Abstract
We have for the first time succeeded in expressing in vitro-synthesized mRNAs in both the sinistral and the dextral Lymnaea stagnalis early embryos by microinjecting the mRNAs into the eggs before the first polar body stage. Translation of exogenous mRNA in developing embryos was confirmed by expressing various fluorescent proteins; mCherry, DsRed-Express, and enhanced green fluorescent protein. We have found that the protein expression derived from the introduced exogenous mRNA largely depends on the elapsed time after the microinjection and not on the developmental stage of injection, and also on the amount of injected mRNA. Developmental abnormalities were hardly observed. The first notable fluorescent signal was detected within 2–3 h after the injection while the embryos were still in uncleaved stage. Fluorescence gradually increased until 8–9 h and was stable up to 24 h. From these results, it is suggested that there is enough translation machinery necessary for early development and the translation of injected mRNA proceeds immediately and constantly in the early embryos. This is true for both the sinistral and dextral L. stagnalis embryos. Application of the developed method to other freshwater pond snails, dextral Lymnaea peregra, sinistral Physa acuta, and sinistral Indoplanorbis exustus revealed that their early expression mechanisms to be similar to that of L. stagnalis. Thus, in vitro-synthesized mRNA expression is expected to be important for the understanding of evolutional process and the molecular mechanism underlining the handedness determination in these freshwater snail embryos.
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Acknowledgements
We thank Prof. Guss Smit and Mr. Carool Popelier for the kind gift of our original stock of sinistral and dextral L. stagnalis, Prof. Gary Freeman for kindly providing us our original stock of the dextral L. peregra and the detailed protocol of L. peregra injection, and Prof. Makoto Asashima for generous gifts of the pCS2-EGFP and pSP64T vectors. We also thank Hitoshi Kuwata for his first effort to do microinjection, Hiroyo Kuwata, Yoko Ozawa, and Keita Miyoshi for rearing snails in our laboratory.
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Communicated by D. A. Weisblat
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Abe, M., Shimizu, M. & Kuroda, R. Expression of exogenous fluorescent proteins in early freshwater pond snail embryos. Dev Genes Evol 219, 167–173 (2009). https://doi.org/10.1007/s00427-009-0278-8
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DOI: https://doi.org/10.1007/s00427-009-0278-8