Worm Breeder's Gazette 10(1): 72

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.

Transcription in Early Embryos of C. elegans

I.E. Schauer and W.B. Wood

We are characterizing early transcription in embryos of C.  elegans.  
While general embryonic transcription appears to begin at about the 
100-cell stage, midway through gastrulation (Hecht et al.), there is 
evidence for some transcription as early as the four-cell stage (L 
Edgar, personal communication).  We have used run-on transcription in 
extracts of staged N2 embryos and gel blot analysis of separated RNAs 
from adults, oocytes, L1's and staged embryos to analyze the pattern 
of embryonic transcription and to look for genes that are 
preferentially expressed during early embryogenesis.  To obtain 
embryos, L1 hermaphrodites are synchronized by hatching overnight in 
M9 without food.  The starved L1s are then plated at 10-20,000/plate 
on NGM plates and kept well fed for 34 days at 16-20 C.  The worms are 
observed as they reach adulthood.  'Early'' embryos are harvested by 
hypochlorite treatment when the most mature worms contain 4-6 eggs.  
The result is a low yield of embryos of which 90-95% are pregastrular, 
with <30 cells.  'late' embryos are obtained by hypochlorite treatment 
of heavily gravid adults followed by 2 hr of aeration of the embryos 
in M9.  Of these embryos, 95-99% have >30 cells; usually about half 
the embryos are premorphogenesis and half are in the process of 
Extracts prepared from early and late embryos are able to 
incorporate [32P]-UTP into run-on transcripts.  In vitro transcription 
is 100% rifampicin-resistant and ~80% amanitin-sensitive, indicating 
that most of the incorporation results from eukaryotic pol II activity.
The transcripts include sequences homologous to the actin gene act-1,
but not to the vitellogenin gene vit-5, consistent with normal 
developmental regulation.
Labeled run-on transcripts from early and late embryonic extracts 
were used to probe duplicate filter lifts from an N2 genomic library (
prepared by C.  Link).  With the reservations that there may be 
artifacts in in vitro transcription and that some later embryos are 
present in the early embryo preparations, the following observations 
suggest that substantial transcription occurs prior to the 30-cell 
stage.  1) Early extracts incorporate labeled nucleotide at about 50% 
of the rate seen in late extracts, on a per nucleus basis.  2) When 
library filters are probed, early run-on transcripts hybridize as 
strongly as late run-on transcripts to 25-50% of the plaques.  3) RNA 
gel blot analysis of actin gene expression indicates that act-1 and 
act-3, but interestingly not act-4, are already being expressed in 
early embryos.
When duplicate library filters are probed with early and late 
running transcripts, about 0.02% of the plaques hybridize more 
strongly to early transcripts.  Several such phage have been isolated 
and further characterized by gel blot analysis of RNAs from the 
following developmental stages: adults with oocytes (temperature 
sensitive fer-1 adults raised at the non-permissive temperature), 
early embryos, late embryos, and L1's.  Nine independently isolated 
phage clones have been found to recognize transcripts that are present 
primarily in early embryos.  Three of these transcripts were 
recognized by more than one of the clones, suggesting that the number 
of primarily early transcripts may not be large.  Six different 
transcripts were recognized by the nine clones.  These six transcripts 
fall-roughly into the following three classes:  1) Low abundance in 
adults with oocytes, high in early embryos, low in late embryos (one 
transcript).  2) Absent from adults with oocytes, high in early 
embryos, barely detectable in late embryos (one transcript).  3) 
Absent in adults with oocytes, high in early embryos, 2- to 5-fold 
lower in late embryos (four transcripts).  None of the transcripts 
persist as far as early L1.  All six are small, ranging from 0.7 to 2 
kb.  We are currently looking for cDNA clones that correspond to these 
transcripts and planning to use the cDNA's for in situ hybridization 
and sequencing.  Five of the six sequences have been placed in contigs 
(J.  Sulston and A.  Coulson, personal communication), but none of the 
relevant contigs are mapped or marked.  Plans to begin genetic 
analysis must, therefore, await further developments.