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The control of cell numbers and the establishment of cell types are two processes that are essential in early embryonic development. We have a reasonable understanding of how these processes occur individually, but we have considerably less sophisticated understanding of how these processes are linked. Tunicates have fixed cell lineages with predictable cell cycles, making them well suited to investigate these processes. In the ascidian Ciona , we show that the transcription factor Zic-r.b, known to be involved in establishing several cell types in early development also activates the expression of the cell cycle inhibitor CDKN1B. Zic-r.b is a major missing component of the cell division clock establishing specific cell numbers. We also show that a larvacean homolog of Zic-r.b is expressed one cell cycle earlier than its Ciona counterpart. The early expression in larvaceans may explain why they have half as many notochord cells as ascidians and may illustrate a general mechanism to evolve changes in morphology. [Display omitted] • Ciona embryo notochord/muscle tissues have precise cell numbers. • Zic-r.b has a key role blocking cell division by upregulating CDKN1B. • Oikopleura embryos have fewer cells and express these genes earlier. [ABSTRACT FROM AUTHOR]

Phase separation underlies the organization of the nucleus, including the biogenesis of nucleoli and the packaging of heterochromatin. Here we explore the regulation of transcription factor condensates involved in gene repression by ERK signaling in gastrulating embryos of a simple proto-vertebrate (Ciona). ERK signaling induces nuclear export of the transcriptional repressor Ets-2 repressive factor (ERF), which has been linked to various human developmental disorders. Using high-resolution imaging, we show that ERF is localized within discrete nuclear condensates that dissolve upon ERK activation. Interestingly, we observe dynamic pulses of assembly and dissociation during interphase, providing visualization of a nuclear phase separation process regulated by cell signaling. We discuss the implications of these observations for producing sharp on/off switches in gene activity and suppressing noise in cell–cell signaling events. [ABSTRACT FROM AUTHOR]

Vertebrate-like sex steroid hormones have been widely detected in mollusks, and numerous experiments have shown the importance of steroids in gonad development. Nevertheless, their signaling pathways in invertebrates have not been uncovered yet. Steroid receptors are an ancient class of transcription factors with multiple roles in not only vertebrates but also invertebrates. Estrogen signaling is thought to have major roles in mollusk physiology, but the full repertoire of estrogen receptors is unknown. We presented the successful cloning of two novel forms of estrogen receptor-like genes. These receptors are present in two closely related species of Mytilus : Mytilus edulis and Mytilus galloprovincialis , commonly known and widely distributed sentinel species. Our phylogenetic analysis revealed that one of these receptors is an estrogen receptor (ER) and the other one is an estrogen-related receptor (ERR). Studies of expression analysis showed that both receptor mRNAs were localized in the oocytes and follicle cells in contact with developing oocytes in the ovary and Sertoli cells in the testis, and in the ciliated cells of the gill. In addition, we have evidence that one (ER) of these may have a capacity to autoregulate its own expression in the gonadal cells by estrogen (E 2 ) and that this gene is responsive to estrogenic compounds. [ABSTRACT FROM AUTHOR]

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis. [ABSTRACT FROM AUTHOR]

Abstract: Gonadotropin-releasing hormone (GnRH) is a neuropeptide that has an essential role in the neural regulation of vertebrate reproduction. Over the past two decades there has been increasing evidence strongly indicating that members of the GnRH superfamily, which includes GnRH, adipokinetic hormone (AKH), corazonin (Crz) and adipokinetic hormone/corazonin-related peptides (ACP), are almost ubiquitous amongst bilateral animals. Moreover GnRH possibly has origins in even more ancient, non-bilateral ancestors. Current knowledge about molluscan GnRH has been accumulated regarding immunological identification, physiological function and sequence analysis. In the present review we summarized a current status of molluscan GnRH research and focus on its role in the reproduction of the molluscs. In cephalopods and gastropods the presence of a GnRH-like peptide was detected with heterologous antibodies and the identified GnRH was suggested to be involved with behavior and reproduction. Reproductive roles for GnRH have been confirmed in both bivalve and cephalopod molluscs. These findings will provide useful insights into the evolution of reproductive endocrinology. [Copyright &y& Elsevier]

Abstract: Gonadotropin-releasing hormone (GnRH) is central to the control of vertebrate reproductive cycles and since GnRH orthologs are also present in invertebrates, it is likely that the common ancestor of bilateral animals possessed a GnRH-like peptide. In order to understand the evolutionary and comparative biology of GnRH peptides we cloned the cDNA transcripts of prepro GnRH-like peptides from two species of bivalve molluscs, the Yesso scallop Patinopecten yessoensis and the Pacific oyster Crassostrea gigas. We compared their deduced uncleaved and mature amino acid sequences with those from other invertebrates and vertebrates, and determined their sites of expression and biological activity. The two molluscan GnRH sequences increased the number of known protostome GnRHs to six different forms, indicating the current classification of protostome GnRHs requires further revision. In both molluscs, RT-PCR analysis showed that the genes were highly expressed in nervous tissue with lower levels present in peripheral tissues including the gonads, while immunocytochemistry, using anti-octopus GnRH-like peptide, demonstrated the presence of GnRH-like peptide in neural tissue. Putative scallop GnRH-like peptide stimulated spermatogonial cell division in cultured scallop testis, but the scallop GnRH-like peptide did not stimulate LH release from cultured quail pituitary cells. This is the first report of the cloning of bivalve GnRH-like peptide genes and of molluscan GnRH-like peptides that are biologically active in molluscs, but not in a vertebrate. [Copyright &y& Elsevier]

Summary Most animal embryos display a delay in the activation of zygotic transcription during early embryogenesis [ 1 ]. This process is thought to help coordinate rapid increases in cell number during early development [ 2 ]. The timing of zygotic genome activation (ZGA) during the maternal-to-zygotic transition (MZT) remains uncertain despite extensive efforts. We explore ZGA in the simple protovertebrate, Ciona intestinalis . Single-cell RNA sequencing (RNA-seq) assays identified Cyclin B3 ( Ccnb3 ) as a putative mediator of ZGA. Maternal Ccnb3 transcripts rapidly diminish in abundance during the onset of zygotic transcription at the 8-cell and 16-cell stages. Disruption of Ccnb3 activity results in precocious activation of zygotic transcription, while overexpression abolishes normal activation. These observations suggest that the depletion of maternal Cyclin B3 products is a critical component of the MZT and ZGA. We discuss evidence that this mechanism might play a conserved role in the MZT of other metazoans, including mice and humans. [ABSTRACT FROM AUTHOR]

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona . This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona . [ABSTRACT FROM AUTHOR]

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. • Ciona intestinalis Type A has the orange-pigmented organ at the sperm duct, which is a species-specific characteristic. • Hox13 is necessary for the formation of the orange-pigmented organ at the sperm duct terminus. • Hox13 is expressed in the neurons and epithelium at the sperm duct terminus. • Hox13 is likely to regulate the migration of orange pigmented cells toward the sperm duct terminus. • C. savignyi Hox13 is expressed at the sperm duct terminus; however, the terminus of this ascidian is not orange-pigmented. [ABSTRACT FROM AUTHOR]

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. • The neurotransmitter GABA is a key regulator of Ciona metamorphosis • Gonadotropin-releasing hormone (GnRH) is the downstream neuropeptide of GABA • GABA positively regulates secretion of GnRH through the metabotropic GABA receptor Metamorphosis is a dramatic body change in metazoans that allows the use of different ecological niches. Hozumi et al. show that GABA is a key regulator of metamorphosis in the ascidian Ciona. During this process, GABA, generally thought of as an inhibitory neurotransmitter, positively regulates secretion of the neuropeptide GnRH. [ABSTRACT FROM AUTHOR]

Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timing of gene induction during the earliest phases of embryogenesis. Here we used high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate Ciona with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by ERK-mediated derepression of the ERF repressor. Depletion of ERF results in ectopic activation of FGF target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the eight- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also used by vertebrates., (© 2023 Treen et al.; Published by Cold Spring Harbor Laboratory Press.)

Targeted mutagenesis of genes-of-interest is a powerful method of addressing the functions of genes. Genome editing techniques, such as transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have enabled this approach in various organisms because of their ease of use. In the ascidian, Ciona intestinalis, recent studies show that TALEN-based knockout can be applied to establishing both mutant lines and tissue-specific knockout for addressing gene functions. Here, we introduce recent updates to the TALEN toolkit that facilitate detailed functional analysis of genes in ascidians.

The chordate pharynx, possessing gill slits and the endostyle, is a complex of multiple tissues that are highly organized along the anterior-posterior (AP) axis. Although Hox genes show AP coordinated expression in the pharyngeal endoderm, tissue-specific roles of these factors for establishing the regional identities within this tissue have not been demonstrated. Here, we show that Hox1 is essential for the establishment of AP axial identity of the endostyle, a major structure of the pharyngeal endoderm, in the ascidian Ciona intestinalis We found that knockout of Hox1 causes posterior-to-anterior transformation of the endostyle identity, and that Hox1 represses Otx expression and anterior identity, and vice versa. Furthermore, alteration of the regional identity of the endostyle disrupts the formation of body wall muscles, suggesting that the endodermal axial identity is essential for coordinated pharyngeal development. Our results demonstrate an essential role of Hox genes in establishment of the AP regional identity in the pharyngeal endoderm and reveal crosstalk between endoderm and mesoderm during development of chordate pharynx., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The ascidian Ciona intestinalis is an important model animal for studying developmental mechanisms for constructing the chordate body. Although molecular and embryological techniques for manipulating Ciona genes were developed a long time ago, recent achievements of genome editing in this animal have innovated functional analyses of genes in Ciona. Particularly, knockout of genes in the G0 generation coupled with tissue-specific expression of TALENs enables us to rapidly address gene functions that were difficult using previous methods.

Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis., (© 2014 Wiley Periodicals, Inc.)