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[
Methods Cell Biol,
1995]
Geneticists like to point out that the ultimate test of a proposed function for a gene and its encoded product (or products) in a living organism involves making a mutant and analyzing its phenotype. This is the goal of reverse genetics: a gene is cloned and sequenced, its transcripts and protein coding sequence are analyzed, and a function may be proposed; one must then introduce a mutation in the gene in a living organism to see what the functional consequences are. The analysis of genetic mosaics takes this philosophy a step further. In mosaics, some cells of an individual are genotypically mutant and other cells are genotypically wild type. One then asks what the phenotypic consequences are for the living organism. This is not the same as asking what cells transcribe the gene or in what cells the protein product of the gene is to be found, but rather it is asking in what cells the wild-type gene is needed for a given function...
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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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[
1980]
Largely through the efforts of Sydney Brenner, many investigators have been attracted to Caenorhabditis elegans as a model organism for asking questions about the genetic basis of eucaryotic development and animal behavior. The philosophy underlying this interest is that of molecular genetics, which has relied heavily on the analysis of single-step mutants to elucidate such genetically controlled processes as metabolic pathways, the regulation of procaryotic gene expression, and the in vivo assembly of bacteriophages. Development and behavior also make use of genetic programs, and it may help to mutate the program steps to discern their nature. At present this seems an enormous undertaking because morphogenesis and behavior, as we now understand them, seem very remote from the genes that control them. Clearly other techniques--physiological, biochemical, anatomical--will be required. But the philosophy behind much C. elegans research is that mutant analysis will ultimately play a valuable role in understanding development and behavior. With this philosophy in mind, the attraction of C. elegans is twofold: suitability for genetic analysis and relative cellular simplicity. The latter feature is reviewed by others in this volume. The purpose of this review is to summarize the current status of the genetics of C. elegans; the applications of genetic analysis to the problems of development and behavior will be left to other reviewers and future work.
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[
Bioscience,
1984]
A small roundworm Caenorhabditis elegans has been the subject of extensive studies on the structure, function, and development of the nervous system. It is the only multicellular organism for which both the cellular anatomy (morphology and connectivity) and cell lineage origin for each of its 302 nerve cells are known. These data and the ability to obtain mutants that are defective in nerve-cell origin, structure, or function allow a detailed examination of the genetic control of nerve-cell production and differentiation. The use of touch-insensitive mutants to study the development of the six touch-receptor neurons of C. elegans is an example of such an analysis.
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[
J Neurogenet,
1989]
The use of genetic mosaics and chimeras is a well established tool in developmental and behavioral genetics. One aim in work with Drosophila mosaics and mouse chimeras has been to elucidate cell lineages. This application of mosaics is largely unnecessary for the nematode Caenorhabditis elegans because the complete wild-type cell lineage has been worked out directly by Nomarski microscopy of living animals. Mosaics have in some cases provided verification of the correctness and invariance of parts of the wild-type C. elegans cell lineage, and it is conceivable that mosaics could be useful in the clonal analysis of mutant lineages, which may be quite variable. But the major goal of mosaic analysis in C. elegans so far has been to address questions about the cell specificity of gene function. These questions arise in two contexts. In the first case, a mutant gene is known to cause a detectable change in a cellular phenotype. One can then use mosaics to ask whether or not the gene is cell autonomous in its action; if it is not, then an interaction with one or more other cells is involved and one can use mosaics again to ask what other cells might be responsible. In the second case, nothing is known at the cellular level about the consequences of a mutant gene; mutant animals may have been identified by virtue of being deficient for an enzyme, for example, or being inviable or showing aberrant behavior or morphology. The general question in this case is: what is the anatomical focus of action of the gene, i.e., what cell or cells are responsible for the phenotype conferred by the gene? In this review I shall first introduce the method by which all of the C. elegans genetic mosaics to be discussed have been generated, and then I shall review the mosaic analysis of 18 genes, eight that were known at the outset to affect cellular phenotypes and ten that were first studied on the basis of their effects on behavior, overall morphology or some other non-cellular phenotype. The positions on the C. elegans genetic map of the loci to be discussed are indicated in Figure 2. Some discussion of C. elegans cell lineage will be necessary for understanding the mosaic analysis of each gene. The derivation of the early embryonic cleavage products called founder cells and a summary of the numbers and types of cells derived from them are given in Figure 2. For several genes it will also be necessary to discuss aspects of C. elegans nervous system structure and function.
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[
Methods,
2016]
With the development of bio-imaging techniques, an increasing number of studies apply these techniques to generate a myriad of image data. Its applications range from quantification of cellular, tissue, organismal and behavioral phenotypes of model organisms, to human facial phenotypes. The bio-imaging approaches to automatically detect, quantify, and profile phenotypic changes related to specific biological questions open new doors to studying phenotype-genotype associations and to precisely evaluating molecular changes associated with quantitative phenotypes. Here, we review major applications of bioimage-based quantitative phenotype analysis. Specifically, we describe the biological questions and experimental needs addressable by these analyses, computational techniques and tools that are available in these contexts, and the new perspectives on phenotype-genotype association uncovered by such analyses.
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[
Mol Neurobiol,
1990]
The established observations and unresolved questions in the assembly of myosin are outlined in this article. Much of the background information has been obtained in classical experiments using the myosin and thick filaments from vertebrate skeletal muscle. Current research is concerned with problems of myosin assembly and structure in smooth muscle, a broad spectrum of invertebrate muscles, and eukaryotic cells in general. Many of the general questions concerning myosin assembly have been addressed by a combination of genetic, molecular, and structural approaches in the nematode Caenorhabiditis elegans. Detailed analysis of multiple myosin isoforms has been a prominent aspect of the nematode work. The molecular cloning and determination of the complete sequences of the genes encoding the four isoforms of myosin heavy chain and of the myosin-associated protein paramyosin have been a major landmark. The sequences have permitted a theoretical analysis of myosin rod structure and the interactions of myosin in thick filaments. The development of specific monoclonal antibodies to the individual myosins has led to the delineation of the different locations of the myosins and to their special roles in thick filament structure and assembly. In nematode body-wall muscles, two isoforms, myosins A and B, are located in different regions of each thick filament. Myosin A is located in the central biopolar zones, whereas myosin B is restriced to the flanking polar regions. This specific localization directly implies differential behavior of the two myosins during assembly. Genetic and structural experiments demonstrate that paramyosin and the levels of expression of the two forms are required for the differential assembly. Additional genetic experiments indicate that several other gene products are involved in the assembly of myosin. Structural studies of mutants have uncovered two new structures. A core structure separate from myosin and paramyosin appears to be an integral part of thick filaments. Multifilament assemblages exhibit multiple nascent thick filament-like structures extending from central paramyosin regions. Dominant mutants of myosin that disrupt thick filament assembly are located in the ATP and actin binding sites of the heavy chain. A model for a cycle of reactions in the assembly of myosin into thick filaments is presented. Specific reactions of the two myosin isoforms, paramyosin, and core proteins with multifilament assemblages as possible intermediates in assembly are proposed.
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[
J Neurobiol,
2002]
The nicotinic acetylcholine receptor is among the most thoroughly characterized molecules in the nervous system, and its role in mediating fast cholinergic neurotransmission has been broadly conserved in both vertebrates and invertebrates. However, the accessory molecules that facilitate or regulate nicotinic signaling remain mostly unknown. One approach to identify such molecules is to use molecular genetics in a simple, experimentally accessible organism to identify genes required for nicotinic signaling and to determine the molecular identity of the mutant genes through molecular cloning. Because cellular signaling pathways are often highly conserved between different animal phyla, the information gained from studies of simple organisms has historically provided many critical insights into more complex organisms, including humans. Genetic screens essentially make no prior assumptions about the types of molecules involved in the process being studied; thus, they are well suited for identifying previously unknown components of cell signaling pathways. The sophisticated genetic tools available in organisms such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster have also proven extremely powerful in elucidating complex biologic pathways in the absence of prior biochemical information and for assessing a molecule's in vivo function of in the context of an intact nervous system. This review describes how genetic analysis has been used to investigate nicotinic signaling mechanisms in worms and flies, and the prospects for using these studies to gain insight into nicotinic receptor function
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[
WormBook,
2013]
The proper understanding and use of statistical tools are essential to the scientific enterprise. This is true both at the level of designing one's own experiments as well as for critically evaluating studies carried out by others. Unfortunately, many researchers who are otherwise rigorous and thoughtful in their scientific approach lack sufficient knowledge of this field. This methods chapter is written with such individuals in mind. Although the majority of examples are drawn from the field of Caenorhabditis elegans biology, the concepts and practical applications are also relevant to those who work in the disciplines of molecular genetics and cell and developmental biology. Our intent has been to limit theoretical considerations to a necessary minimum and to use common examples as illustrations for statistical analysis. Our chapter includes a description of basic terms and central concepts and also contains in-depth discussions on the analysis of means, proportions, ratios, probabilities, and correlations. We also address issues related to sample size, normality, outliers, and non-parametric approaches.