Characterization of calcium transients during early embryogenesis in ascidians Ciona robusta (Ciona intestinalis type A) and Ciona savignyi

The calcium ion (Ca²⁺) is an important second messenger, and a rapid increase in Ca²⁺ level (Ca²⁺ transient) is involved in various aspects of embryogenesis. Although Ca²⁺ transients play an important role in early developmental stages, little is known about their dynamics throughout embryogenesis. Here, Ca²⁺ transients were characterized by visualizing Ca²⁺ dynamics in developing chordate embryos using a fluorescent protein-based Ca²⁺ indicator, GCaMP6s in combination with finely tuned microscopy. Ca²⁺ transients were detected in precursors of muscle cells in the late gastrula stage. In the neurula stage, repetitive Ca²⁺ transients were observed in left and right neurogenic cells, including visceral ganglion (VG) precursors, and the duration of Ca²⁺ transients was 39±4 s. In the early tailbud stage, Ca²⁺ transients were observed in differentiating precursors of nerve cord neurons. A small population of VG precursors showed rhythmical Ca²⁺ transients with a duration of 22±4 s, suggesting a central pattern generator (CPG) origin. At the mid tailbud stage, Ca²⁺ transients were observed in a wide area of epidermal cells and named CTECs. The number and frequency of CTECs increased drastically in late tailbud stages, and the timing of the increase coincided with that of the relaxation of the tail bending. The experiment using Ca²⁺ chelator showed that the CTECs were largely depending on the extracellular Ca²⁺. The waveform analysis of Ca²⁺ transients revealed different features according to duration and frequency. The comprehensive characterization of Ca²⁺ transients during early ascidian embryogenesis will help our understanding of the role of Ca²⁺ signaling in chordate embryogenesis.