Worm Breeder's Gazette 9(1): 58
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Mutations in the genes ced-3 and ced-4 block essentially all of the programmed cell deaths that occur during C. elegans development. These two genes are involved either in the initiation or in an early essential step(s) of the cell death program. We want to know whether ced-3 and ced-4 he dying cells or act within other cells, e.g. by controlling the release of a humoral factor. We have used genetic mosaic analysis to approach this problem. nDp3, a free duplication containing unc-30(+) IV, ced3(+) IV, unc-26( +) IV, dpy-4(+) IV and unc-34(+) V, was derived spontaneously from yDp1 (a duplication isolated by L. DeLong and B. Meyer, WBG Vol.8 No. 3) while we were using yDp1 in a screen for mosaic animals. nDp3 appears to be lost at a high frequency, as about 1% of the Unc of nDp3-carrying animals segregate non-Unc non-Dpy progeny, indicating that those Unc have retained the duplication in germ line cells but have lost it in other cells. By identifying semi-Dpy and semi-Unc progeny from animals of genotypes unc-30 dpy-4: nDp3 or ced-3 dpy-4: nDp3, we have picked 33 mosaic animals. In each of these animals, some cell deaths occurred and some did not. Because cell deaths arise from many parts in the cell lineage, the analysis of such cell-death mosaics allows the identification of the points of duplication loss. Specifically, we have used Nomarski optics to check for the presence of surviving sisters of certain embryonic cells (I2s, MCL, mlvs, NSMs, CEMs, g2 and M4) as well as for the presence of surviving cells in the posterior lateral ganglia. The expression of the Ced-3 phenotype in those mosaic animals we picked is consistent with the hypotheses that ced-3 acts cell autonomously and that the mosaicism resulted from duplication loss at one (29/33) or two (4/33) cell divisions. We observed a number of instances in which only one of two cells located close to each other displayed a Ced-3 phenotype, making it unlikely that ced-3 controls a humoral factor. We conclude that the expression of ced-3 is probably cell autonomous, although we cannot rule out from these experiments the possibility that ced-3 acts in cells very closely related by lineage to the dying cells (e.g., sister cells). However, previous experiments (Sulston and Horvitz, Devel. Biol. 56, 110, 1977) in which cells of the postdeirid lineage have been ablated without altering the fates of the unablated cells suggest that ced-3 does not act via the close relatives of dying cells. In a similar way, we have used sDp3, kindly provided by D. Baillie, to analyze the expression of ced-4. unc-36 and dpy-17 were used as markers. By looking for Unc non-Dpy and Dpy non-Unc progeny from animals of the genotype ced-4 unc-36: identified 26 mosaic animals. As in ced-3 mosaic animals, the patterns of cell deaths in ced-4 mosaic animals are strictly correlated with cell lineage. We conclude that the expression of ced-4 is also most likely cell autonomous. From these experiments, we have also identified the sites of action of several genes used as markers. Duplication loss in the AB lineage ( recognized by the Ced-3 phenotype) is sufficient to make adult dpy-4 animals express a Dpy phenotype, but dpy-4 may also act in the P1 lineage as well, since L4 animals with duplication loss in the AB lineage are slightly longer than true dpy-4 homozygotes. unc-26 acts mostly in the ABp lineage but probably slightly in the ABa lineage. The expression of a mutant unc-30 allele in the ABp lineage appears to be responsible for most of the Unc-30 phenotype, but animals with mutant unc-30 expression only in the ABa lineage move more slowly than N2 (particularly when backing up). These results are consistent with the observation that unc-30 affects the ventral cord D motoneurons (J. White et al., C. elegans Meeting Abstracts, 1985; S. McIntire, personal communication), all of which are generated from the ABp lineage, although the data also suggest that unc-30 is expressed in cells from the ABa lineage as well. unc-36 acts in the ABp lineage, as previously found by C. Kenyon (personal communication). dpy-17 appears to act in both the AB and P1 lineages. Specifically, those Dpy non-Unc progeny derived from animals of the genotype ced-4 unc-36: ell death survivors in the AB or MS lineages, suggesting that the duplication had been lost in the P2 lineage; however, these animals were a little longer than true dpy-17 animals, suggesting that P1 might not be the only lineage in which dpy-17 acts. This hypothesis is supported by one semi-Dpy non-Unc animal in which the duplication had been lost from the ABa lineage. Furthermore, 13 Unc non-Dpy progeny from animals of the genotype ced-4 unc-36: lost the duplication from the ABp lineage, suggesting that the loss of the duplication from the AB lineage would make the animal both Dpy and Unc. We conclude that both ced-3 and ced-4 probably act within cells that undergo programmed cell death. Although it remains possible that the activation of ced-3 and ced-4 requires cell extrinsic signals, all evidence to date is consistent with the hypothesis that the determination of cell death is cell autonomous.