Takeo Horie

D -Serine, a free amino acid synthesized by serine racemase, is a coagonist of N -methyl- d -aspartate–type glutamate receptor (NMDAR). d -Serine in the mammalian central nervous system modulates glutamatergic transmission. Functions of d -serine in mammalian peripheral tissues such as skin have also been described. However, d -serine’s functions in nonmammals are unclear. Here, we characterized d -serine–dependent vesicle release from the epidermis during metamorphosis of the tunicate Ciona . d -Serine leads to the formation of a pocket that facilitates the arrival of migrating tissue during tail regression. NMDAR is the receptor of d -serine in the formation of the epidermal pocket. The epidermal pocket is formed by the release of epidermal vesicles’ content mediated by d -serine/NMDAR. This mechanism is similar to observations of keratinocyte vesicle exocytosis in mammalian skin. Our findings provide a better understanding of the maintenance of epidermal homeostasis in animals and contribute to further evolutionary perspectives of d -amino acid function among metazoans.

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 Ca ²⁺ oscillation with an 80-sec interval at the mid-tailbud stage and 25 sec at the late tailbud stage. The Ca ²⁺ oscillation occurred independently in a dissociated single cell. In the late tailbud stage, the Ca ²⁺ 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.

In embryos of distantly related bilaterian phyla, their lateral neural borders give rise to the peripheral nervous system elements, including various mechanosensory cells derived from migratory precursors, such as hair cells and dorsal root ganglion (DRG) neurons in vertebrates, bipolar tail neuron (BTN) in Ciona, chordotonal organ in Drosophila, and AVM/PVM in Caenorhabditis elegans. Developmental genetics studies had revealed a couple of transcription factors (TFs) regulating differentiation of mechanosensory cells shared by vertebrates and arthropods. However, unbiased systematic profiling of regulators is needed to demonstrate conservation of differentiation gene batteries for mechanosensory cells across bilaterians. At first, we observed that in both C. elegans Q neuroblasts and Drosophila lateral neuroectoderm, conserved NPB specifier Msx/vab‐15 regulates Atoh1/lin‐32, supporting the homology of mechanosensory neuron development in lateral neural border lineage of Ecdysozia. So we used C. elegans as a protostomia model. Single‐cell resolution expression profiling of TFs and genetic analysis revealed a differentiation gene battery (Atonh1/lin‐32, Drg11/alr‐1, Gfi1/pag‐3, Lhx5/mec‐3, and Pou4/unc‐86) for AVM/PVM mechanosensory neurons. The worm‐gene battery significantly overlaps with both that of placode‐derived Atonh1/lin‐32‐dependent hair cells and that of NPB‐derived Neurogenin‐dependent DRG neurons in vertebrates, supporting the homology of molecular mechanisms underlying the differentiation of neural border‐derived mechanosensory cells between protostome and deuterostome. At last, Ciona BTN, the homolog of vertebrate DRG, also expresses Atonh1/lin‐32, further supporting the homology notion and indicating a common origin of hair cells and DRG in vertebrate lineage.

The CNS of the protovertebrate Ciona intestinalis contains a single cluster of dopaminergic (DA) neurons, the coronet cells, which have been likened to the hypothalamus of vertebrates. Whole-embryo single-cell RNA sequencing (RNA-seq) assays identified Ptf1a as the most strongly expressed cell-specific transcription factor (TF) in DA/coronet cells. Knockdown of Ptf1a activity results in their loss, while misexpression results in the appearance of supernumerary DA/coronet cells. Photoreceptor cells and ependymal cells are the most susceptible to transformation, and both cell types express high levels of Meis. Coexpression of both Ptf1a and Meis caused the wholesale transformation of the entire CNS into DA/coronet cells. We therefore suggest that the reiterative use of functional manipulations and single-cell RNA-seq assays is an effective means for the identification of regulatory cocktails underlying the specification of specific cell identities.

Placodes and neural crests represent defining features of vertebrates, yet their relationship remains unclear despite extensive investigation^1,2,3. Here we use a combination of lineage tracing, gene disruption and single-cell RNA-sequencing assays to explore the properties of the lateral plate ectoderm of the proto-vertebrate, Ciona intestinalis. There are notable parallels between the patterning of the lateral plate in Ciona and the compartmentalization of the neural plate ectoderm in vertebrates⁴. Both systems exhibit sequential patterns of Six1/2, Pax3/7 and Msxb expression that depend on a network of interlocking regulatory interactions⁴. In Ciona, this compartmentalization network produces distinct but related types of sensory cells that share similarities with derivatives of both cranial placodes and the neural crest in vertebrates. Simple genetic disruptions result in the conversion of one sensory cell type into another. We focused on bipolar tail neurons, because they arise from the tail regions of the lateral plate and possess properties of the dorsal root ganglia, a derivative of the neural crest in vertebrates⁵. Notably, bipolar tail neurons were readily transformed into palp sensory cells, a proto-placodal sensory cell type that arises from the anterior-most regions of the lateral plate in the Ciona tadpole⁶. Proof of transformation was confirmed by whole-embryo single-cell RNA-sequencing assays. These findings suggest that compartmentalization of the lateral plate ectoderm preceded the advent of vertebrates, and served as a common source for the evolution of both cranial placodes and neural crest^3,4.

Significance: The lateral neural plate border (NPB) gives rise to the neural crest, one of the precursors of the peripheral nervous system (PNS) and generally considered an evolutionary innovation of the vertebrate lineage. Recently, it has been reported that a rudimentary neural crest exists in protovertebrate Ciona , but whether this is true in other invertebrates and there is conserved molecular machinery specifying the NPB lineage are unknown. We present evidence that orthologs of the NPB specification module specify lateral neural progenitor cells in several invertebrates, including worm, fly, and tunicate. We propose that an ancient lateral neuroblast gene regulatory module was coopted by chordates during the evolution of PNS progenitors.

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.

Background: Gonadotropin‐releasing hormones (GnRHs) are neuropeptides that play central roles in the reproduction of vertebrates. In the ascidian Ciona intestinalis, GnRHs and their receptors are expressed in the nervous systems at the larval stage, when animals are not yet capable of reproduction, suggesting that the hormones have non‐reproductive roles. Results: We showed that GnRHs in Ciona are involved in the animal′s metamorphosis by regulating tail absorption and adult organ growth. Absorption of the larval tail and growth of the adult organs are two major events in the metamorphosis of ascidians. When larvae were treated with GnRHs, they completed tail absorption more frequently than control larvae. cAMP was suggested to be a second messenger for the induction of tail absorption by GnRHs. tGnRH‐3 and tGnRH‐5 (the “t” indicates “tunicate”) inhibited the growth of adult organs by arresting cell cycle progression in parallel with the promotion of tail absorption. Conclusions: This study provides new insights into the molecular mechanisms of ascidian metamorphosis conducted by non‐reproductive GnRHs. Developmental Dynamics 243:1524–1535, 2014.