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.

Embryonic Gut-Specific Expression and DNA Synthesis

L. Edgar and J. McGhee

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 
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.