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

Single motor neuron regulates rhythmic tail flick in prehatching Ciona embryo.

No abstract

Swimming aquatic animals exhibit spontaneous motor behaviors before the maturation of swimming locomotion. In this study, we demonstrated that a single pair of motor neurons, A10.64/MN2, which exhibits periodic bursting, is essential for early spontaneous motor behaviors in the invertebrate chordate Ciona intestinalis type A (Ciona robusta). The Ca2+ oscillation was first observed only in a pair of cells at mid tailbud II (St.22) with an 80-sec interval, which shortened to 25 sec at late tailbud II (St.24). A dissociation experiment revealed that the Ca2+ oscillation occurred in a single cell independently, without contact with other cells. The start of the Ca2+ oscillation coincided with the Ca2+ elevation in ipsilateral tail muscle cells at late tailbud I (St.23). Cell lineage tracking revealed that a pair of cells exhibiting Ca2+ oscillation corresponded to A10.64/MN2 motor neurons. Simultaneous imaging of Ca2+ and membrane potential demonstrated that the Ca2+ oscillation coupled with a burst firing of membrane potential. Interestingly, the number and frequency of tail muscle contractions initially coincided with those of the burst, but gradually came to coincide with those of spikes in the burst toward late tailbud II (St.24). Finally, single-cell photoablation of A10.64/MN2 abolished early spontaneous motor behaviors until late tailbud II (St.24), suggesting that the early spontaneous motor behavior of Ciona is directly regulated by only a single pair of A10.64/MN2 motor neurons. These findings revealed that the chordate early spontaneous motor behavior was generated by a minimum motor circuit that consist of a pair of motor neurons, exhibiting spontaneous periodic bursts.