and birds. This article reviews the responses of anoxia/hypoxia-tolerant animals when subjected to environmental and metabolic stresses leading to oxygen limitation. Abrupt changes in metabolic rate in ground squirrels arousing from hibernation, as well as snails arousing from estivation, may also set up a condition of increased ROS formation. Comparing the responses from these diverse animals, certain patterns emerge. The most commonly observed response is an enhancement of the antioxidant defense. The increase in the baseline activity of key antioxidant enzymes, as well as ''secondary'' enzymatic defenses, and/or glutathione levels in preparation for a putative oxidative stressful situation arising from tissue reoxygenation seem to be the preferred evolutionary adaptation. Increasing the overall antioxidant capacity during anoxia/hypoxia is of relevance for species such as garter snakes (Thamnophis sirtalis parietalis) and wood fogs (Rana sylvatica), while diving freshwater turtles (Trachemys scripta elegans) appear to rely mainly upon high constitutive activities of antioxidant enzymes to deal with oxidative stress arising during tissue reoxygenation. The possibility that some animal species might control post-anoxic ROS generation cannot be excluded.
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Methods Cell Biol,
1995]
Although Caenorhabditis elegans was originally chosen as a model organism for cell biology with serial section electron microscopy (EM) methods in mind, these methods have remained a daunting challenge. There is an apocryphal story that Nichol Thomson originally advised Sydney Brenner that C. elegans was unsuitable for electron microscopy and that Brenner should choose another species. Other experienced microscopists have probably shared similar dark thoughts from time to time. Nonetheless, the worm's very small size, simple organization, and cablelike nervous system have permitted Brenner's colleagues to characterize every cell and cell contact in the wild-type animal, potentiating the genetic characterization of cellular development in remarkable detail. We attempt to provide an adequate background for anyone to initiate EM studies of C. elegans. Two decades ago, as the first of Brenner's postdoctoral fellows left his laboratory to establish new worm laboratories, it was standard practice to include an EM component in their studies. Their combined efforts to characterize the adult animal's cell types and the essential steps in its development helped to erect a lovely scaffold of key manuscripts, capped by the description of the "Mind of the Worm" in some 600 micrographs and 175 drawings. Many of these works required technical heroics or suffered long delays before publication. Most people later chose to leave electron microscopy behind in pursuit of molecular quarry. The fruits of their molecular and genetic studies should soon stimulate a renewed flowering of electron microscopy. We hope to smooth your entry or reentry into these techniques. We also summarize our methods for three-dimensional (3D) image reconstruction, based largely on film techniques introduced by John White and Randle Ware. Digital imaging techniques seem poised to make 3D reconstruction more accessible, and may simplify the exchange of morphological data between laboratories. We discuss several computer systems that the C. elegans community could adopt for high-resolution studies of structure and function. In addition, we briefly cover several specialized specimen preparation techniques for electron microscopy, including freeze fracture and electron microscopic immunocytochemistry.