Volume 244, Issue 11 p. 1375-1393

Research Article

Free Access

Neuronal map reveals the highly regionalized pattern of the juvenile central nervous system of the ascidian Ciona intestinalis

Akiko Hozumi

Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan

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Takeo Horie

Japan Science and Technology Agency, PRESTO, Honcho, Kawaguchi, Saitama, Japan

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Yasunori Sasakura

Corresponding Author

Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan

Correspondence to: Yasunori Sasakura, Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415‐0025, Japan. E‐mail: sasakura@shimoda.tsukuba.ac.jp Search for more papers by this author

Akiko Hozumi

Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan

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Takeo Horie

Japan Science and Technology Agency, PRESTO, Honcho, Kawaguchi, Saitama, Japan

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Yasunori Sasakura

Corresponding Author

Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan

First published: 06 August 2015

https://doi.org/10.1002/dvdy.24317

Citations: 10

Abstract

Background: The dorsally located central nervous system (CNS) is an important hallmark of chordates. Among chordates, tunicate ascidians change their CNS remarkably by means of a metamorphosis from a highly regionalized larval CNS to an oval‐shaped juvenile CNS without prominent morphological features. The neuronal organization of the CNS of ascidian tadpole larvae has been well described, but that in the CNS of postmetamorphosis juveniles has not been characterized well. Results: We investigated the number of neural cells, the number and position of differentiated neurons, and their axonal trajectories in the juvenile CNS of the ascidian Ciona intestinalis. The cell bodies of cholinergic, glutamatergic, and GABAergic/glycinergic neurons exhibited different localization patterns along the anterior–posterior axis in the juvenile CNS. Cholinergic neurons extended their axons toward the oral, atrial and body wall muscles and pharyngeal gill to regulate muscle contraction and ciliary movement. Conclusions: Unlike its featureless shape, the juvenile CNS is highly patterned along the anterior–posterior axis. This patterning may be necessary for exerting multiple roles in the regulation of adult tissues distributed throughout the body. This basic information of the juvenile CNS of Ciona will allow in‐depth studies of molecular mechanisms underlying the reconstruction of the CNS during ascidian metamorphosis. Developmental Dynamics 244:1375–1393, 2015. © 2015 Wiley Periodicals, Inc.

Introduction

The developmental and evolutionary mechanisms of chordates are fascinating topics in zoology. Chordates are characterized by their tadpole body, which includes the notochord, pharyngeal gill, endostyle/thyroid gland, and dorsally located central nervous system (CNS). Chordates are classified into three groups: cephalochordates, tunicates, and vertebrates. A fundamental question regarding chordate evolution is how these three groups evolved from their common ancestor (Dehal et al., 2002; Holland et al., 2008; Putnam et al., 2008; Delsuc et al., 2006, Satoh et al., 2014).

A major group of tunicates is the ascidians (Lemaire, 2011). It is not obvious that adult‐stage ascidians are chordates, because of their immotile lifestyle; at the larval stage however, most ascidians possess the typical tadpole body that includes the notochord and dorsally located CNS. In other words, ascidians convert their body structure dramatically from the larval stage to the adult stage by a series of metamorphic events (Cloney, 1982).

During this metamorphosis, the ascidian CNS is subjected to extensive rearrangements. At the larval stage, the ascidian CNS is subdivided into several regions from anterior to posterior; the sensory vesicle, motor ganglion and nerve cord (Fig. 1A; Nicol and Meinertzhagen, 1991; Nishino et al., 2011; Sasakura et al., 2012a). The sensory vesicle is regarded as the ascidian brain, as it contains sensory organs such as the otolith and ocellus (Horie et al., 2009). The motor ganglion includes five pairs of cholinergic neurons, some of which are thought to act as motor neurons innervating tail muscles (Horie et al., 2010; Nishino et al., 2011; Stolfi and Levine, 2011). The nerve cord is present along the dorsal region of the tail, which contains four rows of nonneuronal neural (ependymal) cells; neurons are barely present (Nicol and Meinertzhagen, 1991; Horie et al., 2010). The organization of the larval CNS of ascidians is quite similar to those of vertebrates, and thorough comparisons of the CNS between ascidians and vertebrates have been done at the molecular level (Wada et al., 1998; Ikuta and Saiga, 2007; Imai et al., 2009).

**Figure 1**
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The number of neural cells in the juvenile *Ciona* CNS. A: A larva of *β2tubulin*>*Kaede* line whose CNS was labeled by Kaede‐green fluorescence. Lateral view. The upper panel is a differential interference contrast (DIC) image, and the bottom panel is a pseudo‐colored fluorescence image of Kaede. SV, sensory vesicle; MG, motor ganglion; NC, nerve cord. B: A schematic illustration of *Ciona* juvenile. AS, atrial siphon. BM, body wall muscle. CG, cerebral ganglion (juvenile and adult CNS); ES, endostyle; Eso, esophagus (a part of the digestive tube); Gi, gill. OS, oral siphon. St, stomach. B′: A juvenile of the *β2tubulin*>*Kaede* line. Lateral view. Kaede was expressed in the gill, endostyle and digestive tube in addition to the CNS. C: 3D reconstructed fluorescent images of a juvenile CNS at 4 days postfertilization (dpf). Upper, middle and bottom images respectively represent immunofluorescence of Kaede (green), nuclei labeled by PI (magenta) and their merged images. Anterior is left. CF, ciliated funnel; CG, cerebral ganglion (CNS). The CNS and ciliated funnel are highlighted with white and gray dashed lines, respectively. D: The 3D reconstructed fluorescent images of a juvenile CNS at 7 dpf. E: The number of cells in the juvenile CNS. Each circle corresponds to the result from an individual sample. Blue circles illustrate outliers. In this and following figures, the values of individual numbers less than “first quartile − 1.5 × interquartile range” or more than “third quartile + 1.5 × interquartile range” are labeled as outliers. The median and average are shown by black and red bars, respectively. Left two plots: The results of *β2tubulin*>*Kaede* line at 4 dpf (left) and 7 dpf (right). The remaining plots are the results of *β2tubulin*>*CFP* line. Total, the results of the whole CNS at 7 dpf. The right three are the results of the anterior, middle, and posterior parts of the CNS. The numbers at the bottom illustrate the number of observed specimens. F: A juvenile of the *β2tubulin*>*CFP* line and magnified images of its CNS (bottom). ant, middle and post represent three parts of the CNS according to the length along the A‐P axis.

The highly organized structure of the larval CNS is thought to be lost during metamorphosis. The resultant CNS of juvenile ascidians is oval‐shaped, and no outstanding regionally specific characteristics along the anterior–posterior (A‐P) axis can be recognized from its morphology, unlike the adult vertebrate CNS (Willey, 1893). Therefore, less interest might have been paid to the juvenile/adult CNS of ascidians compared with the larval CNS. Indeed, the neuronal organization of the juvenile/adult CNS of ascidians has not been reported, except for expression profiles and immunoreactivities of some peptide hormones (Johnsen and Rehfeld, 1990; Tsutsui et al., 1998; Satake et al., 2004; Kawada et al., 2011). Two studies, one from our group, indicated that the adult CNS of ascidians is also important to elucidate the evolution of chordate CNS (Dufour et al., 2006; Horie et al., 2011). The findings of those two studies can be summarized as follows: by identifying the cellular changes of the CNS during metamorphosis, it was revealed that the juvenile ascidian CNS conserves the A‐P axis pattern of the larval CNS. Moreover, the expression of some transcription factor genes suggests that the juvenile CNS of ascidians has similarities to the craniofacial nervous system of vertebrates. We are interested in whether the juvenile CNS of the ascidian Ciona intestinalis does or does not harbor a pattern along the A‐P axis, like the patterns of their larvae and vertebrates, in their structure without prominent morphological features.

Thus, it is important to deepen our understanding of the CNS of ascidians at postmetamorphosis stages to elucidate the evolution of chordates. However, our knowledge about the CNS of juvenile ascidians is still limited. Knowledge of the organization of neurons, which is the basic information to understand the nature of nervous systems, is particularly lacking for ascidians. Here we investigated the position, number and axonal trajectories of major neurons in the juvenile CNS of the ascidian C. intestinalis by means of transgenic technologies. Our neuronal map suggests that the juvenile CNS of Ciona possesses highly organized neuronal patterns that exceed our expectations from its superficially featureless morphology.

Results

Cell Number Constituting the CNS of Ciona intestinalis

For the creation of the map of the neurons in the juvenile CNS of Ciona intestinalis, the number and position of cells in the CNS must be estimated. For this purpose, we used a transgenic line that expresses Kaede reporter gene under the control of the cis element of Ci‐β2tubulin, the gene encoding β2tubulin (Kusakabe et al., 2004; Horie et al., 2011). The cis element of β2tubulin drives reporter gene expression in the CNS (Fig. 1A,B′). At the juvenile stage (Fig. 1B), the β2tubulin>Kaede line also exhibited Kaede fluorescence in the tissues with cilia (Fig. 1B′). Juveniles of the transgenic line were fixed after metamorphosis and then immunostained with anti‐Kaede antibody and propidium iodide (PI) for nuclei counterstaining. The fluorescence of the specimens was observed with a confocal microscope (Fig. 1C,D).

We counted the number of nuclei embedded in Kaede‐positive cytoplasmic signals in the three‐dimensional images reconstructed from confocal plane images. At 4 days postfertilization (dpf), an average of 56 cells in the CNS were Kaede‐ and PI‐ double‐positive (Fig. 1C,E). The number of neural cells gradually increased during development, and an average of 73 Kaede and PI‐positive cells were present at 7 dpf (Fig. 1D,E).

The neural cells were clearly unevenly distributed throughout the CNS. We quantified the bias of the number of neural cells along the A‐P axis. We divided the juvenile CNS at 7 dpf into three parts according to its length, and then counted the number of neural cells in each part (Fig. 1E,F). For this analysis, we used a β2tubulin>CFP transgenic line that expresses cyan fluorescent protein (CFP) under control of the β2tubulin promoter (Fig. 1F), because we planned to use this transgenic line for the counterstaining of neural cells in our analyses of the distribution of differentiated neurons in the CNS (see the following sections).

The number of CFP‐positive cells were counted with the help of nuclear counterstaining with sytox blue (e.g., Fig. 2F). As shown in Figure 1E, the anterior part of the CNS had the highest number of neural cells, and the middle part had slightly reduced numbers of neural cells compared with the anterior part. The posterior part had approximately one‐third the number of neural cells compared with those in the anterior and middle parts (Fig. 1E). This is consistent with the characteristic finding that the posterior part of the juvenile Ciona CNS is somewhat narrowed (Fig. 1C,D,F). The total number of CFP‐positive cells was identical to that estimated with the β2tubulin>Kaede line (Fig. 1E).

**Figure 2**
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The number and position of cholinergic neurons in the juvenile CNS. A: A larva of the *VACHT*>*Kaede* transgenic line. B: A juvenile of the *VACHT*>*Kaede* transgenic line. C: The 3D‐reconstructed fluorescent images of a juvenile CNS of the *VACHT*>*Kaede* line at 4 dpf. ant, anterior; post, posterior. The CNS is highlighted with a dashed line. The cell bodies of cholinergic neurons with strong and weak Kaede signals are shown with orange and white arrows, respectively. D: The 3D‐reconstructed fluorescent images of a juvenile CNS of the *VACHT*>*Kaede* line at 7 dpf. Anterior is left. E: The number of cholinergic neurons in the juvenile CNS. The results of the whole CNS at 4 dpf and 7 dpf and the results of three parts (anterior, middle, posterior) at 7 dpf are shown. F: The 3D‐reconstructed fluorescent images of a juvenile CNS of the *VACHT*>*Kaede*;*β2tubulin*>*CFP* double‐transgenic line at 7 dpf. The panels illustrate immunofluorescence of Kaede (green), immunofluorescence of CFP (magenta), nuclei labeled by sytox blue (blue), and their merged image from the top. A neuron that is outside of the CNS is shown by the red arrowhead. The neuron may correspond to the cholinergic neuron derived from the larval stage.

Number and Position of Differentiated Neurons in the Juvenile CNS

We next mapped the differentiated neurons in the juvenile CNS. Because the major neurons constituting the CNS of Ciona intestinalis are cholinergic neurons, glutamatergic neurons and GABA/glycinergic neurons (Horie et al., 2009), we focused on these three neuron types. We created transgenic lines that mark these neurons with Kaede reporter protein using cis elements of marker genes representing specific neuron types, and we used the marker transgenic lines to map neurons in the juvenile CNS.

Cholinergic Neurons

Cholinergic neurons use acetylcholine as the neurotransmitter (Eiden, 1998). Cholinergic neurons can be marked with the cis element of the gene encoding vesicular acetylcholine transporter (VACHT; Yoshida et al., 2004). The transgenic line expressing Kaede under the control of the Ci‐VACHT cis element (Tg[MiCiVACHTK]5 in Horie et al., 2011) was used in this analysis (Fig. 2A,B), and the juvenile CNS of the transgenic line was observed after nuclear staining with PI. We counted the number of PI‐positive nuclei surrounded by Kaede‐green in the cytoplasm to estimate the number of cholinergic neurons.

At 4 dpf, the number of cholinergic neurons in the CNS was 5.63 on average (Fig. 2C,E). The observed cholinergic neurons could be divided into two groups: the neurons with a strong Kaede‐immunostained signal (Fig. 2C, orange arrows) and those with a weaker signal (Fig. 2C, white arrows). The difference in signal intensity may have been due to different timing of the differentiation of the cholinergic neurons; that is, the neurons that differentiate at an earlier time point may have more Kaede protein than the cells that differentiated at later time points. The increase in the average number of cholinergic neurons to 7.86 at 7 dpf (Fig. 2D,E) supports this explanation, because an increase in their number indicates the presence of newly born cholinergic neurons that might have weaker fluorescence. Another possible explanation for the different intensity of Kaede protein signals is that cholinergic neurons include two subgroups with strong and weak VACHT expression levels, even after their full maturation. These two possibilities are not mutually exclusive.

We next attempted to determine the distribution of cholinergic neurons in the juvenile CNS. For this purpose, we used the β2tubulin>CFP line as the background marker, and we mapped cholinergic neurons (that were labeled by Kaede) onto CFP‐positive neural cells (Fig. 2F). We used the β2tubulin>CFP line as a shared marker among the neuron marker lines to achieve a precise comparison of the distribution of different neuron types. This analysis was done at 7 dpf, when the construction of the basic juvenile body is completed. We observed that the middle part of the CNS had the highest number of cholinergic cell bodies, whereas fewer neurons were present at the anterior and posterior parts (Fig. 2E,F).

In our previous study, we found that a few cholinergic neurons in the motor ganglion of the larval CNS remain after metamorphosis (Horie et al., 2011). The precise position of these neurons in the juvenile CNS has not been described. In the present study we determined the position of larval cholinergic neurons inherited by the juvenile CNS. For this purpose, we used the photoconvertibility of Kaede from green to red (Ando et al., 2002). Larval cholinergic neurons in the VACHT>Kaede line were irradiated by ultraviolet (UV) to convert their Kaede to emit red fluorescence (Fig. 3A). This process enabled us to distinguish larval cholinergic neurons from those newly differentiated after metamorphosis, because Kaede photoconversion is an irreversible event; therefore, latter neurons emit exclusively Kaede‐green fluorescence.

**Figure 3**
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Tracing of larval cholinergic neurons during metamorphosis, using the photoconvertibility of Kaede. A: Labeling larval cholinergic neurons with Kaede‐red fluorescence. Left, an alive larva of the *VACHT*>*Kaede* line without UV irradiation. Right, an alive larva of *VACHT*>*Kaede* line after UV irradiation. The two larvae were different individuals. From the top down, the panels illustrate merged images, Kaede‐green + Kaede‐red fluorescence, Kaede‐green fluorescence, Kaede‐red fluorescence, and DIC images. B: Position of cholinergic neurons derived from larval CNS at the juvenile stage. From the top down, the panels illustrate the merged images, Kaede‐green fluorescence, Kaede‐red fluorescence, and DIC images. The cholinergic neurons derived from the larval CNS are shown by arrowheads. The position of the CNS is highlighted with a dashed line. The left two animals had cells labeled by Kaede‐red fluorescence, whereas the animal on the right did not. Autofluorescence (AF) appears around the pigment cells in the juvenile CNS.

As a result, 50% (n = 18) of 4‐dpf‐old juveniles had Kaede‐red cells (Fig. 3B, orange arrowheads). However, all of these cells were located outside of the CNS. The cholinergic neurons were round‐shaped, and we could not see their axons. This suggests that the larval cholinergic neurons remaining after metamorphosis might not have a function as neurons in the juvenile CNS. At 8 dpf, only one of eight examined juveniles had a Kaede‐red cell. There are two possible reasons for the disappearance of larval CNS‐derived cholinergic neurons during the four days after 4 dpf. One is that Kaede‐red fluorescence was diminished by turnover while cells remained, and the other is that the Kaede‐red cells disappeared due to cell death. We were unable to distinguish these two possibilities due to technical limitations.

Glutamatergic Neurons

To visualize glutamatergic neurons in the juvenile CNS, we used transgenic lines harboring the reporter construct expressing Kaede under the control of the cis element of VGLUT, the gene encoding vesicular glutamate transporter (Fig. 4A,B; Horie et al., 2009). As in the case of cholinergic neurons, we made VGLUT>Kaede transgenic lines and then observed the Kaede‐positive cells in the juvenile CNS of the lines.

At 4 dpf, the number of Kaede‐positive glutamatergic neurons in the CNS was 2.75 on average (Fig. 4C,E), which is approximately half the number of cholinergic neurons. Unlike cholinergic neurons, glutamatergic neurons show no clear difference in the intensity of Kaede‐green fluorescence. The number of glutamatergic neurons increased to an average of 3.53 at 7 dpf (Fig. 4D,E).

We next investigated the distribution of glutamatergic neurons along the A‐P axis of the CNS in the genetic background of the β2tubulin>CFP line (Fig. 4F, orange arrows). This analysis showed that strong fluorescence corresponding to glutamatergic neurons was localized at the anterior part of the CNS at 7 dpf (Fig. 4E). During the observation, we noticed that glutamatergic neurons were also present at the peripheral regions including the inside of the ciliated funnel and at the edges of the oral and atrial siphons (Fig. 4B,D,G; Horie et al., 2011).

GABAergic/Glycinergic Neurons

For the labeling of GABAergic/glycinergic neurons, we used the cis element of VGAT that encodes vesicular GABA transporter (Fig. 5A,B; Yoshida et al., 2004). Because this transporter functions in the secretion of both GABA and glycine (Chaudhry et al., 1998), we were unable to distinguish GABAergic neurons and glycinergic neurons with VGAT>Kaede lines (Horie et al., 2011). Because both GABAergic and glycinergic neurons are inhibitory neurons, we analyzed the two neuron types simultaneously.

**Figure 5**
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The number of GABA/glycinergic neurons in the juvenile CNS. A: A larva of the *VGAT*>*Kaede* transgenic line. B: A juvenile of the *VGAT*>*Kaede* transgenic line. C: The 3D‐reconstructed fluorescent images of a juvenile CNS of the *VGAT*>*Kaede* line at 4 dpf. The upper, middle and bottom images represent immunofluorescence of Kaede (green), nuclei labeled by PI (magenta) and their merged images, respectively. Anterior is left. The CNS is highlighted with a dashed line. The cell bodies of GABA/glycinergic neurons with strong and weak Kaede signals are shown with orange and white arrows, respectively. D: The 3D‐reconstructed fluorescent images of a juvenile CNS of the *VGAT*>*Kaede* line at 7 dpf. E: The number of GABA/glycinergic neurons in the juvenile CNS. The results in the whole CNS at 4 dpf and 7 dpf and the results of three parts (anterior, middle, posterior) at 7 dpf are shown. F: The 3D‐reconstructed fluorescent images of a juvenile CNS of the *VGAT*>*Kaede*;*β2tubulin*>*CFP* double‐transgenic line at 7 dpf. From the top down, the panels illustrate the immunofluorescence of Kaede (green), immunofluorescence of CFP (magenta), nuclei labeled by sytox blue (blue) and their merged image.

At 4 dpf, the number of Kaede‐positive GABAergic/glycinergic neurons in the CNS was 5.44 on average (Fig. 5C,E). The average number of GABAergic/glycinergic neurons at 7 dpf was roughly doubled to 10.17 (Fig. 5D,E). Like the cholinergic neurons, the GABAergic/glycinergic neurons showed a clear difference with respect to the intensity of Kaede immunostaining (Fig. 5C,D,F, orange and white arrows).

We then investigated the distribution of GABAergic/glycinergic neurons along the A–P axis of the 7‐day‐old CNS in the genetic background of the β2tubulin>CFP line. We observed that GABAergic/glycinergic neurons were preferentially located at both the anterior and posterior parts of the CNS, but only a small number was present in the middle part (Fig. 5D,E). Because the number of neural cells in the posterior part is approximately one‐third that of the other parts (Fig. 1E), GABAergic/glycinergic neurons are the major neuron type occupying the posterior part.

Axon Trajectories and Their Putative Targets of Cholinergic Neurons

The trajectories of axons reflect the function of neurons. To elucidate the roles of neurons in the juvenile CNS, we traced the axons of juvenile neurons using Kaede fluorescence. Our transgenic lines express the reporter gene in many corresponding neurons in a nonmosaic manner. It was difficult to trace the axon from a neuron when many neurons in the same specimen emitted fluorescence, and we therefore used the transient transgenesis of a reporter construct in this experiment. A DNA vector containing a reporter construct of a differentiated neuron type was introduced into one‐cell embryos by means of electroporation (Corbo et al., 1997). We selected juvenile‐stage animals with fluorescence in only a few neurons to chase their axons. When further specific labeling of the axon from a neuron was necessary in the observed specimens, we carried out the photoconversion of Kaede expressed in a subset of neurons by local UV irradiation. This enabled us to distinguish axons from two different populations of neurons with different intensities of Kaede‐green and Kaede‐red fluorescence (e.g., Fig. 7C).

We first observed axons of cholinergic neurons. The results described in the above sections suggested that the cell bodies of cholinergic neurons were present in all three parts of the juvenile CNS, with the maximum number in the middle part. Axons of cholinergic neurons at the anterior part of the CNS passed two routes toward two destinations (Fig. 6A,B). In one route, axon bundles exited the CNS from its anterior end, and then went toward the opening of the oral siphon, ending around the oral siphon muscle (Fig. 6A, right arrowheads). In the other route, axon bundles went toward the posterior part of the CNS (Fig. 6A, left arrowhead). Some of them ended in the CNS, whereas other axons exited the CNS from its posterior end, and the axons then faded.

**Figure 6**
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Axon trajectories of cholinergic neurons in the juvenile CNS. All panels are photographs of live juveniles developed from fertilized eggs into which the *VACHT*>*Kaede* construct was electroporated. Arrows and arrowheads illustrate the positions of cell bodies and axons, respectively. A: A typical pattern of axon trajectory from cholinergic neurons whose cell bodies were located at the anterior part of the CNS (bracket). Left: a Kaede‐green fluorescent image. Right: a merged image with a DIC image. The left panel is shown in black and white for enhanced contrast. The position of the oral siphon (OS) is shown by a dotted line. B: Cholinergic neurons at the middle part of the CNS extending axons toward the body wall muscle through the anterior end of the CNS. Inset: a magnified image of synaptic boutons along the axons (white arrowheads). The right DIC image illustrates the position of the body wall muscle. A body wall muscle (BM) at the focused plane is highlighted with a dashed line. The other BM is out of focus and difficult to see in the photograph. C: Blue arrow and arrowhead illustrate cholinergic neuron(s) at the middle part of the CNS extending axons toward the body wall muscle through the posterior end of the CNS. The orange arrow and arrowhead illustrate the cell body and nerve extending toward the endostyle, respectively. Note that the cell body could not be distinguished from the cell bodies extending axons to the BM. D: Cholinergic nerve from the middle part of the CNS toward the gill. Inset: a branch of the nerve extending toward the anterior wall of the 1st protostigmata. Note that the axon emits very weak fluorescence; the photograph was over‐exposed for the image. Gi, gill. E: Cholinergic neurons at the middle part of the CNS extending axons toward the atrial siphon muscle. The edge of the atrial siphon (AS) is shown by a dotted line. F: Cholinergic neurons at the middle part of the CNS extending axons toward the esophagus (Eso). TB, tail debris. Left and right panels are derived from different photographs of the same specimen. G: Cholinergic neurons at the posterior part of the CNS (bracket) extending axons toward the anterior part (left of this panel) of the CNS. The CNS is highlighted with a dashed line.

**Figure 7**
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Axon trajectories of glutamatergic neurons. All panels are photographs of live juveniles. A–E: Juveniles developed from fertilized eggs into which the *VGLUT*>*Kaede* construct was electroporated. **A,B**: Axon trajectory of glutamatergic neurons at the anterior part of the CNS. The CNS is highlighted with a dashed line. In panel A, the fluorescence ends near the posterior end of the CNS. In panel B, the fluorescence is seen outside the CNS. Orange arrows and arrowheads illustrate the position of cell bodies and axons, respectively. Red arrowheads represent the posterior end of the CNS. C: Axon trajectory of glutamatergic neurons at the middle and posterior parts of the CNS. The image was taken after Kaede photoconversion. Top row, Kaede‐green fluorescence. Second row, Kaede‐red fluorescence preferentially labeling the neuron at the posterior part. Third row, a merged image of Kaede‐green and Kaede‐red fluorescent images. This panel contrasted the different fluorescent patterns of the middle (white arrow) and posterior neurons (orange arrow and arrowhead). Merge, a merged image of Kaede‐green, Kaede‐red, and brightfield images. D: Axon trajectory of glutamatergic neurons at the oral siphon. The position of the oral siphon (OS) is shown by a dotted line. E: Axon trajectory of glutamatergic neurons at the atrial siphon. The position of the atrial siphon (AS) is shown by a dotted line. Insets: images around the CNS of the same specimen.

The majority (23 of 31) of the cholinergic neurons whose cell bodies were at the middle part of the CNS extended axons toward the anterior side of the CNS (Fig. 6B). These axons exited the CNS from its anterior terminal, and they sharply turned toward the ventral side. After that, the axons again turned posteriorly toward the body wall muscle. There was only a single bundle of the body wall muscle at the side of the juvenile body (Fig. 6B, black dashed lines in the differential interference contrast [DIC] panel). After the axons reached the body wall muscle, they usually forked and ran along both orientations of the body wall muscle (Fig. 6B, orange arrowheads). Some synaptic boutons with strong green fluorescent protein (GFP) florescence were seen along the body wall muscle (Fig. 6B, white arrowheads in the inset). These structures may correspond to motor endplates.

Several neurons at the middle part of the CNS showed different patterns of axon trajectories from those described in the above paragraph. We found that a cluster of cholinergic neurons at the middle part extended their axons ventroposteriorly toward the endostyle after the axons exited the CNS from its anterior end (Fig. 6C, orange arrowhead). We also observed that some middle cholinergic neurons extended their axons toward the posterior side of the CNS. Axons of these neurons exited the CNS from its posterior end, and then these axons could be divided into four groups according to their destinations: body wall muscle, gill slit, atrial siphon muscle, and near the tail debris.

The first two of these four groups ran along the anterior wall of the 1st protostigmata (Fig. 6C, blue arrowhead and Fig. 6D, orange arrowheads; Chiba et al., 2004). Some nerves then ran along the posterior half of the body wall muscle (Fig. 6C, blue arrowhead), whereas the others faded near the ventral side of the protostigmata (Fig. 6D, orange arrowheads). The latter nerves had a short branch that extended toward the protostigmata (Fig. 6D, inset). The third group ran toward the atrial siphon, forked, and then ran along the atrial siphon muscle. The nerves covered approximately half the circumference of the atrial siphon (Fig. 6E, orange arrowheads). The fourth group maintained a posterior trajectory after they exited the CNS and ended near the esophagus and tail debris (Fig. 6F, orange arrowhead). The cholinergic neurons whose cell bodies were at the posterior part of the CNS extended axons toward the anterior part of the CNS, and the axons ended near its anterior end without exiting the CNS (Fig. 6G, orange arrowhead).

These results suggest that cholinergic neurons innervate the juvenile oral siphon muscle, body wall muscle, atrial siphon muscle, and gill. These tissues are usually regulated by different neuronal cells, as our observation suggested that a group of cholinergic neurons extends axons to one of the target tissues. One exception to this rule would be that some cholinergic neurons could regulate both the body wall muscle and gill, as we observed the presence of neurons that extended their axons toward the body wall muscle through the gill slit (Fig. 6C, blue arrowhead).

Axon Trajectories and Their Putative Destinations of Glutamatergic Neurons and GABA/Glycinergic Neurons

As mentioned above, the cell bodies of glutamatergic neurons were preferentially present at the anterior side of the CNS. These anterior glutamatergic neurons extended their axons toward the posterior end of the CNS. Most of the axons terminated in the CNS (Fig. 7A, orange arrow and arrowhead), but we noted that one of 18 anterior neurons exited the CNS (Fig. 7B, orange arrow and arrowhead). Only a few cell bodies of the glutamatergic neurons were found at the middle and posterior parts of the CNS (four and two of the total of 24 glutamatergic neuron clusters observed, respectively). Glutamatergic neurons at the middle part also extended axons toward the posterior part of the CNS (Fig. 7C, white arrow and arrowhead). By contrast, the glutamatergic neurons whose cell bodies were at the posterior part extended axons toward the anterior part of the CNS and ended there (Fig. 7C, orange arrow and arrowhead).

We investigated the axonal trajectories of the peripheral glutamatergic neurons at the oral and atrial siphons to determine the relationships between the peripheral nervous system and the CNS. The neurons at the oral siphon extended axons toward the anterior part of the CNS, and they terminated there (Fig. 7D, orange arrow and arrowhead). The glutamatergic neurons at the atrial siphons, by contrast, extended axons to the posterior end of the CNS (Fig. 7E, orange arrow and arrowhead). The axons then penetrated the CNS, and they ended in the anterior part of the CNS (Fig. 7E, orange arrowheads in the inset). These observations suggest that the anterior part of the CNS is the center accumulating inputs from peripheral tissues.

Most of the cell bodies of GABAergic/glycinergic neurons were found at the anterior and posterior parts of the CNS. The anterior and posterior GABAergic/glycinergic neurons extended axons toward the posterior and anterior ends of the CNS, respectively (Fig. 8A,B, orange arrows and arrowheads). We did not find an axon of a GABAergic/glycinergic neuron exiting the CNS. We observed that two GABAergic/glycinergic neurons whose cell bodies were at the middle part of the CNS also extended their axons toward the posterior or anterior part of the CNS (Fig. 8C,D, orange arrows and arrowheads).

**Figure 8**
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Axon trajectories of GABA/glycinergic neurons. All panels are photographs of live juveniles. A–D: Juveniles developed from fertilized eggs into which the *VGAT*>*Kaede* construct was electroporated. A: Axon trajectory of GABAergic/glycinergic neurons at the anterior part, as highlighted by Kaede‐red fluorescence. The CNS is highlighted with a dashed line. B: Axon trajectory of a GABAergic/glycinergic neuron at the posterior part, as highlighted by Kaede‐red fluorescence. C: GABAergic/glycinergic neuron at the middle part extending axons anteriorly. D: GABAergic/glycinergic neuron at the middle part extending axons posteriorly.

Cholinergic Neurons Regulate the Contraction of the Muscles and the Ciliary Movement of the Gill

The patterns of the axon trajectories of the cholinergic neurons suggested that these neurons regulate the behaviors of juvenile muscles and gill. To investigate this possibility, we carried out an optogenetic analysis of cholinergic neurons. Channelrhodopsin 2 (ChR2) of Chlamydomonas reinhardtii absorbs blue light and then opens its channel to allow an influx of sodium ions (Zhang et al., 2006, 2007). The sodium ion influx depolarizes cells, and if this event occurs in a neuron, the neuron starts emitting an action potential and stimulates postsynaptic cells. The function of neurons can thus be examined by expressing ChR2 in a subset of neurons and observing the behavioral changes during blue‐light irradiation. For example, when ChR2 was expressed in the larval CNS with the cis element of β2tubulin, the larvae showed swimming behavior when blue light was irradiated, and they stopped this behavior when the light was turned off (Supp. Movie S1, which is available online), suggesting the efficacy of the method in Ciona.

We made a fusion construct driving ChR2 cDNA under the control of the cis element of VACHT. Although our ChR2 cDNA was fused with YFP, its yellow fluorescent protein (YFP) fluorescence was not strong enough for us to see the expression adequately. As a countermeasure, we joined a reporter cassette of VACHT>Kaede next to VACHT>ChR2::YFP to enhance the green fluorescence emitted by the cholinergic neurons harboring the construct (Fig. 9, left sequence). After electroporation of the ChR2 expression vector, we selected juveniles that expressed green fluorescence in only a few cholinergic neurons to analyze a restricted number of neurons. However, we were unable to eliminate the possibility that we simultaneously analyzed several cholinergic neurons with different axon trajectories.

**Figure 9**
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Cholinergic neurons appear to regulate the contraction of muscles and the ciliary movement of the gill. All transiently transgenic animals were developed from fertilized eggs into which the *VACHT*>*Kaede*;*VACHT*>*ChR2::YFP* construct was electroporated. *ChR2::YFP* and *Kaede* were thus expressed in only a subset of cholinergic neurons that are difficult to see from the panel due to overexposure. Left: fluorescence images of juveniles harboring *VACHT*>*Kaede*;*VACHT*>*ChR2::YFP*. Second row from the left: DIC images of animals before blue‐light irradiation. Third row: DIC images of animals during blue‐light irradiation. Right: DIC images of animals after blue‐light irradiation. **A–A′″**: This animal shows green fluorescence in cholinergic neurons extending axons to the oral siphon muscle. A″: During blue‐light irradiation, the animal contracted its oral siphon. The arrow indicates the length of the oral siphon opening, which is shorter than that in B″. **B–B′″**: This animal shows green fluorescence in cholinergic neurons extending axons to the body wall muscle. B″: Contraction of the body wall during blue‐light irradiation. This animal kept its oral siphon opened during the shrinking of its body. **C–C′″**: This animal shows green fluorescence in cholinergic neurons extending axons to the atrial siphon muscle. Insets: magnified images of atrial siphons. C″: Contraction of the atrial siphon during blue‐light irradiation. **D–D′″**: This animal shows green fluorescence in cholinergic neurons extending axons to the gill. Insets: magnified images of the gill. In D′ and D′″, cilia are recognized as spikes. D″: Arresting ciliary movement during blue‐light irradiation. Cilia are invisible when their movement is arrested.

When juveniles that had green cholinergic axons extending to the oral siphon muscle were irradiated with blue light, the animals closed the oral siphon during the irradiation, and they relaxed the siphon when the blue light irradiation was terminated (Fig. 9A, Supp. Movie S2). This suggests that cholinergic nerves to the oral siphons regulate the contraction of the oral siphon muscle. Likewise, the animals with cholinergic nerves extending to body wall muscle or atrial siphon muscle showed the contraction of the body or atrial siphon, respectively (Fig. 9B,C; Supp. Movies S3,S4). When we investigated juveniles in which green cholinergic nerves extended axons to the pharyngeal gill, we observed that these juveniles stopped ciliary movement during blue‐light irradiation (Fig. 9D; Supp. Movie S5). This result suggests that the cholinergic neurons negatively regulate the movement of the cilia of stigmata cells. From these results, we concluded that cholinergic neurons in the CNS extend axons to the muscle systems and gill slit to regulate their actions.

Discussion

In this study, we mapped the cell bodies of major neurons and chased their axon trajectories in the juvenile CNS of Ciona intestinalis (Fig. 10A–C). The constructed maps provide useful information for studying the neurobiology of the juvenile/adult CNS of ascidians. The shape of the juvenile CNS does not seem to possess prominent features compared with the morphologically regionalized larval CNS; however, our neuronal map suggests that the juvenile CNS is evidently patterned along the A‐P axis according to the positions of differentiated neurons. The highly regionalized manner may be necessary for the juvenile central nervous system's exertion of multiple roles in the control of adult organs, an idea that is supported by our observation of the axon trajectories and optogenetic analyses.

**Figure 10**
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Juvenile neuronal maps of the *Ciona intestinalis* CNS. A: A map of cholinergic neurons. Pink, dark green and blue illustrate neurons at the anterior, middle and posterior parts of the CNS, respectively. Cell bodies are shown by ovals and axons are shown by lines. The large oval indicates the area where the neurons are preferentially located, and the smaller ovals indicate their minor locations. Anterior = left and dorsal = top. B: Glutamatergic neurons. Purple and magenta ovals illustrate peripheral neurons at the siphons. C: GABA/glycinergic neurons. Because the neurons extend axons inside of the CNS, the diagram illustrates only the CNS and a ciliated funnel. D: Schematic showing the similarity between the larval CNS and the juvenile CNS. Black ovals and solid black lines illustrate neurons and their axons, respectively. The major neuron types located in each part are shown by their abbreviated names: GL, glutamatergic neurons; GG, GABA/glycinergic neurons; CH, cholinergic neurons.

Strong Bias of the Position of Neuronal Cell Bodies in the CNS

We found that the cell bodies of the cholinergic neurons, glutamatergic neurons, and GABAergic/glycinergic neurons in the Ciona CNS have their own preferred positions. Most of the glutamatergic neurons gather at the anterior part of the CNS, whereas the populations of the GABAergic/glycinergic neurons are divided into anterior and posterior parts. The cholinergic neurons are rather well distributed to all three parts of the CNS, with the highest number in the middle part. The biases of neuronal positions may reflect the different functions between the regions of the juvenile CNS.

Our observations suggest that the anterior part of the juvenile CNS receives inputs from peripheral glutamatergic neurons at the oral/atrial siphons. Peripheral glutamatergic neurons in Ciona larvae are thought to be sensory neurons (Horie et al., 2008), and this may be true of the glutamatergic neurons at the oral and atrial siphons, both of which are formed from structures similar to placodes (Manni et al., 2004; Kourakis and Smith, 2007; Sasakura et al., 2012b). The anterior part of juvenile CNS collectively thus receives signals from the putative sensory systems to transcend the signals to the other parts of the CNS for starting appropriate reaction(s) for sensed stimuli. Accumulated glutamatergic neurons at the anterior part of the CNS may also function in the direct reception of some stimuli, as in the case of the larval CNS (Horie et al., 2008), and/or they may function as secondary neurons that pass the inputs from peripheral systems to the posterior parts. Cholinergic neurons in the CNS regulate the muscle systems and gill. The position of cholinergic neurons is closely related to the positions of their effectors (see the discussion below). GABAergic/glycinergic neurons at the anterior and posterior parts of the CNS project axons toward the opposite side of the CNS but they do not extend axons outside the CNS, suggesting that these neurons function in the communication inside the CNS. GABAergic/glycinergic neurons are generally inhibitory neurons that arrest the excitation of postsynaptic neurons (Varju et al., 2001). The GABAergic/glycinergic neurons are probably components of negative feedback loops in the CNS: the neurons receive excitatory stimuli from other neuron types and then arrest further transmission of the excitation to terminate the responses.

The unique positioning of neuron types clearly suggests that their progenitor cells are differently distributed in the CNS. Characterization of the precursor cells will enable us to understand how the position of each neuron type is determined and how the terminal differentiation of neurons is conducted at the molecular level. For the identification of neuronal progenitor cells, our neuronal map will be a powerful tool to deduce their positions. The number of total neural cells in the juvenile CNS that was estimated here with the β2tubulin>Kaede and β2tubulin>CFP lines is evidently larger than the total number of differentiated neurons. The unidentified neural cells may include these progenitor (or stem) cells in addition to minor differentiated neurons and glial cells that we could not address in this study.

Axon Trajectories of Neurons in the CNS

Our present findings demonstrated that the orientation of the axon trajectories of neurons in the Ciona CNS was firmly determined according to the position of the cell bodies and neuronal types. This is the best represented by the cholinergic neurons, which extend axons outside of the CNS. All of the axons exit the CNS from its anterior or posterior ends, but no axon exits from the lateral side of the CNS even if its innervating tissue is near the lateral side. This suggests the presence of a mechanism preventing axon projection from the lateral side or a mechanism promoting axons to extend toward either the anterior or posterior end. At the outside of the CNS, most of the cholinergic axons turn toward the ventral side, passing two major routes at the anterior and posterior sides (Fig. 10A), and then their routes are separated to go to each of their destinations. The axons of juvenile neurons are therefore probably guided by a shared mechanism in and near the CNS. When they come closer to their destinations, the axons may be attracted by their destined tissues or may be repulsed by nearby tissues to refine the orientation of axon projections. The full‐grown adult CNS of Ciona is known to possess major routes of the nerves at both the anterior and posterior ends, like those in the juvenile CNS (Willey, 1893), suggesting that the rule of axon guidance is generally unchanged during growth.

Unlike the anterior and middle cholinergic neurons, the posterior cholinergic cell bodies extend axons to the anterior side of the CNS. This is not restricted to cholinergic neurons; posterior glutamatergic and GABAergic/glycinergic neurons also extend axons in the same way. It will be interesting to determine whether all posterior neurons use the same mechanism of axon guidance. The mechanism underlying the inhibition of the posterior neurons' extension of axons toward the posterior side of the body while allowing this extension for anterior/middle neurons should also be identified.

The positions of the cell bodies/axons of cholinergic neurons and the positions of their innervating tissues have a co‐relationship in that the anterior neurons tend to extend axons to anterior tissues, and the middle neurons extend to the middle‐posterior tissues. Indeed, the oral siphon receives axons from anterior cholinergic neurons, and the atrial siphon and gill receive axons from middle neurons. The body wall muscle, although it is only a pair of muscles at the left and right sides of the juvenile body, is long enough to be innervated by both anterior and posterior nerves. Despite the dual regulation, posterior cholinergic nerves preferentially extend to the posterior side of the body wall muscle (Fig. 6C). Although the Ciona CNS seems to be highly compacted after metamorphosis, the CNS is likely to possess polarity so as to efficiently regulate adult tissues distributed along the A‐P axis of the body.

Juvenile Behaviors Regulated by Cholinergic Neurons

Our present results show that juvenile cholinergic neurons regulate behaviors of muscles and gill in Ciona. Cholinergic neurons induce contraction of the oral, atrial and body wall muscles and arrest the ciliary movement of the gill. The dual function of a cholinergic network is reasonable, because ascidians simultaneously contract the siphons and body and stop ciliary movement when they are endangered or stimulated; the synchrony could be achieved more easily if the behaviors are regulated by the same neuronal network rather than by different networks.

The regulation of juvenile muscles by cholinergic neurons indicates that many of these neurons are likely to be motor neurons, like the larval cholinergic neurons that are thought to regulate the contractions of the tail muscle (Horie et al., 2010; Nishino et al., 2011). It was suggested that the plausible motor neurons of the postmetamorphosis stage of Ciona are homologous to the cranial motor system of vertebrates (Dufour et al., 2006). In that study, the authors chased Ciona neural cells expressing the transcription factor gene Phox2, which marks cranial motor neurons in vertebrates (Pattyn et al., 2000; Brunet and Pattyn, 2002). Phox2‐positive Ciona cells are seen in the larval neck region located between the sensory vesicle and motor ganglion. After metamorphosis, the neck cells extend axons to the gill slits and body wall muscle. Moreover, the Phox2‐positive neurons express motor neuron marker genes such as Tbx20 and choline acetyl transporter (ChAT). However, there is no physiological evidence that the Phox2‐positive, putative cholinergic neurons function as motor neurons. Our present results support the idea that the juvenile cholinergic neurons are homologous to the cranial motor neurons by showing their functions of cholinergic neurons in the CNS of metamorphosed Ciona.

Conserved Features Between the Larval CNS and the Juvenile CNS

We realized that the organization of the neurons in the juvenile CNS has some similarities to that in the larval CNS (Fig. 10D). In both cases, the anterior part of the CNS (i.e., the sensory vesicle in the larval CNS) is composed of all three neuron types, and it is where most of the glutamatergic neurons are accumulated. The anterior parts of both the larval and juvenile CNS receive the input from sensory systems (Takamura, 1998; Horie et al., 2008). Cholinergic motor neurons are the major neurons in the motor ganglion of the larval CNS, similar to the middle part of the juvenile CNS. The posterior part of the larval CNS includes GABAergic/glycinergic neurons that extend axons toward the anterior side (Horie et al., 2010; Nishino et al., 2010).

This similarity may support our previous finding that the basic organization of the larval CNS is inherited by the juvenile CNS (Horie et al., 2011). In that study, we found that the juvenile CNS, including the neurons there, is derived from nonneuronal glial cells of the larval CNS, whereas most of the larval neurons disappear during metamorphosis. In addition, the A‐P pattern of the larval CNS could be transferred to the juvenile CNS, because each of the anterior and posterior regions of the larval CNS tends to respectively form the anterior and posterior parts of the juvenile CNS. The A‐P patterns of the larval CNS are specified through transcription factor and signaling molecule genes that are expressed in a region‐specific manner (Wada et al., 1998; Imai et al., 2002; Dufour et al., 2006; Ikuta and Saiga, 2007). The established A‐P patterns of the larval CNS could be “remembered” by the glial cells during metamorphosis, and they may re‐create the similar patterns of the CNS that could result in the conserved neuronal patterns between the larval and juvenile CNS. For example, some larval glial cells might have a property as progenitor cells of a specific neuron type, and their position is prepatterned in the larval CNS. They could produce the same neurons in both the larval and juvenile CNS, which may result in the similar neuronal organizations in the CNS at the two stages.

In contrast to the similarities of the neuronal organizations along the A‐P axis, the functions of the juvenile CNS are quite different from those of the larval CNS. In the larval body, the tail muscle is the only known tissue that needs regulation from the CNS, whereas the juvenile body contains many more tissues that require regulation by the CNS. This remarkable functional difference may be a reason why ascidians adopted the renewal of neurons during metamorphosis (Horie et al., 2011). Curiously, the Ciona juvenile body organization is more similar to those of vertebrates in that both of the bodies contain mature feeding organs homologous to each other (e.g., Ogasawara et al., 1999). It is of interest to determine whether the CNS of the postmetamorphic stage of Ciona possesses properties homologous to those of the vertebrate CNS, as was discussed concerning cholinergic neurons (Dufour et al., 2006). Understanding the mechanisms underlying the formation of the neuronal network in Ciona juveniles may give us clues to the conservation of neuronal patterning that regulates feeding (facial) organs among chordate central nervous systems.

Experimental Procedures

Animals

Wild‐type Ciona intestinalis were cultivated at the ports of Misaki (Kanagawa Prefecture), Maizuru (Kyoto), Mukaishima (Hiroshima), and Usa (Kochi) in Japan. After being transferred to the laboratory, the animals were maintained under constant light to induce oocyte maturation. Transgenic lines were maintained as described (Joly et al., 2007). Eggs and sperm were isolated surgically from the gonoducts, and they were mixed in seawater in petri dishes to obtain fertilized eggs for the experiments described below. When the larvae started adhesion, the seawater of the petri dishes was exchanged several times to remove any unsettled larvae. This treatment was done to synchronize the timing of metamorphosis (Matsunobu and Sasakura, 2015).

Constructs

The cis element of β2tubulin (Kusakabe et al., 2004) was amplified by polymerase chain reaction (PCR) using pMiCiβ2tubulin>Kaede (Horie et al., 2011) as the template, digested with BamHI and inserted into the BamHI site of pSPCFP (Horie et al., 2011) to create pSPCiβ2tubulin>CFP. The β2tubulin>CFP cassette was inserted in pMiDestF (Sasakura et al., 2010) with gateway technology (Invitrogen, Carlsbad, CA, USA) to create pMiCiβ2tubulin>CFP. The fusion of cDNAs of Channelrhodopsin and YFP (Zhang et al., 2007) was PCR‐amplified and replaced with cDNA of GFP in pSPEGFP (Sasakura et al., 2003) to create pSPChR2::YFP. The cis elements of β2tubulin and VACHT were inserted into the BamHI site of pSPChR2::YFP to create pSPβ2tubulin>ChR2::YFP and pSPVACHT>ChR2::YFP. The VACHT>Kaede cassette was inserted into the HindIII and SalI sites of pSPVACHT>ChR2::YFP to create pSPVACHT>Kaede;VACHT>ChR2::YFP. The full names of the constructs based on the nomenclature rule of the ascidian community (Stolfi et al., 2015) are given in Table 1.

Table 1. Full Names of DNA Constructs and Transgenic Lines Used in This Study

Figure or sectiona^a The part of the manuscript in which the construct appears first time.	Name in the manuscript	Full name according to the nomenclature rule	Synomim
Figure 1	β2tubulin>Kaede line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.L116.156051‐160357>Kaede]2
Figure 1	β2tubulin>CFP line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.L116.156051‐160357>ECFP]4
Figure 2	VACHT>Kaede line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.C1.4530860‐4534650>Kaede]5
Figure 4	VGLUT>Kaede line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.C3.4780953‐4783803>Kaede]6
Figure 4	β2tubulin>CFP line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.L116.156051‐160357>ECFP]3
Figure 5	VGAT>Kaede line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.C2.4282950‐4285597>Kaede]1
Figure 5	VGAT>Kaede line	Ciinte.Tg[pMi‐Ciinte.REG.KH2012.C2.4282950‐4285597>Kaede]2
Figure 6	VACHT>Kaede construct	pCiinte.REG.KH2012.C1.4530860‐4534650>Kaede	pSPCiVACHTK (Horie et al., 2011)
Figure 7	VGLUT>Kaede construct	pCiinte.REG.KH2012.C3.4780953‐4783803>Kaede	pSPCiVGLUTK (Horie et al., 2011)
Figure 7	VGAT>Kaede construct	pCiinte.REG.KH2012.C2.4282950‐4285597>Kaede	pSPCiVGATK (Horie et al., 2011)
Supplementary Movie S1	β2tubulin>ChR2::YFP	pCiinte.REG.KH2012.C1.4530860‐4534650>ChR2::YFP	pSPβ2tubulin>ChR2::YFP (Experimental Procedures)
Supplementary Movie S2	VACHT>Kaede;VACHT>ChR2::YFPconstruct	p(Ciinte.REG.KH2012.C1.4530860‐4534650>Kaede; Ciinte.REG.KH2012.C1.4530860‐4534650>ChR2::YFP)	pSPVACHT>Kaede;VACHT>ChR2::YFP (Experimental Procedures)
Experimental Procedures	pSPVACHT>ChR2::YFP	pCiinte.REG.KH2012.C1.4530860‐4534650>ChR2::YFP
Experimental Procedures	pSPβ2tubulin>CFP	pCiinte.REG.KH2012.C1.4530860‐4534650>ECFP
Experimental Procedures	pMiβ2tubulin>CFP	pMi‐Ciinte.REG.KH2012.C1.4530860‐4534650>ECFP

^a The part of the manuscript in which the construct appears first time.

Transgenic Lines

β2tubulin>Kaede, VACHT>Kaede and VGAT>Kaede lines were described (Horie et al., 2011). Other transgenic lines were created by Minos‐meditated transgenesis (Sasakura et al., 2003; Matsuoka et al., 2005). VGLUT>Kaede lines were reported (Horie et al., 2011), but the lines used in that report were lost. We established new lines with the same vector described in the Horie et al. (2011) report. The full names of the transgenic lines based on the nomenclature rule of the ascidian community (Stolfi et al., 2015) are provided in Table 1.

Electroporation

For the labeling of a few neurons in the juvenile CNS, 20–60 μg of DNA was used for electroporation into 1‐cell embryos as described (Corbo et al., 1997; Treen et al., 2014). After electroporation, the embryos were washed once with seawater to decrease the amount of un‐electroporated DNA, and they were then cultured at approximately 18°C until the larval stage. At the larval stage, larvae with fluorescence in the CNS were transferred to new petri dishes filled with seawater to induce metamorphosis. The seawater was exchanged at approximately 2‐day intervals until sampling.

Immunostaining

Juveniles were fixed with formalin 1/10 diluted with seawater for 3 hr or overnight at 4°C. After being washed with phosphate‐buffered saline (PBS) containing Tween20 (PBST), the juveniles were treated with fetal goat serum (FGS) 1/10 diluted with PBST for 3 hr for the blocking of nonspecific binding of antibodies. After blocking, the juveniles were incubated with mouse anti‐Kaede and rabbit anti‐CFP antibodies 1/1,000 diluted with PBST containing FGS overnight at 4°C.

After being washed with PBST, the juveniles were incubated with Alexa 488‐conjugated anti‐mouse antibodies and Alexa 555‐conjugated anti‐rabbit antibodies 1/1,000 diluted with PBST containing FGS overnight at 4°C. After a wash with PBST, the juveniles were further treated with PBST containing 1.7 μg/ml propidium iodide (PI) plus 30 μg/ml RNase A for 3 hr at 4°C or PBST containing 5 μM sytox blue for 3 hr at room temperature before observation with a confocal microscope (LSM700, Carl Zeiss MicroImaging, Jena, Germany). Three‐dimensional images were reconstructed from z‐stack images using IMARIS software (Carl Zeiss). The number of nuclei in the Kaede‐positive cells were counted with the software.

Photo‐conversion

For chasing larval cholinergic neurons during metamorphosis, larvae of the VACHT>Kaede line were cultured in an 11‐cm petri dish with seawater. The petri dish was put on a UV illuminator, and UV (356 nm wavelength) was irradiated for 40–50 min.

For the local labeling of a subset of neurons in the juvenile CNS, fertilized eggs electroporated with a construct driving Kaede in differentiated neurons were developed until the juvenile stage. Juveniles that exhibited Kaede‐green fluorescence in a few neurons were selected, and they were mounted alive on a slide glass and a coverslip. Fluorescence for exciting DAPI (4′,6‐diamidine‐2‐phenylidole‐dihydrochloride) was irradiated with a fluorescent microscope (Axioimager Z, Carl Zeiss) for a few seconds to the restricted area of the juvenile CNS with a diaphragm. Fluorescent images were then obtained.

Optogenetic Analysis

Larvae developed from eggs electroporated with the β2tubulin>ChR2::YFP construct or juveniles developed from eggs electroporated with the VACHT>Kaede;VACHT>ChR2::YFP construct were mounted on a slide glass and covered with a coverslip. GFP excitation fluorescence was irradiated to the juveniles with a fluorescent microscope (Axioimager Z).

Acknowledgments

We thank the members of the Shimoda Marine Research Center at the University of Tsukuba for their kind cooperation during our study. We are grateful to Dr. Atsuo Nishino for his critical reading of the manuscript. We also thank Drs. Shigeki Fujiwara, Nobuo Yamaguchi, Kunifumi Tagawa, Tatsuya Ueki, and all members of the Department of Zoology, Graduate School of Science, Kyoto University, the Misaki Marine Biological Station, University of Tokyo, the Maizuru Fisher Research Station of Kyoto University, the Marine Biological Laboratory, Graduate School of Science, Hiroshima University, Department of Applied Science, Kochi University, for providing us with adult Ciona intestinalis. We thank Dr. Karl Deisseroth for his gift of ChR2 H134R plasmids. This study was supported by Grants‐in‐Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to Y.S and T.H. Y.S. was supported by a Toray Science and Technology Grant. T.H. was supported by the JST PRESTO program. Further support was provided by grants from the National Bioresource Project.

Supporting Information

Additional supporting information may be found in the online version of this article.

Filename	Description
dvdy24317-sup-0001-suppmovie1.wmv583.5 KB	Suppl. Movie S1 Swimming behavior of a larva induced by channelrhodopsin. This larva was developed from a fertilized egg into which β2tubulin>ChR2::YFP was electroporated. Blue‐light irradiation can be seen by the appearance of green fluorescence derived from YFP.
dvdy24317-sup-0002-suppmovie2.mp4844.8 KB	Suppl. Movie S2 Contraction of the oral siphon during blue‐light irradiation. The animal is the same as the one shown in Figure 9A.
dvdy24317-sup-0003-suppmovie3.mp4816 KB	Suppl. Movie S3 Contraction of the body wall muscle during blue‐light irradiation. The animal is the same as the one shown in Figure 9B.
dvdy24317-sup-0004-suppmovie4.mp4784.2 KB	Suppl. Movie S4 Contraction of the atrial siphon during blue‐light irradiation. The animal is the same as the one shown in Figure 9C.
dvdy24317-sup-0005-suppmovie5.mp4442.3 KB	Suppl. Movie S5 Arresting ciliary movement during blue‐light irradiation. The animal is the same as the one shown in Figure 9D.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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Citing Literature

Volume244, Issue11

November 2015

Pages 1375-1393

Neuronal map reveals the highly regionalized pattern of the juvenile central nervous system of the ascidian Ciona intestinalis

Abstract

Introduction

Results

Cell Number Constituting the CNS of Ciona intestinalis