MiR-92 Family Members Form a Cluster Required for Notochord Tubulogenesis in Urochordate Ciona savignyi.

MicroRNAs are frequently clustered in the genome and polycistronically transcribed, regulating targeted genes in diverse signaling pathways. The miR-17-92 cluster is a typical miRNA cluster, playing crucial roles in the organogenesis and homeostasis of physiological processes in vertebrates. Here, we identified three miRNAs (csa-miR-92a, csa-miR-92b, and csa-miR-92c) that belonged to the miR-92 family and formed a miRNA cluster in the genome of a urochordate marine ascidian Ciona savignyi. Except for miR-92a and miR-92b, other homologs of the vertebrate miR-17-92 cluster members could not be identified in the Ciona genome. We further found that the mature sequences of urochordate miR-92 family members were highly conserved compared with the vertebrate species. The expression pattern revealed that three miR-92 family members had consistent expression levels in adult tissues and were predominantly expressed in heart and muscle tissue. We further showed that, at the embryonic and larval stages, csa-miR-92c was expressed in the notochord of embryos during 18-31 h post fertilization (hpf) by in situ hybridization. Knockout of csa-miR-92c resulted in the disorganization of notochord cells and the block of lumen coalescence in the notochord. Fibroblast growth factor (FGF), mitogen-activated protein kinase (MAPK), and wingless/integrated (Wnt)/planar cell polarity (PCP) signaling pathways might be involved in the regulatory processes, since a large number of core genes of these pathways were the predicted target genes of the miR-92 family. Taken together, we identified a miR-92 cluster in urochordate Ciona and revealed the expression patterns and the regulatory roles of its members in organogenesis. Our results provide expression and phylogenetic data on the understanding of the miR-92 miRNA cluster's function during evolution.

Ciona embryonic tail bending is driven by asymmetrical notochord contractility and coordinated by epithelial proliferation.

Ventral bending of the embryonic tail within the chorion is an evolutionarily conserved morphogenetic event in both invertebrates and vertebrates. However, the complexity of the anatomical structure of vertebrate embryos makes it difficult to experimentally identify the mechanisms underlying embryonic folding. This study investigated the mechanisms underlying embryonic tail bending in chordates. To further understand the mechanical role of each tissue, we also developed a physical model with experimentally measured parameters to simulate embryonic tail bending. Actomyosin asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis was faster than that in the ventral counterpart during embryonic tail bending. Genetic disruption of actomyosin activity and inhibition of cell proliferation dorsally caused abnormal tail bending, indicating that both asymmetrical actomyosin contractility in the notochord and the discrepancy of epidermis cell proliferation are required for tail bending. In addition, asymmetrical notochord contractility was sufficient to drive embryonic tail bending, whereas differential epidermis proliferation was a passive response to mechanical forces. These findings showed that asymmetrical notochord contractility coordinates with differential epidermis proliferation mechanisms to drive embryonic tail bending.This article has an associated 'The people behind the papers' interview.

GABA-Induced GnRH Release Triggers Chordate Metamorphosis.

Metamorphosis, a widespread life history strategy in metazoans, allows dispersal and use of different ecological niches through a dramatic body change from a larval stage [1, 2]. Despite its conservation and importance, the molecular mechanisms underlying its initiation and progression have been characterized in only a few animal models. In this study, through pharmacological and gene functional analyses, we identified neurotransmitters responsible for metamorphosis of the ascidian Ciona. Ciona metamorphosis converts swimming tadpole larvae into vase-like, sessile adults. Here, we show that the neurotransmitter GABA is a key regulator of metamorphosis. We found that gonadotropin-releasing hormone (GnRH) is a downstream neuropeptide of GABA. Although GABA is generally thought of as an inhibitory neurotransmitter, we found that it positively regulates secretion of GnRH through the metabotropic GABA receptor during Ciona metamorphosis. GnRH is necessary for reproductive maturation in vertebrates, and GABA is an important excitatory regulator of GnRH in the hypothalamus during puberty [3, 4]. Our findings reveal another role of the GABA-GnRH axis in the regulation of post-embryonic development in chordates.

Hox13 is essential for formation of a sensory organ at the terminal end of the sperm duct in Ciona.

Species-specific traits are thought to have been acquired by natural selection. Transcription factors play central roles in the evolution of species-specific traits. Hox genes encode a set of conserved transcription factors essential for establishing the anterior-posterior body axis of animals. Changes in the expression or function of Hox genes can lead to the diversification of animal-body plans. The tunicate ascidian Ciona intestinalis Type A has an orange-colored structure at the sperm duct terminus. This orange-pigmented organ (OPO) is the characteristic that can distinguish this ascidian from other closely related species. The OPO is formed by the accumulation of orange-pigmented cells (OPCs) that are present throughout the adult body. We show that Hox13 is essential for formation of the OPO. Hox13 is expressed in the epithelium of the sperm duct and neurons surrounding the terminal openings for sperm ejection, while OPCs themselves do not express this gene. OPCs are mobile cells that can move through the body vasculature by pseudopodia, suggesting that the OPO is formed by the accumulation of OPCs guided by Hox13-positive cells. Another ascidian species, Ciona savignyi, does not have an OPO. Like Hox13 of C. intestinalis, Hox13 of C. savignyi is expressed at the terminus of its sperm duct; however, its expression domain is limited to the circular area around the openings. The genetic changes responsible for the acquisition or loss of OPO are likely to occur in the expression pattern of Hox13.

Shared evolutionary origin of vertebrate neural crest and cranial placodes.

Placodes and neural crests represent defining features of vertebrates, yet their relationship remains unclear despite extensive investigation1-3. 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 crest3,4.

The peripheral nervous system of the ascidian tadpole larva: Types of neurons and their synaptic networks.

Physical and chemical cues from the environment are used to direct animal behavior through a complex network of connections originating in exteroceptors. In chordates, mechanosensory and chemosensory neurons of the peripheral nervous system (PNS) must signal to the motor circuits of the central nervous system (CNS) through a series of pathways that integrate and regulate the output to motor neurons (MN); ultimately these drive contraction of the tail and limb muscles. We used serial-section electron microscopy to reconstruct PNS neurons and their hitherto unknown synaptic networks in the tadpole larva of a sibling chordate, the ascidian, Ciona intestinalis. The larva has groups of neurons in its apical papillae, epidermal neurons in the rostral and apical trunk, caudal neurons in the dorsal and ventral epidermis, and a single tail tip neuron. The connectome reveals that the PNS input arises from scattered groups of these epidermal neurons, 54 in total, and has three main centers of integration in the CNS: in the anterior brain vesicle (which additionally receives input from photoreceptors of the ocellus), the motor ganglion (which contains five pairs of MN), and the tail, all of which in turn are themselves interconnected through important functional relay neurons. Some neurons have long collaterals that form autapses. Our study reveals interconnections with other sensory systems, and the exact inputs to the motor system required to regulate contractions in the tail that underlie larval swimming, or to the CNS to regulate substrate preference prior to the induction of larval settlement and metamorphosis.