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

The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution owing to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain.

Vertebrate rhythmic motor behaviour is generated by the central pattern generator (CPG) located in the spinal cord. However, the development of the CPG has not been elucidated at the single-neuron level. We found that a single pair of motor neurons (A10.64/MN2) constitutes the CPG and regulates rhythmic early motor behaviour in the proto-chordate, Ciona. This pair of cells exhibited Ca2+ oscillation with an 80-sec interval at the mid-tailbud stage and 25 sec at the late tailbud stage. The Ca2+ oscillation occurred independently in a dissociated single cell. In the late tailbud stage, the Ca2+ oscillation began to coincide in phase with the ipsilateral tail muscle contraction, which corresponds to rhythmic early motor behaviour. Interestingly, the number and frequency of tail muscle contractions gradually coincided with spikes in the burst of membrane potential as the embryos developed toward late tailbud stage. Photoablation of A10.64/MN2 abolished the rhythmic early motor behaviour. These findings indicate that the early spontaneous rhythmic motor behaviour of Ciona is directly regulated by A10.64/MN2 as an essential component of the CPG. Our findings shed light on the development and evolution of chordate rhythmic behaviours.

The Ciona intestinalis larva has two distinct photoreceptor organs, a conventional pigmented ocellus and a nonpigmented ocellus, that are asymmetrically situated in the brain. The ciliary photoreceptor cells of these ocelli resemble visual cells of the vertebrate retina. Precise elucidation of the lineage of the photoreceptor cells will be key to understanding the developmental mechanisms of these cells as well as the evolutionary relationships between the photoreceptor organs of ascidians and vertebrates. Photoreceptor cells of the pigmented ocellus have been thought to develop from anterior animal (a-lineage) blastomeres, whereas the developmental origin of the nonpigmented ocellus has not been determined. Here, we show that the photoreceptor cells of both ocelli develop from the right anterior vegetal hemisphere: those of the pigmented ocellus from the right A9.14 cell and those of the nonpigmented ocellus from the right A9.16 cell. The pigmented ocellus is formed by a combination of two lineages of cells with distinct embryonic origins: the photoreceptor cells originate from a medial portion of the A-lineage neural plate, while the pigment cell originates from the lateral edge of the a-lineage neural plate. In light of the recently proposed close evolutionary relationship between the ocellus pigment cell of ascidians and the cephalic neural crest of vertebrates, the ascidian ocellus may represent a prototypic contribution of the neural crest to a cranial sensory organ.

The ascidian larva has a pigmented ocellus comprised of a cup-shaped array of approximately 30 photoreceptor cells, a pigment cell, and three lens cells. Morphological, physiological and molecular evidence has suggested evolutionary kinship between the ascidian larval photoreceptors and vertebrate retinal and/or pineal photoreceptors. Rx, an essential factor for vertebrate photoreceptor development, has also been suggested to be involved in the development of the ascidian photoreceptor cells, but a recent revision of the photoreceptor cell lineage raised a crucial discrepancy between the reported expression patterns of Rx and the cell lineage. Here, we report spatio-temporal expression patterns of Rx at single-cell resolution along with mitotic patterns up to the final division of the photoreceptor-lineage cells in Ciona. The expression of Rx commences in non-photoreceptor a-lineage cells on the right side of the anterior sensory vesicle at the early tailbud stage. At the mid tailbud stage, Rx begins to be expressed in the A-lineage photoreceptor cell progenitors located on the right side of the posterior sensory vesicle. Thus, Rx is specifically but not exclusively expressed in the photoreceptor-lineage cells in the ascidian embryo. Two cis-regulatory modules are shown to be important for the photoreceptor-lineage expression of Rx. The cell division patterns of the photoreceptor-lineage cells rationally explain the generation of the cup-shaped structure of the pigmented ocellus. The present findings demonstrate the complete cell lineage of the ocellus photoreceptor cells and provide a framework elucidating the molecular and cellular mechanisms of photoreceptor development in Ciona.

The process of establishing the anterior-posterior axis is an important event in the development of bilateral animals. Otx, which encodes a homeodomain transcription factor, is continuously expressed in the anterior part of the embryo in a wide range of animals. This pattern of expression is thought to be important for the formation of anterior neural structures, but the regulatory mechanism that sustains the expression is not known. Here, using embryos of the ascidian, Halocynthia roretzi, we investigated how the transcription of Otx is maintained in the cells of the anterior neural lineage during embryogenesis. We identified an enhancer region sufficient to mimic the Otx expression pattern from the gastrula to tailbud stages. Several putative transcription factor binding sites that are required for generating the Otx expression pattern were also identified. Distinct sets of sites were required at different developmental stages, suggesting that distinct transcriptional mechanisms regulate Otx transcription in each of the gastrula, neurula and tailbud stages. Along with previous studies on the transcriptional regulatory mechanism of Otx during the pre-gastrula stages, the present results provide the first overview of the mechanism that sustains Otx expression in the anterior neural lineage during ascidian embryogenesis and demonstrate the complexity of a developmental mechanism that maintains Otx transcription.

SummaryAsymmetric localization of RNA is a widely observed mechanism of cell polarization. Using embryos of the ascidian, Halocynthia roretzi, we previously showed that mesoderm and endoderm fates are separated by localization of mRNA encoding a transcription factor, Not, to the future mesoderm-side cytoplasm of the mesendoderm cell through asymmetric positioning of the nucleus. Here, we investigated the mechanism that defines the direction of the nuclear migration. We show that localization of PtdIns(3,4,5)P3 to the future mesoderm region determines the direction of nuclear migration. Localization of PtdIns(3,4,5)P3 was dependent on the localization of PI3Kα to the future mesoderm region. PI3Kα was first localized at the 1-cell stage by the ooplasmic movement. Activity of localized PI3Kα at the 4-cell stage was required for the localization of PI3Kα up to the nuclear migration. Our results provide the scaffold for understanding the chain of causality leading to the separation of germ layer fates.

Establishment of the anterior-posterior axis is an important event in the development of bilateral animals. A homeodomain transcription factor, Otx, is important for the formation of the anterior part of the embryo, and its mRNA is expressed in a continuous manner in a wide range of animals. This pattern of expression is thought to be important for the formation of anterior neural structures, but the mechanism that regulates Otx expression remains largely unknown. Towards understanding how the transcription of Otx is maintained in the cells of anterior neural structure, the sensory vesicle, during embryogenesis, we examined transcription regulatory mechanisms of Otx, using embryos of the ascidian, Ciona intestinalis, from the gastrula to tailbud stages, which have not been studied previously. We identified two genomic regions capable of mimicking the Otx expression pattern from the gastrula to tailbud stages. Putative transcription factor binding sites required for this activity were identified. Notably, distinct sets of transcription factor binding sites were required at different developmental stages for the expression of Otx, suggesting that the continuity of Otx is supported by distinct transcriptional mechanisms in the gastrula and neurula stages. Along with previous studies using Halocynthia roretzi, the present results provide insight into the evolution of transcriptional regulatory mechanism of Otx.