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Fig. 5 Zygotic transcription is prematurely activated by the inhibition of oxidative phosphorylation. A: cDNA prepared from untreated pre-midblastula transition (MBT; unfertilized and 512 cells) and post-MBT (30% epiboly) embryos or from embryos treated with KCN and arrested at 256-512 cells (KCN at 4 cells and KCN at 64 cells), was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for zygotic expression of the transcription factors tbx16 and tbx6. The maternal transcript ampkα1 was amplified for comparison across cDNA samples. B: Schematic of the experimental approach to assess the chemical inhibition of transcription. One-cell embryos were injected with water or the transcription inhibitors actinomycin D (ActD) or α-amanitin (α-am) and beginning at 64 cells were incubated in KCN or egg water. RNA was collected at 64 cells to assess the presence of maternal transcripts and when untreated embryos reached 30% epiboly to detect zygotic transcripts. C: The presence of two zygotic transcripts was assessed by RT-PCR analysis at 64 cells and 30% epiboly in unarrested embryos injected with transcriptional inhibitors as indicated. W, water-injected. D: The transcriptional activation of tbx16 and tbx6 was determined by RT-PCR in embryos treated with KCN beginning at 64 cells and collected when siblings reached 30% epiboly. Each RT-PCR sample constitutes 10 pooled embryos.