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Sorrentino V, Deplancke B, Ouhmad T, Cornaglia M, Gijs MA, Auwerx J, Williams EG, Krishnamani G, Frochaux MV, Nicolet-Dit-Felix AA, Lin T, Mouchiroud L
[
Curr Protoc Neurosci,
2016]
Phenotyping strategies in simple model organisms such as D. melanogaster and C. elegans are often broadly limited to growth, aging, and fitness. Recently, a number of physical setups and video tracking software suites have been developed to allow for accurate, quantitative, and high-throughput analysis of movement in flies and worms. However, many of these systems require precise experimental setups and/or fixed recording formats. We report here an update to the Parallel Worm Tracker software, which we termed the Movement Tracker. The Movement Tracker allows variable experimental setups to provide cross-platform automated processing of a variety of movement characteristics in both worms and flies and permits the use of simple physical setups that can be readily implemented in any laboratory. This software allows high-throughput processing capabilities and high levels of flexibility in video analysis, providing quantitative movement data on C. elegans and D. melanogaster in a variety of different conditions. 2016 by John Wiley and Sons, Inc.
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In the next five years, molecular biology will get its first look at the complete genetic code of a multicellular animal. The Caenorhabditis elegans genome sequencing project, a collaboration between Robert Waterston's group in St. Louis and John Sulston's group in Cambridge, is currently on schedule towards its goal of obtaining the complete sequence of this organism and all its estimated 15,000 to 20,000 genes by 1998. By that time, we should also know the complete genome sequence of a few other organisms as well, including the prokaryote Escherichia coli and the single-celled eukaryote Saccharomyces
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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.
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[
Methods Cell Biol,
1995]
In studying embryos of many species, methods of fragmenting and culturing embryonic tissues or cells have been useful for addressing questions of blastomere autonomy in early and later embryogenesis, for exposure to drugs or other agents that perturb specific processes, and for direct labeling of DNA or RNA. For Caenorhabditis elegans workers, the small size of the embryo and the impermeability of the chitinous eggshell and inner vitelline membrane have made such experiments difficult. A method of permeabilization and blastomere isolation, a culture system that will support further cellular development and differentiation, and assay methods for assaying the degree of development and its relative normality after experimental manipulation are minimal requirements for a satisfactory C. elegans embryonic culture system. Methods of isolating early blastomeres have included crushing of the eggshell and extrusion, laser ablation of neighboring blastomeres within an itact eggshell, laser puncturing of the eggshell producing extrusion, and digestion of the eggshell followed by shearing or manual stripping of the vitelline membrane. This last method is described in detail below. Permeabilization of complete embryos can be achieved by the same methods; in addition, one-cell embryos within the shell can be permeabilized to certain drugs such as cytochalasin D by gentle pressure on an overlying
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[
1987]
Vitellogenins of many insects, vertebrates, nematodes and sea urchins are very similar in size and amino acid composition. We have determined the nucleotide sequences of the genes that encode vitellogenins in nematodes (C. elegans) and sea urchins (S. purpuratus), and compared the deduced amino acid sequences to the published sequences of two vertebrate vitellogenins (X. laevis and G. gallus). This comparison demonstrated unequivocally that the nematode and vertebrate proteins are encoded by distant members of a single gene family. The less extensive sequence data available for the sea urchin gene indicates that this, too, may be a member of this family of genes, as may the vitellogenin genes of locust. On the other hand, we were unable to detect any similarity between these genes and the D. melanogaster yolk protein genes. Thus it appears that while nematodes, vertebrates, sea urchins and at least some insects utilize the same family of genes to encode vitellogenins, Drosophila uses a different gene family. All of the vitellogenin genes are regulated in a tissue-specific manner. They are expressed in the intestine in nematodes, in the liver in vertebrates, in the fat body in insects, and in the intestine and gonad in sea urchins. Their production is limited to adult females in all species except sea urchins, in which they are expressed by adults of both sexes. In nematodes we have identified two heptameric sequence elements repeated multiple times in all eleven of the vitellogenin genes sequenced. One of these elements is also present in the vertebrate promoters and has recently been shown to be required for transcriptional activation. All of the 5' ends of the vitellogenin mRNAs of nematodes, vertebrates and locust can be folded into potentially-stable secondary structures. We present evidence that these structures have been strongly selected for and presumably perform some function in regulation of vitellogenin production.