Timecourse of SpH protease mRNA expression along development.

<p>Expression of SpH transcripts along development was evaluated by Northern blot analysis (<b>A</b>) and semi-quantitative RT-PCR (<b>B</b>). PCR was performed as described in Material and Methods using 1 µg of cDNA and 35 amplification cycles (95°C-1 min, 62°C-1 min, 72°C-1 min). The stability of SpH transcripts was estimated (<b>C</b>) by treating embryos with actinomycin D (50 µg/ml) 3 min post-fertilization and comparing the abundance of mRNAs in control and treated embryos by semi-quantitative RT-PCR (RT on DNA-free total RNA, PCR with 0.2 µg cDNA, 27 cycles). Inhibition of RNA synthesis with actinomycin D blocks development beyond hatching as visualized in (1) 20 hours p.f.. In these conditions the abundance of SpHp mRNAs remains invariant in treated embryos when compared to controls (1, 2). Quantification of 3 different experiments is reported in (3). Hours post-fertilization are indicated.