DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction.

Fertilization constitutes a critical step in the plant life cycle during which the gamete genomes undergo chromatin dynamics in preparation for embryogenesis. In mammals, parental chromatin is extensively reprogrammed through the global erasure of DNA methylation. However, in flowering plants it remains unclear whether and how DNA methylation is remodeled in gametes and after fertilization in the zygote. In this study, we characterize DNA methylation patterns and investigate the function of DNA glycosylases in rice eggs, sperm, and unicellular zygotes and during embryogenesis. We found that DNA methylation is locally reconfigured after fertilization and is intensified during embryogenesis. Genetic, epigenomic, and transcriptomic analysis revealed that three rice DNA glycosylases, DNG702, DNG701, and DNG704, demethylate DNA at distinct genomic regions in the gametes and the zygote, and are required for zygotic gene expression and development. Collectively, these results indicate that active DNA demethylation takes place in the gametes and the zygote to locally remodel DNA methylation, which is critical for egg and zygote gene expression and reproduction in rice.

Acetyl-CoA synthases are essential for maintaining histone acetylation under metabolic stress during zygotic genome activation in pigs.

ACSS1/2 converts acetate into acetyl-coenzyme A, which contributes to histone acetylation in the mitochondria and cytoplasm. Zygotic genome activation (ZGA) is critical for embryo development involving drastic histone modification. An efficient crRNAs-Cas13a targeting strategy was employed to investigate the ACSS1/2 function during ZGA. The results showed that nuclear accumulation of ACSS1 and ACSS2 occurs during ZGA. Knockdown of ACSS1/2 did not affect blastocyst formation when using a normal medium. On culturing embryos in a medium with acetate and no pyruvate (-P + Ace), knockdown of ACSS1 did not affect histone acetylation levels but significantly reduced ATP levels, whereas knockdown of ACSS2 significantly reduced histone acetylation levels in porcine embryos. Inhibition of fatty acid beta-oxidation by etomoxir significantly reduced ATP levels, which could be restored by acetate. The histone acetylation levels in the ACSS1 and ACSS2 knockdown groups both decreased considerably after etomoxir treatment. Moreover, acetate showed dose-dependent effects on SIRT1 and SIRT3 levels when under metabolic stress. The C-terminus of ACSS1 regulated the nuclear translocation. In conclusion, ACSS1/2 helps to maintain ATP and histone acetylation levels in porcine early embryos under metabolic stress during ZGA.

Histone demethylase KDM4A overexpression improved the efficiency of corrected human tripronuclear zygote development.

Human zygotes are difficult to obtain for research because of limited resources and ethical debates. Corrected human tripronuclear (ch3PN) zygotes obtained by removal of the extra pronucleus from abnormally fertilized tripronuclear (3PN) zygotes are considered an alternative resource for basic scientific research. In the present study, eight-cell and blastocyst formation efficiency were significantly lower in both 3PN and ch3PN embryos than in normal fertilized (2PN) embryos, while histone H3 lysine 9 trimethylation (H3K9me3) levels were much higher. It was speculated that the aberrant H3K9me3 level detected in ch3PN embryos may be related to low developmental competence. Microinjection of 1000 ng/µl lysine-specific demethylase 4A (KDM4A) mRNA effectively reduced the H3K9me3 level and significantly increased the developmental competence of ch3PN embryos. The quality of ch3PN zygotes improved as the grading criteria, cell number and pluripotent expression significantly increased in response to KDM4A mRNA injection. Developmental genes related to zygotic genome activation (ZGA) were also upregulated. These results indicate that KDM4A activates the transcription of the ZGA program by enhancing the expression of related genes, promoting epigenetic modifications and regulating the developmental potential of ch3PN embryos. The present study will facilitate future studies of ch3PN embryos and could provide additional options for infertile couples.

Knockdown of CYP301B1 and CYP6AX1v2 increases the susceptibility of the brown planthopper to beta-asarone, a potential plant-derived insecticide.

The cytochrome P450 monooxygenases of insects play crucial roles in the metabolic detoxification of insecticides. Our previous finding showed that two cytochrome P450 genes, both CYP301B1 and CYP6AX1v2, in the BPH underwent overexpression due to ß-asarone. In this study, we investigated the molecular characteristics, expression patterns and functions of these two cytochrome P450 genes. The results showed that CYP301B1 had the highest expression level in the eggs, while CYP6AX1v2 was expressed in macropterous female adults. Moreover, the expression level of CYP301B1 in the head was higher than that in the integument, fat body and gut. The expression level of CYP6AX1v2 in the fat body and gut was higher than that in head and integument. Importantly, silencing CYP301B1 and CYP6AX1v2 separately could increase the sensitivity, resulting in significant higher mortality of BPH following treatment with ß-asarone. Our findings indicated that CYP301B1 and CYP6AX1v2 could contribute to the resistance of BPH to ß-asarone, and these two genes may be involved in the detoxification metabolism of ß-asarone in BPH.

The landscape of RNA Pol II binding reveals a stepwise transition during ZGA.

Zygotic genome activation (ZGA) is the first transcription event in life1. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.

Nuclear accumulation of pyruvate dehydrogenase alpha 1 promotes histone acetylation and is essential for zygotic genome activation in porcine embryos.

Porcine zygotic genome activation (ZGA) occurs along with global epigenetic remodeling at the 4-cell stage. These processes are regulated by histone acetylation, which requires acetyl-coenzyme A (CoA). Pyruvate dehydrogenase complex (PDC) is a crucial enzyme in glucose metabolism that converts pyruvate into acetyl-CoA. In mammalian cells, acetyl-CoA is produced by pyruvate dehydrogenase alpha 1 (PDHA1) translocated into the nucleus in special conditions. To determine whether zygotic PDHA1 plays a critical role in promoting histone acetylation during ZGA, a CRISPR/Cas9 genome editing system using multiple guide RNAs was employed to generate a PDHA1-targeted parthenogenetic embryo model. Results of immunofluorescent staining showed that the nuclear accumulation of PDHA1 during ZGA was significantly inhibited by PDHA1 targeting. Meanwhile, the 4-cell arrest rate significantly increased at 72 h after activation, indicating impeded embryonic development. In addition, nuclear histone acetylation significantly decreased when PDHA1 was targeted, and quantitative PCR showed that expression of several zygotic genes was significantly decreased in the PDHA1-targeting group compared to the control group. Overexpression of PDHA1 recovered the nuclear PDHA1, H3K9Ac and H3K27Ac and EIF1A expression levels. Moreover, the 5-to-8-cell-stage embryo development rate was only partially rescued. In conclusion, expression of zygotic origin PDHA1 contributes to porcine ZGA by maintaining histone acetylation in porcine embryos.

Possible Mechanisms of Hatching from Egg Capsules in the Gastropods Crepipatella dilatata and Crepipatella peruviana, Species with Different Modes of Early Development.

Many invertebrates enclose their embryos within egg capsules, from which the offspring hatch. In marine gastropods that brood their egg capsules, hatching could involve radular activity by the mother or by unhatched stages, increased osmotic concentration of the intracapsular fluid, or production of hatching enzymes. The present research sought to determine whether mechanical action by the brooding female or by the encapsulated embryos was involved in the hatching for two sympatric and closely related species of calyptraeid: Crepipatella dilatata, which exhibits direct development without free-living larvae, and Crepipatella peruviana, which releases free-living veliger larvae. We also considered the role that enzymatic action or osmotic changes in the intracapsular fluid might play in hatching. Using scanning electron micrograph analyses, we found no evidence that the well-developed, pre-hatching juvenile radula of C. dilatata played any role in the hatching process and that the radula of C. peruviana did not even develop until long after hatching; so there was no evidence of radular activity involved in the hatching of either species. For C. peruviana, the intracapsular fluid osmolality was always higher than that of the surrounding seawater, suggesting that there is a strong natural water inflow during development. Moreover, when egg capsules of C. peruviana were exposed to lower ambient salinities, the substantial entry of water correlated well with high percentages of hatching, particularly for egg capsules containing advanced veligers, suggesting that an osmotic mechanism may be involved in the hatching process of this species. In contrast, hatching in C. dilatata appeared to be enzymatically mediated.

Peroxiredoxin as a functional endogenous antioxidant enzyme in pronuclei of mouse zygotes.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

Two Fungicides Alter Reproduction of the Small Brown Planthopper Laodelphax striatellus by Influencing Gene and Protein Expression.

Aside from their intended actions, fungicides can drive pest insect outbreaks due to virtually continuous use and pest evolution. Small brown planthopper (SBPH), Laodelphax striatellus, outbreaks occurred recently in many provinces in China, with devastating rice losses. Because exposure to the fungicide jinggangmycin (JGM) increased reproduction of the brown plant hopper, Nilaparvata lugens, via its influence on fatty acid synthase, we posed the hypothesis that JGM and carbendazim (CBM) influence SBPH reproduction via their influence on enzymes involved in other aspects of lipid metabolism. Exposure to the fungicide CBM stimulated SBPH reproduction (egg-laying up by 78%) and to another fungicide, JGM, led to decreased egg-laying (down by 47.3%). These inverse effects are mediated by down-regulated expression of l-3-hydroxyacyl-coenzyme A dehydrogenase (LCHAD) in JGM-treated females and up-regulated expression of hydroxysteroid dehydrogenase-like protein 2-like (HSD) in CBM-treated females. RNAi knockdown of, separately, LCHAD and HSD led to reduced egg-laying (down by 52% for dsLCHAD and by 73% for dsHSD). dsLCHAD, dsHSD, and JGM treatments also led to severely reduced ovarian development in experimental SBPH, with shorted and thinned valvula and lack of egg cells in ovaries. Valvula of CBM-treated females enlarged, with banana-shaped eggs in ovaries. These data strongly support our hypothesis.

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation.

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh-/- oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH.

Transcriptional integration of paternal and maternal factors in the Arabidopsis zygote.

In many plants, the asymmetric division of the zygote sets up the apical-basal axis of the embryo. Unlike animals, plant zygotes are transcriptionally active, implying that plants have evolved specific mechanisms to control transcriptional activation of patterning genes in the zygote. In Arabidopsis, two pathways have been found to regulate zygote asymmetry: YODA (YDA) mitogen-activated protein kinase (MAPK) signaling, which is potentiated by sperm-delivered mRNA of the SHORT SUSPENSOR (SSP) membrane protein, and up-regulation of the patterning gene WOX8 by the WRKY2 transcription factor. How SSP/YDA signaling is transduced into the nucleus and how these pathways are integrated have remained elusive. Here we show that paternal SSP/YDA signaling directly phosphorylates WRKY2, which in turn leads to the up-regulation of WOX8 transcription in the zygote. We further discovered the transcription factors HOMEODOMAIN GLABROUS11/12 (HDG11/12) as maternal regulators of zygote asymmetry that also directly regulate WOX8 transcription. Our results reveal a framework of how maternal and paternal factors are integrated in the zygote to regulate embryo patterning.

Inhibition of casein kinase 2 blocks G2/M transition in early embryo mitosis but not in oocyte meiosis in mouse.

Casein kinase 2 (CK2) is a highly conserved, ubiquitously expressed serine/threonine protein kinase with hundreds of substrates. The role of CK2 in the G2/M transition of oocytes, zygotes, and 2-cell embryos was studied in mouse by enzyme activity inhibition using the specific inhibitor 4, 5, 6, 7-tetrabromobenzotriazole (TBB). Zygotes and 2-cell embryos were arrested at G2 phase by TBB treatment, and DNA damage was increased in the female pronucleus of arrested zygotes. Further developmental ability of arrested zygotes was reduced, but that of arrested 2-cell embryos was not affected after releasing from inhibition. By contrast, the G2/M transition in oocytes was not affected by TBB. These results indicate that CK2 activity is essential for mitotic G2/M transition in early embryos but not for meiotic G2/M transition in oocytes.

MutSα maintains the mismatch repair capability by inhibiting PCNA unloading.

Eukaryotic mismatch repair (MMR) utilizes single-strand breaks as signals to target the strand to be repaired. DNA-bound PCNA is also presumed to direct MMR. The MMR capability must be limited to a post-replicative temporal window during which the signals are available. However, both identity of the signal(s) involved in the retention of this temporal window and the mechanism that maintains the MMR capability after DNA synthesis remain unclear. Using Xenopus egg extracts, we discovered a mechanism that ensures long-term retention of the MMR capability. We show that DNA-bound PCNA induces strand-specific MMR in the absence of strand discontinuities. Strikingly, MutSα inhibited PCNA unloading through its PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutSα in the retention of the post-replicative MMR capability.

Oxidative stress-induced DNA damage of mouse zygotes triggers G2/M checkpoint and phosphorylates Cdc25 and Cdc2.

In vitro fertilized (IVF) embryos show both cell cycle and developmental arrest. We previously showed oxidative damage activates the ATM → Chk1 → Cdc25B/Cdc25C cascade to mediate G2/M cell cycle arrest for repair of hydrogen peroxide (H2O2)-induced oxidative damage in sperm. However, the mechanisms underlying the developmental delay of zygotes are unknown. To develop a model of oxidative-damaged zygotes, we treated mouse zygotes with different concentrations of H2O2 (0, 0.01, 0.02, 0.03, 0.04, 0.05 mM), and evaluated in vitro zygote development, BrdU incorporation to detect the duration of S phase. We also examined reactive oxygen species level and used immunofluorescence to detect activation of γH2AX, Cdc2, and Cdc25. Oxidatively damaged zygotes showed a delay in G2/M phase and produced a higher level of ROS. At the same time, γH2AX was detected in oxidatively damaged zygotes as well as phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15). Our study indicates that oxidative stress-induced DNA damage of mouse zygotes triggers the cell cycle checkpoint, which results in G2/M cell cycle arrest, and that phospho-Cdc25B (Ser323), phospho-Cdc25C (Ser216), and phospho-Cdc2 (Tyr15) participate in activating the G2/M checkpoint.

Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation.

Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.

Maternally provided LSD1/KDM1A enables the maternal-to-zygotic transition and prevents defects that manifest postnatally.

Somatic cell nuclear transfer has established that the oocyte contains maternal factors with epigenetic reprogramming capacity. Yet the identity and function of these maternal factors during the gamete to embryo transition remains poorly understood. In C. elegans, LSD1/KDM1A enables this transition by removing H3K4me2 and preventing the transgenerational inheritance of transcription patterns. Here we show that loss of maternal LSD1/KDM1A in mice results in embryonic arrest at the 1-2 cell stage, with arrested embryos failing to undergo the maternal-to-zygotic transition. This suggests that LSD1/KDM1A maternal reprogramming is conserved. Moreover, partial loss of maternal LSD1/KDM1A results in striking phenotypes weeks after fertilization; including perinatal lethality and abnormal behavior in surviving adults. These maternal effect hypomorphic phenotypes are associated with alterations in DNA methylation and expression at imprinted genes. These results establish a novel mammalian paradigm where defects in early epigenetic reprogramming can lead to defects that manifest later in development.

Maternal TET3 is dispensable for embryonic development but is required for neonatal growth.

The development of multicellular organisms is accompanied by reprogramming of the epigenome in specific cells, with the epigenome of most cell types becoming fixed after differentiation. Genome-wide reprogramming of DNA methylation occurs in primordial germ cells and in fertilized eggs during mammalian embryogenesis. The 5-methylcytosine (5mC) content of DNA thus undergoes a marked decrease in the paternal pronucleus of mammalian zygotes. This loss of DNA methylation has been thought to be mediated by an active demethylation mechanism independent of replication and to be required for development. TET3-mediated sequential oxidation of 5mC has recently been shown to contribute to the genome-wide loss of 5mC in the paternal pronucleus of mouse zygotes. We now show that TET3 localizes not only to the paternal pronucleus but also to the maternal pronucleus and oxidizes both paternal and maternal DNA in mouse zygotes, although these phenomena are less pronounced in the female pronucleus. Genetic ablation of TET3 in oocytes had no significant effect on oocyte development, maturation, or fertilization or on pregnancy, but it resulted in neonatal sublethality. Our results thus indicate that zygotic 5mC oxidation mediated by maternal TET3 is required for neonatal growth but is not essential for development.

Maternal and Zygotic Sphingosine Kinase 2 Are Indispensable for Cardiac Development in Zebrafish.

Sphingosine 1-phosphate (S1P) is synthesized from sphingosine by sphingosine kinases (SPHK1 and SPHK2) in invertebrates and vertebrates, whereas specific receptors for S1P (S1PRs) selectively appear in vertebrates, suggesting that S1P acquires novel functions in vertebrates. Because the developmental functions of SPHK1 and SPHK2 remain obscure in vertebrates, we generated sphk1 or sphk2 gene-disrupted zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases. Both zygotic sphk1 and sphk2 zebrafish mutants exhibited no obvious developmental defects and grew to adults. The maternal-zygotic sphk2 mutant (MZsphk2), but not the maternal-zygotic sphk1 mutant and maternal sphk2 mutant, had a defect in the cardiac progenitor migration and a concomitant decrease in S1P level, leading to a two-heart phenotype (cardia bifida). Cardia bifida in MZsphk2, which was rescued by injecting sphk2 mRNA, was a phenotype identical to that of zygotic mutants of the S1P transporter spns2 and S1P receptor s1pr2, indicating that the Sphk2-Spns2-S1pr2 axis regulates the cardiac progenitor migration in zebrafish. The contribution of maternally supplied lipid mediators during vertebrate organogenesis presents as a requirement for maternal-zygotic Sphk2.

Generation of Foxo3-targeted Mice by Injection of mRNAs Encoding Transcription Activator-like Effector Nucleases (TALENs) into Zygotes.

In this study, for exploring the mechanism of forkhead box O3(Foxo3) participating in regulation of the activation of primordial oocytes, Foxo3-targeted mice were generated by injection of mRNAs encoding transcription activator-like effector nucleases (TALENs) into mouse zygotes. The TALEN sites were designed with high conservative homologous region among pig, bovine, buffalo and mouse by commercial bio-companies. The TALENs mutagenic non-homologous end-joining (NHEJ) repair activity were determined to be 31.3% in human embryonic kidney 293T (HEK-293T) cells by dual luciferase reporter assay system. Then, we firstly injected TALEN-mRNAs into the cytoplasm of mouse zygotes by micromanipulation, and four of 48 mouse blastocysts were identified as mutation by sequencing. Subsequently, by the method of TALEN-mRNAs injected into the zygotes with pronucleus micromanipulation technique, we obtained seven Foxo3 mutants of 20 FVB/NJ backgrounds mice which were Foxo3-independent alleles with frameshift and deletion mutations. It was very interesting that all seven were heterozygous mutants (Foxo3(-/+) ), and the gene mutagenesis rates of the mice reached 35%. The five Foxo3 mutant females were all infertile in the following 6 months after birth. The histological examination results showed that there were rare primordial follicles and primary follicles in the ovary of Foxo3 mutant compared to that of wide-type female mice. Moreover, one of two mutant males was subfertile and another was fertile normally. Those results suggested that the mutant of Foxo3 severely affected the fertile ability of female and perhaps male in some degree; furthermore, an even more efficient TALENs-based gene mutation method has been established to be poised to revolutionize the study of mouse and other species genetics.

The expression of an immune-related phenoloxidase gene is modulated in Ciona intestinalis ovary, test cells, embryos and larva.

Two distinct Ciona intestinalis phenoloxidases (CinPO1, 2) had previously been cloned and sequenced. The CinPO2 is involved in innate immunity and is expressed by inflammatory hemocytes that populate the tunic and pharynx vessels as a response to LPS inoculation. In situ hybridization and immunohistochemistry assays on histological section, showed that the expression of this gene and the produced protein are shared with oogenesis, embryogenesis and larval morphogenesis. Intriguingly, upregulation of gene transcription was found in the test cell layer that envelopes the ovary follicle, ovulated egg, and gastrula, as well as it was modulated in the zygotic nucleus of outer balstomers of 32-cell embryo, neurula presumptive epidermis tissue and larval mesenchyme. The anti-CinPO2 antibodies, specific for adult inflammatory cells, recognize epitopes in the cytoplasm of ovarian oocytes, ovulated eggs, development stages and larval mesenchyme. The overall findings disclose the precocious activation of the CinPO2 immunity-related gene, and show a developmentally programmed expression of this phenoloxidase. Furthermore, these findings support the multifunctional roles of immunity-related genes and allows us to explore new perspectives on ascidian development and immunity.