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.
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 morphogenesis. 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.