Worm Breeder's Gazette 13(1): 65 (October 1, 1993)

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.


Shai Shaham, Bob Horvitz

HHMI, Dept. Biology, MIT, Cambridge, MA 02139

We have been analyzing the ability of constructs containing fusions of the ced-3 and ced-4 cDNAs to different promoters to kill cells in which these promoters are active. We have used promoters for the mec-7 gene (expressed in the touch cells and a few other cells; A. Fire, M. Hamelin, and J. Culotti, personal communication), the unc-30 gene (expressed in the VD and DD neurons; Y. Jin, personal communication), and the heat-shock promotor (expressed throughout the animal; A. Fire, P. Candido, personal communication). We initially examined integrated lines containing both a mec-7 - ced-3 and a mec-7 - ced-4 construct in ced-9 ( n2812 ); ced-3 ( n717 )animals to avoid any potential inhibitory effects of ced-9 .We found that the touch cells were missing in these animals. By observing the cell lineages of QL and its progeny we found that PVM and occasionally SDQL undergo a cell death process that is morphologically indistinguishable from normal programmed cell deaths. The mec-7 promoter we have been using is active in both PVM and SDQL. These results support the idea suggested by mosaic analysis of ced-3 and ced-4 (Yuan and Horvitz, Developmental Biology, 138, 33-41 1992) that these genes act cell-autonomously to kill cells, since we never observed any dying cells near PVM or SDQL. These results also suggest that the machinery necessary for engulfment of cell corpses is either constitutively present or is induced by the activities of ced-3 and ced-4 ,since engulfment of corpses was generally normal. In one case we directly observed the hypoderm engulfing a dying PVM.

To elucidate interactions among ced-3 , ced-4 ,and ced-9 ,we have begun to analyze the effects of the constructs described above in various genetic backgrounds. So far we have found that introducing these constructs into animals of different genetic backgrounds has different effects. Killing of target cells is more efficient in the absence of endogenous ced-9 ,since ced-3 fusions kill more efficiently in ced-9 ( n2812 ); ced-3 ( n717 )animals than in ced-3 ( n717 )animals, and ced-4 fusions kill more efficiently in ced-4 ( n1162 ) ced-9 ( n2812 )animals than in ced-4 ( n1162 )animals. We also found that the mec-7 - ced-3 fusions kill less efficiently in the ced-4 ( n1162 ) ced-9 ( n2812 )background than in the ced-9 ( n2812 ); ced-3 ( n717 )background and that mec-7 - ced-4 fusions kill less efficiently in the ced-9 ( n2812 ); ced-3 ( n717 )background than in the ced-4 ( n1162 ) ced-9 ( n2812 )background. These results suggest that ced-3 function is augmented by ced-4 function and vice versa and also suggest that ced-3 and ced-4 are normally expressed in the touch cells even though these cells normally do not die.

Finally, we would like to suggest that ced-3 and ced-4 can be used to genetically ablate cells in which a given promoter is active, and in the near future we hope to make available convenient vectors that can be used with any promoter.