Worm Breeder's Gazette 9(2): 66
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.
To determine whether a particular round of DNA synthesis is required for lineage-specific gene expression, we have been treating early embryos with aphidicolin, a specific inhibitor of DNA polymerase alpha. The relation between DNA synthesis and aphidicolin concentration was quantitated in DAPI stained embryos using a Zeiss photocytometric microscope. An aphidicolin dose of 10 g/ml was sufficient to hold DNA at 1.3-1.5x the initial level (assayed at 1 to 6 hours following drug addition), without preventing expression of gut granules and gut- localized esterase in older embryos. Experiments were done by permeabilizing embryos with hypochlorite and gentle pressure, timing each embryo with respect to the 2nd or 3rd AB cleavage (+/- 1 min), adding the drug, and then incubating overnight before scoring for gut granules and esterase staining. Cytochalasin D (5 g/ml) was also added both to verify permeabilization and to reduce chromosome fragmentation due to abortive attempts at division. Events at the third cleavage appear to be essential for proper gene expression in the gut lineage. If aphidicolin is added before completion of the 4 to 8 cell round of DNA synthesis, gut granules and esterase are not expressed. However, drug addition beyond 2 to 3 minutes after the EMS to E + MS cleavage allows the expression of both gut markers at approximately the normal time, 3-4 hours after first cleavage. The above results are not likely to be due simply to a toxic effect of aphidicolin on early embryos. If embryos are blocked during the 2 to 4 cell stage and the drug removed 30 minutes later, DNA synthesis resumes and the embryo produces several hundred nuclear equivalents of DNA; yet esterase and gut granules are not expressed. alpha-amanitin sensitivity in the presence or absence of aphidicolin continues until the 4E cell stage for the esterase. This is 2 cell divisions and an hour or more later than the aphidicolin-sensitive event, indicating that the aphidicolin does not affect transcription of the gene but some earlier process. Interestingly, gut granule expression shows resistance to alpha-amanitin beginning at the 16-cell (2E) stage, suggesting that this marker results from zygotic transcription earlier than that of the esterase. Thus DNA replication during the cell cycle that clonally establishes the E lineage appears to be critical for the later expression of at least 2 lineage-specific products. No further DNA replication seems necessary for this expression. Thus the permission as well as the timing for subsequent gene expression do not depend either on the normal number of rounds of DNA synthesis, or on a normal nucleocytoplasmic ratio.