Developmental stage | Treatment | Observation | Conclusion |
---|---|---|---|
2-cell stage | Arrest of DNA synthesis for 2 h with aphidicolin, 2 μg/ml | No combs formed (0/30) | DNA synthesis is essential for comb formation. |
16-cell stage | |||
32-cell stage | |||
60-cell stage | 27/30 (90%) formed combs, comb formation was delayed by 2Â h | ||
4 hpf | 29/30 (97%) formed combs, comb formation was delayed by 2Â h | ||
5 hpf | 29/30 (97%) formed combs, comb formation was delayed by 2Â h | ||
60-cell stage and 4 hpf | Arrest of DNA synthesis and cytokinesis with aphidicolin, 2 µg/ml, plus cytochalasin B, 1 to 2 μg/ml | Immediately arrested, single nuclei observed, neither combs nor photoprotein were formed in any embryo (0/30) | DNA synthesis is essential for comb formation even in cleavage-arrested embryos. |
1- to 2-cell stage | Blocking of transcription with actinomycin D, 10 μg/ml | 0/30 gastrulated, development arrests at 32- to 60-cell stage | Zygotic transcription begins around the 32- to 60-cell stage and the specification of the combs requires more than only maternal RNAs. |
4- to 8-cell stage | 0/30 gastrulated, development arrests at 32- to 60-cell stage | ||
16- to 32-cell stage | 10/30 (33%) gastrulate, 20/30 (67%) arrest at 32- to 60-cell stage | ||
60-cell stage | 28/30 (93%) gastrulate but no pharynx is formed, 2/30 (7%) do not gastrulate | ||
5 hpf (post-gastrulation) | No pharynx is formed | ||
2-cell stage | Arrest of translation with puromycin, 125 μg/ml, 25 μg/ml | 0/30 formed combs | Comb protein is translated prior to 6 hpf, comb formation requires protein that is translated in the embryo and not maternally loaded. |
16-cell stage | 0/30 formed combs | ||
60-cell stage | 0/30 formed combs | ||
6 hpf (post-gastrulation) | 32/30 (97%) formed combs |