[
1977]
The soil nematode Caenorhabditis elegans was selected 11 years ago by Sydney Brenner as an experimental organism suitable for the isolation of many behavioral mutants and small enough for anatomical analysis of such mutants with the electron microscope. Two distinct goals motivated the initial studies of this organism: first, the hope that some of the mutants would have simple anatomical alterations that could be directly correlated with their behavioral defects, allowing the assignment of specific functions to specific neurons, and second, the hope that the detailed analysis of the kinds of alterations induced by individual mutations and the classes of cells affected by given mutations would reveal general features of the genetic program that specifies the development of the organism. Over the past 11 years the number of investigators working on C. elegans has increased to about 75 and is still growing. Nearly 3,000 different mutants have been isolated and different investigators are pursuing their effects on different cells. My own research is in the development of the nervous system. In particular, I would like to learn something about the workings of the complex black box that connects individual genes to the determination of the morphology of developing neurons. Are there gene products whose specific function is to determine the morphology of cells? If so, what are these gene products and how do they act in the developing cell? One would anticipate that mutations in such hypothetical genes would cause specific morphological alterations in cells. Because the morphology of a neuron determines its function, by selecting behavioral mutants altered in the function of the nervous system one might commonly find mutants that alter the morphology of neurons, and some of these might be in specific morphological genes. It is my hope that it will be possible to compare such mutants to the wild type in order to identify the defective gene products and thereby learn something about the role of normal gene products in determining the development of neurons. In this paper I will first summarize the results of several years' work on one specific class of mutants in the nematode, sensory mutants, work performed both in my laboratory and that of my colleagues Jim Lewis and Jonathan Hodgkin. Second, I will discuss frankly some of the difficulties and frustrations we have experienced in trying to interpret the effects of these specific mutants. Some of these difficulties illustrate problems endemic to genetic studies of development. Third, I will describe the more recent work performed in my labortory that is being directed toward genetic analysis of the structure and function of a
[
1994]
Nematodes have been cultured continuously in the laboratory since 1944 when Margaret Briggs Gochnauer isolated and cultured the free-living hermaphroditic species Caenorhabditis briggsae. Work with C. briggsae and other rhabditid nematodes, C. elegans, Rhabditis anomala, and R. pellio, demonstrated the relative ease with which they could be cultured. The culturing techniques described here were developed for C. elegans, but are generally suitable (to varying degrees) for other free-living nematodes. Whereas much of the early work involved axenic culturing, most of these techniques are no longer in common use and are not included here. In the 1970s C. elegans became the predominant research model due to work by Brenner and co-workers on the genetics and development of this species. An adult C. elegans is about 1.5 mm long, and under optimal laboratory conditions has a life cycle of approximately 3 days. There are two sexes, males and self-fertile hermaphrodites, that are readily distinguishable as adults. The animals are transparent throughout the life cycle, permitting observation of cell divisions in living animals using differential interference microscopy. The complete cell lineage and neural circuitry have been determined and a large collection of behavioral and anatomical mutants have been isolated. C. elegans has six developmental stages: egg, four larval stages (L1-L4), and adult. Under starvation conditions or specific manipulations of the culture conditions a developmentally arrested dispersal stage, the dauer larva, can be formed as an alternative third larval stage. Many of the protocols included here and other experimental protocols have been summarized in "The Nematode Caenorhabditis elegans". We also include a previously unpublished method for long-term chemostat cultures of C. elegans. General laboratory culture conditions for nematode parasites of animals have been described, but none of these nematodes can be cultured in the laboratory through more than one life cycle. Marine nematodes and some plant parasites have been cultured xenically or with fungi. Laboratory cultivation of several plant parasites on Arabidopsis thaliana seedlings in agar petri plates has also been reported.