Yasunori Sasakura

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on‐target site. CRISPR/Cas9‐mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

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.

Maternal mRNAs play crucial roles during early embryogenesis of ascidians, but their functions are largely unknown. In this study, we developed a new method to specifically knockdown maternal mRNAs in Ciona intestinalis using transposon-mediated transgenesis. We found that GFP expression is epigenetically silenced in Ciona intestinalis oocytes and eggs, and this epigenetic silencing of GFP was used to develop the knockdown method. When the 5[prime] upstream promoter and 5[prime] untranslated region (UTR) of a maternal gene are used to drive GFP in eggs, the maternal gene is specifically knocked down together with GFP. The 5[prime] UTR of the maternal gene is the major element that determines the target gene silencing. Zygotic transcription of the target gene is unaffected, suggesting that the observed phenotypes specifically reflect the maternal function of the gene. This new method can provide breakthroughs in studying the functions of maternal mRNAs.

Summary: 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. genesis 52:431–439, 2014.

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.

The discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified. Here we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites. We conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body.

Background: Transposon-mediated transgenesis is an excellent method for creating stable transgenic lines and insertional mutants. In the chordate Ciona intestinalis, Minos is the only transposon that has been used as the tool for germline transformation. Adding another transposon system in this organism enables us to conduct genetic techniques which can only be realized with the use of two transposons. Results: In the present study, we found that another Tc1/mariner superfamily transposon, sleeping beauty (SB), retains sufficient activity for germline transformation of C. intestinalis. SB shows efficiencies of germline transformation, insertion into gene coding regions, and enhancer detection comparable to those of Minos. We have developed a system for the remobilization of SB copies in the C. intestinalis genome by using transgenic lines expressing SB transposase in the germ cells. With this system, we examined the manner of SB mobilization in the C. intestinalis genome. SB shows intrachromosomal transposition more frequently than Minos. Conclusions: SB-based germline transformation and the establishment of a new method that uses its frequent intrachromosomal transposition will result in breakthroughs in genetic approaches that use C. intestinalis together with Minos. Developmental Dynamics 242:30–43, 2013.

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.