Worm Breeder's Gazette 10(3): 80

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.

Early Embryonic Transcription in C. elegans

Irene Schauer and W.B. Wood

We have been analyzing transcriptional activity in embryos by 
preparing extracts and assaying run-on transcription in the presence 
of [32P]-UTP (WBG Nov.  1987).  We reported that early embryos (<30 
cells) are transcriptionally active, having about 50% of the activity 
of later embryos.  We have now further characterized these reactions 
with regard to linearity, developmental timing of specific gene 
transcripts, amanitin sensitivity, and comparative rates of 
incorporation in extracts from different embryonic stages.  
Incorporation of [32P]-UTP is linear to about 15-20 minutes and 
plateaus by about 60 minutes.  The time course of incorporation does 
not differ significantly between extracts made from embryos at 
different stages.  Amanitin-sensitive incorporation is consistently 80-
90% of the total, but this has not yet been checked for extracts of 
the earliest embryos analyzed (99% <30 cells).  Early and late run-on 
transcripts (from extracts of 95% <30-cell embryos and >95%o >30-cell 
embryos, respectively) have been used to probe Southern blots of a 
collection of genes with known or predicted patterns of developmental 
regulation during embryogenesis.  Histone, tubulin, and actin genes 
are already being transcribed in the early extracts, and transcription 
increases significantly in the late extracts.  In contrast, unc-54 and 
col-1 are expressed in late but not in early extracts.  Finally, vit-5 
transcripts are not detected in either extract.  Thus run-on 
transcription in vitro appears to follow the expected patterns of 
developmental regulation, at least for these genes.  Further 
quantitation of incorporation by extracts of embryos at different 
stages suggests that very early embryos are as active 
transcriptionally as later embryos on a per nucleus basis.  Extracts 
of early embryos (99% <30 cells) incorporate at least as much [32P]-
UTP per nucleus as extracts of post-gastrulation embryos.  These 
incorporation rates at 22 C correspond to 300-900 kb of RNA (or about 
100-300 mRNA molecules) per nucleus per min.  The variation in this 
number probably reflects the difficulty of accurately determining 
concentrations of nuclei rather than reflecting stage-specific 
variations in incorporation rates.  Incorporation in these extracts 
was also normalized to DNA concentration, determined by fluorimetry, 
and again no significant stage-specific variation of incorporation 
rates was found.  However, DNA readings were 2-3 fold higher than 
expected based on nuclear estimates.  This is presumably due at least 
in part to high levels of mitochondrial DNA in embryos.  Because of 
this discrepancy the two methods of normalization give different 
absolute values for [32P]-UTP incorporation/nucleus.  However, by both 
methods early embryos are as active transcriptionally as later embryos.
This conclusion appears to be at odds with those of Hecht et al.  (
Dev.  Biol.  83:374, 1981), who used in situ hybridization of a [3H]-
poly(U) probe to squashes of embryos at different stages to estimate 
levels of nuclear poly(A)+ mRNA per embryo compared to total cellular 
poly(A)+ mRNA.  These authors reported that nuclear poly(A)+ is first 
detectable around the 90-cell stage, suggesting that  transcription of 
the embryonic genome begins at this time.  However, their data (Fig.  
3) also show that the average number of grains per nucleus stays 
approximately constant from the 100-cell to about the 500-cell stage, 
consistent with our results.  Furthermore the level of about 100 poly(
A)+ mRNA molecules per nucleus that can be estimated from their data 
during this time is roughly consistent with our incorporation levels, 
assuming a short time for transit of nuclear poly(A)+ molecules to the 
cytoplasm.  Therefore, the principal discrepancy between our results 
and theirs is simply that we find evidence for equivalent levels of 
transcription in early and late embryos, whereas they do not.  One 
possible explanation is that in our experiments, despite the evidence 
presented above for apparently normal control of selected transcripts, 
the earliest incorporation we observe in extracts is an artifact not 
representative of in vivo transcription.  If our results do reflect 
the in vivo situation, then the discrepancy most probably results from 
the substantial differences between the two studies in what is being 
measured: nucleotide incorporation rates vs levels of accumulated 
nuclear poly(A) present, respectively.  Other explanations could be, 
for example, that: 1) early transcription does not produce mature 
mRNA's, or produces only poly(A)- mRNA's; or 2) early transcription 
produces poly(A)+ mRNA's but their transit time to the cytoplasm is 
shorter than in later embryos, so that the steady state level of 
nuclear poly(A)+ is below the level of detection by the methods of 
Hecht et al.  We are attempting to distinguish between the various 
alternatives by analyzing RNA synthesis in permeablized whole early