Abstract
Evolutionary developmental biology often combines methods for examining morphology (e.g., scanning electron microscopy, SEM) with analyses of gene expression (e.g., RNA in situ hybridization). Due to differences in tissue preparation for SEM and gene expression analyses, the same specimen cannot be used for both sets of techniques. To aid in the understanding of morphological variation, it would be particularly useful to have a high-magnification image of the very same sample in which gene expression is subsequently analyzed. To address this need, we developed a method that couples extended depth of field (EDF) epi-illumination microscopy to in situ hybridization in a sequential format, enabling both surface microscopy and gene expression analyses to be carried out on the same specimen. We first created a digital image of inflorescence apices using epi-illumination microscopy and commercially available EDF software. We then performed RNA in situ hybridizations on photographed apices to assess the expression of two developmental genes: Knotted1 (Kn1) in Zea mays (Poaceae) and a PISTILLATA (PI) homolog in Musa basjoo (Musaceae). We demonstrate that expression signal is neither altered nor reduced in the imaged apices as compared with the unphotographed controls. The demonstrated method reduces the amount of sample material necessary for developmental research, and enables individual floral development to be placed in the context of the entire inflorescence. While the technique presented here is particularly relevant to floral developmental biology, it is applicable to any research where observation and description of external features can be fruitfully linked with analyses of gene expression.
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Acknowledgements
We especially acknowledge Sankar Sridaran and Solomon Stonebloom for their help in developing these techniques. We also thank Sarah Hake and her lab (Plant Gene Expression Center, United States Department of Agriculture) for providing in situ protocols and the Kn1 probe and members of the Specht lab for insightful comments on earlier versions of this manuscript.
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Communicated by K. Schneitz
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Bartlett, M.E., Kirchoff, B.K. & Specht, C.D. Epi-illumination microscopy coupled to in situ hybridization and its utility in the study of evolution and development in non-model species. Dev Genes Evol 218, 273–279 (2008). https://doi.org/10.1007/s00427-008-0211-6
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DOI: https://doi.org/10.1007/s00427-008-0211-6