[
Worm Breeder's Gazette,
1994]
Recently, we fortuitously revealed phalloidin-stained, ring-shaped structures shared by sister cells in the early embryo. The detection of these structures requires formaldehyde fixation followed by 100% acetone extraction, a treatment that extracts other phalloidin-binding components including most of the cortical actin cytoskeleton in early nematode embryos. The time of appearance, position, morphology and number of phalloidin-positive rings within embryos suggest these organelles are persistent remnants from prior cell divisions. We refer to these rings as cell division remnants (CDRs). Preliminary characterization of these structures reveals the following: (i) A given embryo always has one fewer CDR than the total number of cells or a number equal to the number of cell divisions. (ii) The CDRs persist through many cell cycles and appear to be shared by all sister cells. (iii) The dimensions and position of the CDRs are similar to the initial size and location of the transient, CP-actin complexes that coincide with the onset of centrosome rotation in the P(1) lineage(1). For example, the CDR from first cleavage is located at a central site in the midfocal plane between AB and P(1) early in the 2-cell stage. Later, nuclear movements in the dividing AB cell push the CDR off-center in a manner which resembles the movement of the transient CP-actin complex during the 2-3 cell transition. (iv) The diameter of CDRs is variable (0.25-2.0 m) and unpredictable. In general, older CDRs are smaller but there is no obvious correlation between ring diameter and the cell cycle. (v) The CDR derived from first cleavage is invariably shared by EMS and ABa at the 4-cell stage, EMS and ABal at the 6-cell stage and MS and ABal at the 8-cell stage. At least some of the above observations suggest that once a CDR is formed, it is not divisible by subsequent cell divisions. Are CDRs involved in cell fate decisions? At the 4-cell stage, a signal from the EMS blastomere is required to instruct the anterior AB daughter to express a fate different from its sister, this interaction requires maternal expression of theglp-1 gene product (2,3). Theglp-1 protein is found in the membrane of both AB-derived cells at the 4-cell stage(4) in agreement with previous experiments which show these two cells have equivalent potential. Paradoxically, the EMS cell contacts both AB-derived cells at the 4-cell stage, yet only the anterior AB daughter expresses the EMS-induced fate change. How an EMS-derived signal is directed to the appropriateGlp-1 expressing AB descendant remains unclear. The invariant "path" of the persistent CDR from first cleavage to the 6-cell stage provides an internal, asymmetrically-positioned structure which could be exploited to distinguish between equivalent sites of contact between AB-derived cells and EMS at the 4 and 6-cell stages. The AB-EMS lineage interactions specify the dorsal-ventral(2) and left-right(5,6) embryonic axes at the 4 and 6-cell stages, respectively. We propose a model whereby the asymmetrically positioned CDR could be exploited to deliver a signal from EMS to specific AB descendants despite apparently equivalent contacts. A CDR model for the specification of the d-v embryonic axis in C. elegans. Fig 1. shows a schematic view of an experiment done by Priess and Thompson(2). At the 2-cell stage, segregation of determinants (shaded region) along the a-p axis in P(1) define the EMS and P(2) "ends" of the cell. Left unperturbed, embryos develop as shown on the left. On the right, the spindle in the AB cell is reoriented by physical manipulation with a blunt micropipette. One consequence of this manipulation is reorientation of the P(1) spindle. We propose that because the centrosome in P(1) is tethered to a CDR from first cleavage (bold circle between cells) the CDR, its associated cortex and determinants, and the anterior centrosome are repositioned as a unit. As a result, the absolute position of the EMS cell changes. At the stage, a signal from EMS is received by the AB daughter that shares the CDR from first cleavage; this interaction defines the d-v axis. A CDR model for the specification of the l-r embryonic axis in C. elegans. Fig. 2 shows a schematic view of an experiment done by Woods(5). EMS and its descendant MS are shaded to emphasize orientation. In all cases, anterior is left and ventral comes out of the page. In unoperated embryos, nuclear movements in the AB cells are skewed such that AB daughters on the left are slightly more anterior than their sisters on the right. If the skew of the AB spindles is reoriented prior to cytokinesis (right column), the l-r axis is switched giving rise to an enantiomeric embryo and adults. Reorientation of the AB cell positions after cytokinesis does not reverse the l-r axis(5). We propose the physical manipulation changes the absolute position of the CDR derived from first cleavage. This change might reroute an EMS-derived signal that makes ABal different from ABar and reverse the l-r axis. A correlation between the position of the CDR and cell fate specification can now be tested. The prediction is that the embryo manipulations described in Figs. 1 and 2 should invariably reposition the CDR upon successful reversal of the embryonic axes. We plan to test the models diagrammed in Figs. 1 and 2 and to investigate the composition of the CDRs using existing antibodies to various cytoskeletal components. REFERENCES: (l) Waddle et al, 1993 Worm Mtg, Abs#2. (2) Priess and Thompson, 1987 (3) Priess et al, 1987 (4) Kimble et al, 1992, CSH Symp Quant Biol 57:401 (5) Wood, 1991 Nature 349:536. (6) Schnabel, 1991, Mech Dev 34:85.
[
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.