Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes

Abstract

Asymmetric RNA probes, which contain only the mRNA coding strand, provide a large increase in hybridization efficiency in situ over that observed with either symmetric (both strands represented) RNA or DNA probes. Asymmetric RNA probes are synthesized in vitro by transcription from recombinants formed between sequences encoding sea urchin mRNAs and the transcription vector R7 delta 7. Using a probe representing early variant histone mRNA sequences we have characterized hybridization to sections of sea urchin embryos with respect to thermal stability of the hybrids formed, optimum temperature, effect of sequence divergence on hybrid thermal stability, and dependence of the hybridization signals on probe concentration and hybridization time. Estimates from the observed signals indicate that a large fraction of target RNAs is both retained in sections and hybridized with probe at saturation. Coupled with measurements of nonspecific background binding of heterologous probes, these data indicate that the method has sufficient sensitivity to detect many moderately abundant mRNAs (20-75 molecules per cell in the 1500-cell pluteus). In situ hybridizations to embryos at different developmental stages show that while histone mRNAs are uniformly distributed in cleaving embryos, different cell lineages of older embryos show large differences in accumulation of these mRNAs.