The C. elegans Male:Postembryonic Development of Nongonadal Structures

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Table of contents  -  Abstract  -   Introduction  -   Materials & Methods  -   Results  -   Discussion  -   References

Material and Methods

  Caenorhabditis elegans, var Bristol, strain N2, was cultured as described by Brenner (1974).

  The movements of cells were observed in living animals by Nomarski differential interference contrast microscopy. The animals were mounted on layers of 5% agar, as described by Sulston and Horvitz (1977) except that polyvinylpyrrolidone was not added to the S medium; a green interference filter was always inserted

  (1) Standart Mount.The agar was trimmed around the cover slip, and the mount was simply flooded with immersion oil without the application of grease. Unfortunately, this convenient technique cannot be used with the immersion oil currently obtainable (Zeiss 518C), because of its toxicity to the nematodes. For this reason, we have continued to use the old type of oil, containing polychlorinated biphenyls (PCB). If the new type of oil is used, it must be kept out of contact with the agar. A layer of grease does not provide a sufficient barrier. One approach is to employ an 18 X 18 mm cover slip and to keep the oil away from theedges, which are sealed with grease or Voltalef oil (Judith Kimble, personal communication); another, applicable for periods of a few hours, is to use the quick mount, described below.

  (2) Quick mount, used when the animal was to be retrieved after a brief examination. A 13-mm-diameter circular cover slip, coated with bacteria in the usual way, was placed over the nematodes; the agar was not trimmed, but covered with a 25-mm square of Saran Wrap having an 11-mm- diameter hole. Only a tiny drop of immersion oil was added, so that the plastic was not wetted by it. For recovery, the Saran Wrap was peeled off, and then the cover slip was raised, the animal being watched to ensure that it remained on the agar; if necessary, the cover slip was lowered and raised until it did so (extra fluid helped).

 (3) Anaesthetic mount, used for laser surgery and for drawing with the camera lucida. The nematode was anaesthetised to the required extent in 0.5-1% l-phenoxy-2- propanol and then mounted on agar containing 0.2% l-phenoxy-2-propanol; by this procedure, anaesthesia is rapid (0.5-5 min,depending upon the age of the animal) yet controllable, and the animals do not deteriorate as fast as they do when mounted in more concentrated l-phenoxy-2-propanol. The quick mount procedure was followed, except that no bacteria were applied to the cover slip.

 (4) Invertible mount, used to follow certain cells through lethargi (the periods of inactivity during moults), when animals frequently turn over. It was particularly useful in tracking the muscle cells, which are obscured contralaterally by the intestine, through L3 lethargus.

To allow inversion, it is necessary to mount the animal in a very thin layer of aqueous medium; this in turn necessitates a plastic cover film, to permit gas exchange. The cover film was prepared in advance by coating a horizontal 38 x 76 mm microscope slide with 1.5 ml of 2% celloidin (Gurr, London) in amyl acetate. The slide was left for several days in a dustfree place, and the film was then cut into 10 x 10 mm squares; when required, a square was peeled off and the centre was lightly smeared with bacteria. In order to prepare the mount itself, a 22 x 50 mm coverslip was placed between two others raised 60 micrometer with Ofrex tape, all three being held onto a sheet of plate glass by films of water. A drop of hot 1% agarose was placed on the central coverslip and flattened by means of a siliconised slide supported on the outer coverslips. After cooling, the slide was slid away and the agarose was allowed to dry to about 40 micrometer (judged by experience; the final thickness of the mount was measured using the calibrated fine focus control). A very small drop of S medium containing the nematode was added, followed by the cover film. The edges of the agarose were allowed to dry completely, and then the mount was flooded with immersion oil. For convenience of handling, it was clamped in a metal frame (35 x 75 x 3 mm). Such mounts lasted for several hours before dehydration began to compress the nematode unduly; by that time the animal had moulted and was remounted in the normal way. PCB immersion oil was used for invertible mounts, but, perhaps on account of the limited exposure period, the new oil can also be employed. Under PCB oil, the upper surface of the celloidin slowly becomes beaded with water droplets; these can be cleared by wiping very lightly with a shred of lens tissue.

Individual animals were always handled by pipette; 1.5 mm tubing was drawn in a flame, and cut to give a tip diameter of 0.2-0.3 mm. The pipette was attached to a mouth tube and kept partly filled with buffer during use. Provided that animals were not sucked into the wide part of the pipette, they never became trapped inside.

 Drawings of anaesthetised animals were made with the help of a Zeiss camera lucida. Approximate depths of the nuclei in the animal were recorded by means of a colour code. Stereo pairs of the proctodeal nuclei were made by manual tracing, the spacing of the images of each nucleus being adjusted according to the colour code.

 Developmental ages of animals are given in hours from the time of hatching at 20° C, according to the time scale of Sulston and Horvitz (1977).

 Animals were prepared for electron microscopy in various ways. For bulk fixation, a male culture plate from which the bacte­ria had cleared was kept at 4°C overnight and then left for a few minutes in a covered dish containing some dry ice. The plate was inverted over 2% osmium tetroxide for 1-2 min, and the nematodes were washed off into 1% osmium tetroxide. After fixation for 1 hr, straight animals were selected and dehydrated and embedded as described by Ward et al. (1975). Series 4 and 5 (Table 1) were obtained in this way. For series 3 the animal was treated similarly, but was straightened manually after anaesthesia with carbon dioxide. For series 1 and 2 the animals were transferred directly from the Nomarski microscope to 1% osmium tetroxide, to ensure that development was ar­rested at a known stage.



Series Age of animal Cells lineaged
1 42 hr Left R3-R9; right R5-R9; dorsorectal ganglion
2 43 hr -
3 Young adult Preanal ganglion
4 Old adult -
5 Old adult -

 In areas where the morphology and arrangement of cells is well defined (mesoderm, most of the proctodeum, part of the preanal ganglion), the assignment of cell fate did not require the electron micro­scopic reconstruction of animals of known lineage. This was helpful, because the lineage of only a limited number of cells can be watched in a single individual, and anatomical reconstruction is a timeconsuming operation. To supply the missing information, two animals were analysed by electron microscopy after particular lineages had been followed (Table 1).

 A laser microbeam system developed by John White was used to kill individual cells, as described in the following paper.

 The nomenclature used to describe cell lineages is the same as that employed by Sulston and Horvitz (1977). After a cell division, each daughter is given its parent's name followed by a letter representing its relative position: a, anterior; p, posterior; d, dorsal; v, ventral; l, left; r, right. Thus, Rn.aa is the anterior daughter of the anterior daughter of Rn. In the lineage charts, divisions are anterior (to the left) posterior (to the right) unless otherwise designated.

 Because many cell lineages are unknown, and some are ambiguous, it is desirable to name cells also in terms of their differentiated characters. The nomenclature for neurons was devised by John White. In his system, every cell is given a name consisting either of three upper case letters or of two upper case letters and a number; the second alternative is used only for groups of similar cells, each cell in a given group having the same letters but a different number. The three-character name is unique except in the case of radially symmetric groups, of which the members appear to be identical: in such a group, every cell has the same three-character name, followed by L (left), R (right), D (dorsal), or V (ventral). Supporting cells in the nervous system, and some other structural cells, are named by a slight modification of the neuron system. In the figures, these names are distin­guished from lineage designations by being printed in Gothic type. For all the components of a given sensil­lum, the same initial pair of letters is used, i.e.-HO, hook sensillum; PC, postcloacal sensillum; PH, phasmid; Rn, ray number n; SP, spicule. The next one or two letters indicate the nature of the component, as follows-sh, sheath cell; so, socket cell; st, structural cell; A, B, C, D or V, neuron. For bilaterally paired sensilla, the name ends in L (left) or R (right). The correspondence between lineal names and systematic names is shown in the lineage charts and drawings.

Adapted by Yusuf KARABEY for WORMATLAS, 2003