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±4s. 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±4s, 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.

MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos.

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light-oxygen-voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A ( Ciona robusta) , a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera. Graphical abstract.

A single motor neuron determines the rhythm of early motor behavior in Ciona.

Recent work in tunicate supports the similarity between the motor circuits of vertebrates and basal deuterostome lineages. To understand how the rhythmic activity in motor circuits is acquired during development of protochordate Ciona, we investigated the coordination of the motor response by identifying a single pair of oscillatory motor neurons (MN2/A10.64). The MN2 neurons had Ca2+ oscillation with an ~80-s interval that was cell autonomous even in a dissociated single cell. The Ca2+ oscillation of MN2 coincided with the early tail flick (ETF). The spikes of the membrane potential in MN2 gradually correlated with the rhythm of ipsilateral muscle contractions in ETFs. The optogenetic experiments indicated that MN2 is a necessary and sufficient component of ETFs. These results indicate that MN2 is indispensable for the early spontaneous rhythmic motor behavior of Ciona. Our findings shed light on the understanding of development and evolution of chordate rhythmical locomotion.