Correlative light and electron microscopic analyses of mitochondrial distribution in blastomeres of early fish...

Early embryos of vertebrates undergo remarkable dynamic molecular events, such as embryonic gradient, cellular polarity, and asymmetry necessary for cell fate decisions. Correlative light and electron microscopy (CLEM) is a powerful tool to investigate rare or dynamic molecular events and has been developed for relatively small cells in culture and tissues but is not yet available for large cells of early development stage embryos. Here we report the capability of CLEM in blastomeres of medaka fish by using the mitochondria detection system. A short N-terminal signal peptide of the mitochondrial protein Tom20 was linked to green fluorescent protein (GFP), resulting in a fusion protein termed Tom20:GFP. The subcellular location of Tom20:GFP in medaka blastomeres reveals the lack of mitochondrial distribution in pseudopodia as well as inconspicuous redistribution during divisions. Blastomeres, after sample preparation procedures including high-pressure freezing and freeze substitution, are able to preserve fluorescence, antigenicity, and fine structures, which allows for precise correlation between the Tom20:GFP fluorescence and mitochondria on merged light and electron micrographs. Furthermore, nanogold immunostaining for Tom20:GFP and endogenous Tom20 revealed their specific localization on the mitochondrial outer membrane. Our results extend the CLEM approach to early development stage embryos of a vertebrate.