Curr Protoc Neurosci,
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.
Methods Cell Biol,
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
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.