Abstract
Intracellular calcium ion (Ca2+) signaling is heavily involved in development, as illustrated by the use of a number of Ca2+ indicators. However, continuous Ca2+ patterns during morphogenesis have not yet been studied using fluorescence resonance energy transfer to track the Ca2+ sensor. In the present study, we monitored Ca2+ levels during zebrafish morphogenesis and differentiation with yellow cameleon, YC2.12. Our results show not only clear changes in Ca2+ levels but also continuous Ca2+ patterns at 24 hpf and later periods for the first time. Serial Ca2+dynamics during early pharyngula period (Prim-5-20; 24–33 hpf) was successfully observed with cameleon, which have not reported anywhere yet. In fact, high Ca2+ level occurred concurrently with hindbrain development in segmentation and pharyngula periods. Ca2+ patterns in the late gastrula through segmentation periods which were obtained with cameleon, were similar to those obtained previously with other Ca2+sensor. Our results suggested that the use of various Ca2+ sensors may lead to novel findings in studies of Ca2+ dynamics. We hope that these results will prove valuable for further research in Ca2+ signaling.
Highlights
Intracellular calcium ions (Ca2+) act as second messengers in organism cellular signaling pathways
Since the Ca2+ monitoring had been well studied with aquorin by Créton, Speksnijder & Jaffe (1998), we mainly focused on novel findings here
Ca2+ involves with neural network in zebrafish and Ca2+ sensors were used for studying neuronal activity and reflexive behavior (Higashijima et al, 2003; Muto et al, 2013; Portugues et al, 2014)
Summary
Intracellular calcium ions (Ca2+) act as second messengers in organism cellular signaling pathways. To present, ‘continuous’ Ca2+ patterns such as long-term time lapse imaging in zebrafish morphogenesis after 24 hpf (hour post fertilization) have not been reported yet. Stable Ca2+ signals are expected with fluorescent Ca2+ sensors such as yellow cameleon YC2.12 because the sensor molecule is integrated into cells. This is advantageous in longterm measuring since Ca2+ sensor is synthesized in vivo and does not require a substrate like luminescent Ca2+ sensor does. Fluorescence emits stronger light than luminescence in general requiring an excitation light, which enables us to measure real-time and to detect subtle signals. The purpose of the present study was to analyze serial Ca2+ patterns for long-term periods, from late gastrula to pharyngula periods, using cameleon
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