Real-time monitoring of dynamic intracellular Ca(2+) movement during early embryogenesis through expression of yellow cameleon.

ABSTRACT

Intracellular calcium ion (Ca2+) is important in a variety of developmental events in the vertebrate embryo through wide-ranging signal transduction pathways. The purpose of this study was to detect dynamic changes in Ca2+ during gastrulation using yellow cameleon 2.12 (YC2.12), a Ca2+ indicator protein based on cyan and yellow fluorescent proteins. mRNA for YC2.12 was synthesized in vitro, and injected into early embryos at the single-cell stage. Fluorescence images were obtained using a fluorescence microscope equipped with a cyan fluorescent protein-yellow fluorescent protein (CFP-YFP) filter. Image acquisition and analysis were executed using the fluorescence resonance energy transfer (FRET) software. Embryos injected with YC2.12 mRNA became larvae normally, indicating that YC2.12 may not be toxic. FRET analysis enabled us to trace dynamic changes of Ca2+ up to 30 hours postfertilization (hpf). The highest concentration of Ca2+ was observed during gastrulation. Although the fertilized eggs were symmetrical by microscopic observation, FRET images clearly indicated asymmetrical distribution and dynamic movement of Ca2+. When Fluo-3, a chemical Ca2+ indicator, was used to monitor Ca2+, fluorescence images could be obtained only during gastrulation (<6 hpf), and the images were not clear compared to when YC2.12 was used. These results suggested that YC2.12 is useful for noninvasive and real-time detection of dynamic Ca2+ change throughout gastrulation.