[
Worm Breeder's Gazette,
1994]
LOW TEMPERATURE AFFECTS EMBRYONIC HANDEDNESS REVERSAL Bill Wood, Arny Florance and Dominique Bergmann Department of MCD Biology, University of Colorado, Boulder C. elegans embryos become left-right asymmetric at the 6-cell stage witlf a handedness which is essentially invariant among N2 animals reared at 20, and which persists into adulthood, determining the asymmetric placement of the gonad, intestine, the coelomocytes, and several other cells. Reversal of embryonic handedness at the 6-cell stage by micromanipulation results in development of healthy adults with all left-right asymmetries reversed. l Spontaneous reversal occurs at a frequency of a few percent among animals developing embryos treated with chitinase at the 2-cell stage to remove the egg shell.2 We have now found that when N2 are reared at 10, the frequency of animals with reversed gonad handedness as adults increases to above 0.5%. Those examined by Nomarski microscopy exhibited handedness reversal of the ventral nerve cord (cell bodies on the left) and reversed placement of the coelomocytes as well as the intestine and gonad, suggesting that these animals developed from reversed embryos. To determine the cold-sensitive period for handedness reversal, young gravid adults reared at 20 were placed at 10 for 2 hours, then transferred to new 20 plates and subsequently transferred to fresh 20 plates at 2-hr intervals. Eggs laid during the cold pulse (interval l; exposed to 10 after fertilization), the first 2 hr after return to 20 (interval 2; exposed to 10 either just before or just after fertilization), and each of the four subsequent 2-hr intervals at 20 (3-6, exposed to 10 before fertilization) were allowed to hatch and develop at 20 into adults, which were then scored for gonad handedness. Among animals developing from embryos exposed to 10 after fertilization (interval 1), no reversals were' observed (N=1,956), whereas among animals developing from oocytes and sperm exposed to 10 before fertilization (intervals 3-6), the frequency of reversal was 0.21% + 0.09% (N=9,223). Surprisingly, although left-right asymmetry first appears at the 6-cell stage, and the embryo's left and right are highly unlikely to be specified until the 4-cell stage, I the cold-sensitive period for reversal is before fertilization. This result suggests that the effect is on the gametes. We can imagine two possible explanations: 1) Production of some eggshell precursor component, synthesized during oogenesis, could be cold sensitive, such that embryos with "soft" eggshells are produced at 10, leading to a low frequency of reversal as seen in chitinased embryos.2 2) Some handed structure in a gamete, such as the sperm centriole for example, may normally dictate the skewing of the ABa and ABp spindles that leads to handed asymmetry in the embryo;l when this structure is exposed to 10 its subsequent ability to dictate handedness could be impaired, leading to a low frequency of reversal. We are currently carrying out experiments with chilled males to determine whether egg or sperm is the cold-sensitive gamete, as well as examining effects of other treatments on handedness reversal. Finally, we are continuing small scale screens for maternal-effect mutations that result in a high frequency of embryonic reversal. None have been found, but so far we have screened only about 1000 EMS-mutagenized genomes. I Wood, W. B. (1991) Nature 349: 536-538. 2 Wood, W. B. and Kershaw, D. (1991) In Biological Asymmetry and Handedness, CIBA Foundation Symposium 162: 143-164.
[
Worm Breeder's Gazette,
1988]
I've written a little primer on how to use Word (version 3.0 or higher) to automatically generate references (reference number or author-year citation) and cross-references (to page number) within a document using a combination of Word's merge function and table of contents generation facility. Also included in the article are directions for using reference information stored in a database to automatically generate a reference list according to a given style using Word's merge function. The utility of all this is likely to be superseded by future versions of Word and commercially available add- on programs. However, if you are interested, drop me a line and I'll send you a copy of my article.
[
Worm Breeder's Gazette,
1986]
In the past, we have used the standard method for decontaminating stocks, which involves washing worms off a plate, spinning them in a centrifuge, etc The following procedure is much quicker and requires fewer worms. Also, it can be used to directly decontaminate the progeny of a mating and thus accelerate strain constructions. Using a platinum wire with a glob of bacteria on the bottom, pick up several gravid adults and/or eggs from the contaminated plate and transfer them to a seeded plate. Then put a drop of the usual sodium hypochlorite solution (2-4% NaOCl, .4M NaOH) onto the eggs. About half of the eggs will hatch. Occasionally, a very tough bacterial contaminant will survive to form a few colonies, so it is wise to transfer the survivors on the following day.
[
Worm Breeder's Gazette,
1984]
We have written a computer program for storage and retrieval of information about the recombinant DNA collection held by a laboratory. For each clone, information such as clone name, cloning vector, cloner, date cloned, notebook page, storage location, etc., is entered. The cloned insert is identified by a serially-assigned fragment number and by a restriction map. Wherever a given fragment occurs in the collection it is identified by its fragment number, and all restriction mapping data for that fragment is held together in a single storage location and kept updated. Data regarding interrelationships among fragments in the collection, such as which are subclones of others, are also held. The stored information can be searched and listed in a variety of ways. The program is modular in design and can be readily modified and expanded. It is written in Basic for a Dec RT-11 operating system ( PDP-11 computer or equivalent) and should be easily adaptable to other systems. We encourage other laboratories to use it. The program anticipates multiple laboratory use by prefixing each fragment number with a laboratory number. By this means each cloned segment of the genome is given a unique name. Eventually there will undoubtedly be a need for a centralized listing of information about cloned fragments of the C. elegans genome. We suggest that a centralized listing could be a subset of the information stored in laboratory based listings such as this one. For a free interchange of information among laboratories and between laboratories and a centralized data base to be possible, a uniform system of nomenclature and computerized format will have to be adopted. We have written this program in order to gain some experience with the use of a laboratory based data bank that hopefully will be helpful in designing a widely used system. Please contact us if you are interested in using this program. We would be glad to help you adapt it to your computer system.