Review of: "Disruption of left-right axis specification in Ciona induces molecular, cellular, and functional defects in asymmetric brain structures"

The L−R asymmetric features of animals are easily recognized by the positions of the organs. Many organs, such as the gut, liver, and heart, are asymmetrically located in a determinative manner. L−R asymmetries are a shared feature of metazoans; various aspects of animal characteristics are indeed asymmetric. For example, the winding direction of snail shells is usually clockwise, with a few examples of counterclockwise. Male grasshoppers have asymmetric wing shapes that create sounds to attract females. In sea urchins, whose adult morphology is basically radially symmetric, the adult rudiment forms on one side near the gut of Pluteus larvae.


Left−right asymmetries of animals
The L−R asymmetric features of animals are easily recognized by the positions of the organs. Many organs, such as the gut, liver, and heart, are asymmetrically located in a determinative manner. L−R asymmetries are a shared feature of metazoans; various aspects of animal characteristics are indeed asymmetric. For example, the winding direction of snail shells is usually clockwise, with a few examples of counterclockwise. Male grasshoppers have asymmetric wing shapes that create sounds to attract females.
In echinoderm sea urchins, whose adult morphology is basically radially symmetric, the adult rudiment forms on one side near the gut of Pluteus larvae.
L−R asymmetry is not limited to morphology. The left and right sides of the cerebellum are known to have different roles (reviewed in [2]). This is associated with the different features of neurons, such as neuronal subtypes, gene expression, and axon trajectories. The L−R asymmetries of animals are also reflected in behavior. A famous example is the handedness of humans [3][4]. Behavioral asymmetries may be associated with the cellular and structural asymmetries of our bodies, including the nervous systems, because these factors determine behaviors. Thus, studies of L−R axis formation include molecular, cellular, and behavioral backgrounds to elucidate animal asymmetries, as the title of this manuscript suggests.
To this end, it's necessary to consider that certain left-or right-biased characteristics, particularly behavioral ones, could be predominantly acquired features more than a result of genetic background. these acquired features must be distinguished from those determined by the genetic system. Because animals that have simple behavioral mechanisms are less influenced by acquired modifications, they will be valuable for thorough studies of the L−R axis.

Ascidians and their embryogenesis
Phylogenetic studies have shown that ascidians are the closest living relatives of vertebrates [5]. In this chordate group, it is not easy to recognize the phylogenetic position at the sessile adult stage; however, ascidians have a tadpole-shaped larval body, and the tadpole larva swims actively by swinging or beating its tail. The overall larval morphology and the tissue organization in the larval body, having a notochord and a dorsally located, hollow CNS, are features shared among chordates [6].
In contrast to the phylogenetic position of ascidians as the sister group of vertebrates, the mechanisms underlying early embryogenesis in ascidians are very different from those in vertebrates.
Ascidian eggs are called mosaic eggs, in which the determinants of cell fate specification are prelocalized [7][8]. A blastomere that receives a determinant is destined to differentiate into the cell type specified by the determinant. This mosaic mode of embryogenesis is in contrast to the regulative mode of vertebrate embryogenesis. Another characteristic of ascidian embryogenesis is its simplicity. In the model ascidian

The L−R asymmetry of ascidian larva
Ascidian larvae have L−R asymmetric features [13][14]. In Ciona, an easy example is the location of the pigmented ocellus at the brain-corresponding region of the CNS; this structure is clearly visible only from the right side. This bias occurs because the larval brain of Ciona is twisted and the ocellus is located on the right side [reviewed in 15]. The otolith is the other pigmented sphere near the ocellus. The midline of the otolith is somewhat biased to the right. In larvae with the twisted CNS, many brain regions, which can be specified by the distribution of neuronal subtypes, are also asymmetrically located.
The L−R asymmetric features of ascidian larvae are established during embryogenesis. During cleavage stages, ascidian embryos are perfectly symmetric. For example, the left and right blastomeres of two-cell embryos respectively form the left and right sides of the body [16]. Therefore, the bilateral symmetry is disrupted at a certain developmental period. The earliest sign of L−R asymmetry is observed at the twocell stage, in which asymmetric inositol triphosphate (IP3)-mediated calcium ion signaling occurs [17].
Then, the famous signature of L−R asymmetry is seen at the neurula stage. At this stage, the gene encoding Nodal, the key signaling molecule responsible for L−R asymmetry formation, is expressed only in the left side of the ectoderm [18][19]. These relays between molecules are suggested to create the L−R axis at the larval stage.
Removal of the chorion disrupts L−R asymmetries of larvae. This is because the chorion is necessary to specify theL−R axis. In Halocynthia, the physical contact between the chorion and the left side of the embryo induces the left-side expression of nodal [20]. A similar phenomenon is observed in Ciona, suggesting that a conserved mechanism underlies the breakage of L−R symmetry. Larvae developed from dechorionated eggs exhibit randomization of the L−R axis; larvae with normal asymmetry, complete reversal, partial reversal, and the absence of some asymmetric features are all seen in a single experiment.
L−R disruption by dechorionation persists after metamorphosis, which converts tadpole-like swimming larvae into immotile adults. At the larval stage, many organs are at the premature state and are not functional in solitary ascidians such as Ciona [12; 21]. After metamorphosis, however, ascidian juveniles have developed organs such as the heart and digestive tube. The location and structure of these adult organs are asymmetric, like those in vertebrates. The asymmetric features are disrupted in juveniles that developed from dechorionated eggs, suggesting that the mechanism that specifies the L−R axis during embryogenesis also determines this axis after metamorphosis. The rudiments of the adult organs are specified during embryogenesis, and some organs start to form at the larval stage [22][23]. In this way, the effect of L−R axis disruption by dechorionation could persist after metamorphosis.

How does the disruption of the L−R axis influence Ciona?
Kourakis and colleagues observed detailed changes in L−R disrupted larvae by dechorionation. They employed a new way of removing the chorion after the L−R axis is established, and they compared larvae subjected to dechorionation at this later stage (late-dechorionated larvae) with larvae developed from dechorionated eggs (early-dechorionated larvae). The early-dechorionated larvae exhibited various ranges of morphological and cellular abnormalities that seemed to be beyond the mere morphological disruption of the characteristics along the L−R axis.
In normal and late-dechorionated larvae, for example, the ocellus is cup-shaped while the otolith looks like a complete sphere. In early-dechorionated larvae, on the other hand, two cup-shaped or two sphereshaped pigments, or a single large pigment, is frequently observed, and the pigment or pigments are misoriented. These results suggest that L−R axis disruption is not merely a simple impairment of the arrangement of properly specified cells, tissues, or organs; rather, this disruption interferes with the specification processes. Accordingly, those authors showed that several sensory organs were absent iñ 50% of the early-dechorionated larvae. The rate of loss of a neural structure in early-dechorionated larvae differs among the structures. This difference is probably caused by the different strengths of the association between the mechanisms of specification of structures and L−R axis formation.
In addition, those authors found that the bilateral structures along the L−R axis are also affected by dechorionation at the early stage. Pitx is the key transcription factor responsible for the specification of the L−R axis as a downstream factor of Nodal [24,[18][19]. Pitx is expressed only on the left side of the motor ganglion, where motor neurons are aligned bilaterally [25]. Moreover, the gene encoding AMPA-type glutamate receptor (AMPAR) is also expressed only on the left side of the motor ganglion. Therefore, left and right motor neurons could have different functions by the left-side-specific reception of the neurotransmitter L-glutamate. Left-side-specific pitx and AMPAR expressions were impaired by early dechorionation, suggesting that the effects of dechorionation are not limited to the morphologically recognizable asymmetric characteristics but also influence the hidden asymmetric features of cells and tissues at the molecular level.

Behavioral changes in dechorionated larvae
The disruption of the L−R axis by dechorionation affects larval behavior. Ciona larvae exhibit negative gravitaxis at the initial stage after hatching [26][27]. By this activity, larvae swim toward the surface of the ocean; this movement is suggested to enhance the diffusion of siblings by the tidal movement. This gravitaxis is reproducibly observed in normally developed larvae. In addition to the gravitaxis, larvae start tail beating by responding to the dim conditions from a certain period after hatching [28]. Therefore, by using dimness to induce swimming, the authors observed swimming trajectories in relation to gravity. They found that, in contrast to the control groups, early-dechorionated larvae exhibited a much lower frequency of negative gravitaxis; many early-dechorionated larvae showed downward or sideways swimming in addition to upward. As mentioned above, L−R axis disruption causes various changes in the nervous systems beyond the positioning of structures along the L−R axis. Therefore, the variety of changes in swimming behavior is suggested to reflect the sum of the complexity of the effects caused by dechorionation.
The authors of the cited manuscript found that a portion of larvae from the early-dechorionation experiment had an axis that was a perfectly mirror image reversal of the normal axis. Using these mirrored larvae, the authors observed behavioral asymmetry in gravitaxis. When Ciona larvae with a normal L−R axis swam upward, they exhibited counterclockwise trajectories when viewed from above. In contrast, the L−R reversed larvae swam clockwise, suggesting that the mirroring of the body also causes the perfect reversal of swimming behavior with respect to gravity. This reversal means that the asymmetry in swimming behavior is based on the L−R asymmetric features in the body and could not be adjusted in an acquired manner.

Perspectives
L−R axis disruptions influence molecular, cellular, morphological, and behavioral aspects of ascidian larvae. Owing to this study and previous studies, we have accumulated knowledge about L−R asymmetric characteristics in ascidian larvae, the mechanisms underlying how the asymmetries are formed, and how the asymmetries influence animals. After reading the manuscript cited in this review, I have realized that there remains a big question: What is the significance of the rigid selection of L−R asymmetry? As the mirrored larvae suggest, the reversed construction of the L−R axis seems not to cause a deleterious effect on animals. Despite that, the L−R axis is rigidly determined. The molecules underlying L−R asymmetry construction, such as Nodal and Pitx, show conserved patterns of left-biased expressions and functions throughout animals, with the exceptions seen in echinoderms [29,30]. The simplicity of the Ciona larva, in both morphological and behavioral aspects, provides an ideal system with which to uncover this question by addressing how Ciona benefits by selecting, for example, a counterclockwise way of swimming in the negative gravitaxis.
Technically, chemical dechorionation is useful and widely used among Ciona researchers to introduce exogenous molecules, to simultaneously remove obstructive chorions before fixation for sampling of in situ hybridization, and so on. We have empirically learned the deleterious effects of this treatment, particularly on L−R axis formation. This study shows many concrete examples of how the L−R axis is disrupted by dechorionation. This information is helpful for judging whether early dechorionation will be appropriate or not in an experiment a researcher is planning to do. Moreover, this manuscript shows that dechorionation after embryogenesis is possible without impairing the L−R axis. The hesitation toward chemical dechorionation at a later stage exists because the dechorionation of ascidians uses a protease that breaks cell−cell adhesions [31][32][33]. Kourakis