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Nematode strain. Wild-type C. elegans var. Bristol, designated N2, is from the University of Colorado, Boulder, stock. The nematodes were handled as described by Brenner (1974) and Hirsh et al. (1976). Worms for lineage studies were kept at either 16 or 20°C until they were picked for observation.
Technique to obtain cell lineages. We used the technique of Sulston and Horvitz (1977) for mounting live worms for observation over long periods of time. Briefly, a worm is placed on a thin agar pad on a microscope slide. A slurry of E. coli is applied in a small disk to a coverslip. The coverslip is placed so that the worm is trapped between the agar surface and the coverslip in a thin layer of S medium (0.1 M NaCl and 0.05 M KH2PO4, pH 6.0). The worm is able to crawl on the agar surface and feed on the bacteria supplied. The edge of the coverslip is sealed with Vaseline or immersion oil so that the preparation does not dry out.
Our observations were made using a Zeiss Universal microscope equipped with Plan 100 objective and Nomarski differential interference contrast optics. One limit of this technique is that cell boundaries are indistinct. Thus, the bulk of the lineage data reflects the behavior of cell nuclei. Drawings were made as frequently as possible during periods of active migration and rapid division, and as frequently as necessary (every 0.5 to 2 hr) when nuclei were less active. The complete lineages were compiled from observations of over 50 hermaphrodites and 20 males. Photomicrographs were taken with a Zeiss microflash illuminator.
Nomenclature. We have used the nomenclature of Sulston and Horvitz (1977) for naming cells. A cell's daughters are each named by adding to the name of the mother cell a single lower case letter describing the position of each daughter relative to the other just after division (a = anterior, p = posterior, d = dorsal, v = ventral, 1 = left, r = right). Thus, if the cleavage plane of Z1 is oriented perpendicular to the anterior-posterior axis, the resulting anterior daughter is identified as Z1.a and the posterior daughter as Z1.p. If a division occurs obliquely, the resulting daughters are identified by adding a single letter signifying one axis only.
Lineage charts. The lineage trees presented here follow the rules formulated by Sulston and Horvitz (1977) as far as possible. A division is depicted by a branch point in which the left branch represents an anterior, dorsal, or left daughter and the right branch represents a posterior, ventral, or right daughter. Branches are marked by the same letters used in naming cells except that combinations of letters are used to describe oblique divisions. In the case of simple anterior-posterior divisions, the letters are omitted. In each lineage chart, the vertical coordinate represents time. Since the lineages are obtained at varying temperatures (19-23°C), the times divisions take place are normalized to 20°C. The times at which individual molts occur have been used as time standards for this correction. The horizontal coordinate of the lineage chart represents the anterior-posterior axis with anterior to the left for the entire hermaphrodite lineage and the first half of the male lineage. In the male lineage chart, the horizontal coordinate system in the last half of the divisions represents the distal-proximal axis with proximal to the left. The proximal end of the gonad is the opening at the cloaca; the distal is the farthest from the opening. Anterior, then, is to the left in the chart until the gonad reflexes, and it is to the right for the rest of the divisions. This is necessary, because in males, unlike hermaphrodites, divisions occur in the bend of the gonad. Since these divisions take place at variable points in the bend, the positions of the daughters vary with respect to the coordinates of the worm. However, the divisions are invariant when viewed with respect to the distal-proximal axis of the gonad.
Some variability is observed in the times at which divisions occur and in the orientation of cleavage planes. For example, in some worms Z1 divides first, in others Z4 divides first and, in a rare worm, they divide at the same time. Since they almost always divide within an hour of each other, they are represented in the lineage chart as dividing at the same time. A similar limited variability is observed in the cleavage planes, and here too, an average is shown in the lineage charts.
One-micron sections. Adult hermaphrodites were cut posterior to the pharynx or anterior to the anus in a drop of 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). A good cut will release the anterior or posterior gonadal arm intact into the fixative, though it remains attached to the worm by the somatic structures. The dissected animals were fixed in 2% glutaral-dehyde for 1 hr, postfixed in 1% OsO4 for 1 hr, stained en bloc in 1% uranyl acetate for 1 hr, dehydrated in increasing concentrations of ethanol, transferred to propylene oxide, and embedded in Epon. Serial sections of 1micrometer thickness stained with toluidine blue were examined by light microscopy.
Adapted by Yusuf KARABEY for WORMATLAS, 2003