Distribution of cionin, a cholecystokinin/gastrin family peptide, and its receptor in the central nervous system of Ciona intestinalis type A.

The cholecystokinin (CCK)/gastrin family peptides are involved in regulation of feeding and digestion in vertebrates. In the ascidian Ciona intestinalis type A (Ciona robusta), cionin, a CCK/gastrin family peptide, has been identified. Cionin is expressed exclusively in the central nervous system (CNS). In contrast, cionin receptor expression has been detected in the CNS, digestive tract, and ovary. Although cionin has been reported to be involved in ovulation, its physiological function in the CNS remains to be investigated. To elucidate its neural function, in the present study, we analyzed the expression of cionin and cionin receptors in the CNS. Cionin was expressed mainly in neurons residing in the anterior region of the cerebral ganglion. In contrast, the gene expressin of the cionin receptor gene CioR1, was detected in the middle part of the cerebral ganglion and showed a similar expression pattern to that of VACHT, a cholinergic neuron marker gene. Moreover, CioR1 was found to be expressed in cholinergic neurons. Consequently, these results suggest that cionin interacts with cholinergic neurons as a neurotransmitter or neuromodulator via CioR1. This study provides insights into a biological role of a CCK/gastrin family peptide in the CNS of ascidians.

Extrinsic apoptosis participates to tail regression during the metamorphosis of the chordate Ciona.

Apoptosis is a regulated cell death ubiquitous in animals defined by morphological features depending on caspases. Two regulation pathways are described, currently named the intrinsic and the extrinsic apoptosis. While intrinsic apoptosis is well studied and considered ancestral among metazoans, extrinsic apoptosis is poorly studied outside mammals. Here, we address extrinsic apoptosis in the urochordates Ciona, belonging to the sister group of vertebrates. During metamorphosis, Ciona larvae undergo a tail regression depending on tissue contraction, migration and apoptosis. Apoptosis begin at the tail tip and propagates towards the trunk as a polarized wave. We identified Ci-caspase 8/10 by phylogenetic analysis as homolog to vertebrate caspases 8 and 10 that are the specific initiator of extrinsic apoptosis. We detected Ci-caspase 8/10 expression in Ciona larvae, especially at the tail tip. We showed that chemical inhibition of Ci-caspase 8/10 leads to a delay of tail regression, and Ci-caspase 8/10 loss of function induced an incomplete tail regression. The specificity between apoptotic pathways and initiator caspase suggests that extrinsic apoptosis regulates cell death during the tail regression. Our study presents rare in vivo work on extrinsic apoptosis outside mammals, and contribute to the discussion on its evolutionary history in animals.

The neuroendocrine system of Ciona intestinalis Type A, a deuterostome invertebrate and the closest relative of vertebrates.

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuropeptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona. In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona, and their evolutionary aspects.

CatSper mediates not only chemotactic behavior but also the motility of ascidian sperm.

Introduction: Sperm motility, including chemotactic behavior, is regulated by changes in the intracellular Ca2+ concentration, and the sperm-specific Ca2+ channel CatSper has been shown to play an important role in the regulation of intracellular Ca2+. In particular, in mammals, CatSper is the only functional Ca2+ channel in the sperm, and mice deficient in the genes comprising the pore region of the Ca2+ channel are infertile due to the inhibition of sperm hyperactivation. CatSper is also thought to be involved in sea urchin chemotaxis. In contrast, in ascidian Ciona intestinalis, SAAF, a sperm attractant, interacts with Ca2+/ATPase, a Ca2+ pump. Although the existence of CatSper genes has been reported, it is not clear whether CatSper is a functional Ca2+ channel in sperm. Results: We showed that CatSper is present in the sperm flagella of C. intestinalis as in mammalian species, although a small level of gene expression was found in other tissues. The spermatozoa of CatSper3 KO animals were significantly less motile, and some motile sperms did not show any chemotactic behavior. These results suggest that CatSper plays an important role in ascidians and mammals, and is involved in spermatogenesis and basic motility mechanisms.

Characterization of a novel species-specific 51-amino acid peptide, PEP51, as a caspase-3/7 activator in ovarian follicles of the ascidian, Ciona intestinalis Type A.

Invertebrates lack hypothalamic-pituitary-gonadal axis, and have acquired species-specific regulatory systems for ovarian follicle development. Ascidians are marine invertebrates that are the phylogenetically closest living relatives to vertebrates, and we have thus far substantiated the molecular mechanisms underlying neuropeptidergic follicle development of the cosmopolitan species, Ciona intestinalis Type A. However, no ovarian factor has so far been identified in Ciona. In the present study, we identified a novel Ciona-specific peptide, termed PEP51, in the ovary. Immunohistochemical analysis demonstrated the specific expression of PEP51 in oocyte-associated accessory cells, test cells, of post-vitellogenic (stage III) follicles. Immunoelectron microscopy revealed that PEP51 was localized in the cytosol of test cells in early stage III follicles, which lack secretory granules. These results indicate that PEP51 acts as an intracellular factor within test cells rather than as a secretory peptide. Confocal laser microscopy verified that activation of caspase-3/7, the canonical apoptosis marker, was detected in most PEP51-positive test cells of early stage III. This colocalization of PEP51 and the apoptosis marker was consistent with immunoelectron microscopy observations demonstrating that a few normal (PEP51-negative) test cells reside in the aggregates of PEP51-positive apoptotic test cells of early stage III follicles. Furthermore, transfection of the PEP51 gene into COS-7 cells and HEK293MSR cells resulted in activation of caspase-3/7, providing evidence that PEP51 induces apoptotic signaling. Collectively, these results showed the existence of species-specific ovarian peptide-driven cell metabolism in Ciona follicle development. Consistent with the phylogenetic position of Ciona as the closest sister group of vertebrates, the present study sheds new light on the molecular and functional diversity of the regulatory systems of follicle development in the Chordata.

Multi-regional expression of pancreas-related digestive enzyme genes in the intestinal chamber of the ascidian Ciona intestinalis type A.

Bilateria share sequential steps in their digestive systems, and digestion occurs in a pre-absorption step within a chamber-like structure. Previous studies on the ascidian Ciona intestinalis type A, an evolutionary research model of vertebrate organs, revealed that Ciona homologs of pancreas-related exocrine digestive enzymes (XDEs) are exclusively expressed in the chamber-like bulging stomach. In the development of the gastrointestinal tract, genes for the pancreas-related transcription factors, namely Ptf1a, Nr5a2, and Pdx, are expressed near the stomach. Recent organ/tissue RNA-seq studies on two Ciona species reported that transcripts of the XDE homologs exist in the intestinal regions, as well as in the stomach. In the present study, we investigated the spatial gene expression of XDE homologs in the gastrointestinal region of the C. intestinalis type A. Whole-mount in situ hybridization using adult and juvenile specimens revealed apparent expression signals of XDE homologs in a small number of gastrointestinal epithelial cells. Furthermore, two pancreas-related transcription factor genes, Nr5a2 and Pdx, exhibited multi-regional expression along the Ciona juvenile intestines. These results imply that ascidians may form multiple digestive regions corresponding to the vertebrate pancreas.

Repetitive and zonal expression profiles of absorption-related genes in the gastrointestinal tract of ascidian Ciona intestinalis type A.

Intestinal absorption is essential for heterotrophic bilaterians with a tubular gut. Although the fundamental features of the digestive system were shared among chordates with evolution, the gut morphologies of vertebrates diverged and adapted to different food habitats. The ascidian Ciona intestinalis type A, a genome-wide research model of basal chordates, is used to examine the functional morphology of the intestines because of its transparent juvenile body. In the present study, the characteristic gene expression patterns (GEP) of Ciona absorptive proteins, e.g., brush border membrane enzymes for terminal digestion (lactase, maltase, APA, and APN) and transporters (SGLT1, GLUT5, PEPT1, and B0AT1), were investigated in juveniles and young adults, with a special reference to the absorption of other nutrients by pinocytosis- and phagocytosis-related proteins (megalin, cubilin, amnionless, Dab2, Rab7, LAMP, cathepsins, and MRC1). Whole-mount in situ hybridization revealed that these GEP showed multi-regional and repetitive features along the Ciona gastrointestinal tract, mainly in the stomach and several regions of the intestines. In young adults, many absorption-related genes, including pinocytosis-/phagocytosis-related genes, were also expressed between the stomach and mid-intestine. In the gastrointestinal epithelium, absorption-related genes showed zonal GEP along the epithelial structure. Comparisons of GEP, including other intestinal functions, such as nutrient digestion and intestinal protection, indicated the repetitive assignment of a well-coordinated set of intestinal GEP in the Ciona gastrointestinal tract.

Detection of TALEN-mediated genome cleavage during the early embryonic stage of the starfish Patiria pectinifera.

BACKGROUND: Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish. RESULTS: We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization. CONCLUSION: The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results.

Distribution changes of non-self-test cells and self-tunic cells surrounding the outer body during Ciona metamorphosis.

BACKGROUND: Ascidians significantly change their body structure through metamorphosis, but the spatio-temporal cell dynamics in the early metamorphosis stage has not been clarified. A natural Ciona embryo is surrounded by maternally derived non-self-test cells before metamorphosis. However, after metamorphosis, the juvenile is surrounded by self-tunic cells derived from mesenchymal cell lineages. Both test cells and tunic cells are thought to be changed their distributions during metamorphosis, but the precise timing is unknown. RESULTS: Using a metamorphosis induction by mechanical stimulation, we investigated the dynamics of mesenchymal cells during metamorphosis in a precise time course. After the stimulation, two-round Ca2+ transients were observed. Migrating mesenchymal cells came out through the epidermis within 10 min after the second phase. We named this event "cell extravasation." The cell extravasation occurred at the same time as the backward movement of posterior trunk epidermal cells. Timelapse imaging of transgenic-line larva revealed that non-self-test cells and self-tunic cells temporarily coexist outside the body until the test cells are eliminated. At the juvenile stage, only extravasated self-tunic cells remained outside the body. CONCLUSIONS: We found that mesenchymal cells extravasated following two-round Ca2+ transients, and distributions of test cells and tunic cells changed in the outer body after tail regression.

Zic-r.b controls cell numbers in Ciona embryos by activating CDKN1B.

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.

Improved Genome Editing in the Ascidian Ciona with CRISPR/Cas9 and TALEN.

The ascidian Ciona intestinalis type A (or Ciona robusta) is an important organism for elucidating the mechanisms that make the chordate body plan. CRISPR/Cas9 and TAL effector nuclease (TALEN) are widely used to quickly address genetic functions in Ciona. Our previously reported method of CRISPR/Cas9-mediated mutagenesis in this animal has inferior mutation rates compared to those of TALENs. We here describe an updated way to effectively mutate genes with CRISPR/Cas9 in Ciona. Although the construction of TALENs is much more laborious than that of CRISPR/Cas9, this technique is useful for tissue-specific knockouts that are not easy even by the optimized CRISPR/Cas9 method.

Mechanism underlying retinoic acid-dependent metamorphosis in the starfish.

The evolution of the biphasic life cycle in marine invertebrates has attracted considerable interest in zoology. We recently provided evidence that retinoic acid (RA) is involved in the regulation of metamorphosis in starfish. It also functions in life cycle transitions of jellyfish (cnidaria). Thus, documenting the evolutionarily conserved role of RA in such transitions will help to trace the life cycle evolution of bilaterians and cnidarians. In this study, we examined the molecular mechanisms by which RA signaling is involved in the commencement of metamorphosis in starfish. First, we measured RA levels during the larval and metamorphosis stages by liquid chromatography-tandem mass spectrometry. We found that all-trans RA levels in the larval body are high before larvae acquire competence for metamorphosis, suggesting that the commencement of metamorphosis is not controlled by increased RA synthesis. Furthermore, the suppression of rar gene expression by TALEN-mediated gene knockout revealed that RA receptor (RAR) is essential for metamorphosis. These observations suggest that the initiation of metamorphosis is regulated at the level of synthesized RA to activate RAR. We discuss the divergence of ligand molecules and receptors during the evolution of life cycle regulation.

The TRP channel PKD2 is involved in sensing the mechanical stimulus of adhesion for initiating metamorphosis in the chordate Ciona.

Metamorphosis is the dramatic and irreversible reconstruction of animal bodies transitioning from the larval stage. Because of the significant impact of metamorphosis on animal life, its timing is strictly regulated. Invertebrate chordate ascidians are the closest living relatives of vertebrates. Ascidians exhibit metamorphosis that converts their swimming larvae into sessile adults. Ascidian metamorphosis is triggered by a mechanical stimulus generated when adhesive papillae adhere to a substrate. However, it is not well understood how the mechanical stimulus is generated and how ascidian larvae sense the stimulus. In this study, we addressed these issues by a combination of embryological, molecular, and genetic experiments in the model ascidian Ciona intestinalis Type A, also called Ciona robusta. We here showed that the epidermal neuronal network starting from the sensory neurons at the adhesive papillae is responsible for the sensing of adhesion. We also found that the transient receptor potential (TRP) channel PKD2 is involved in sensing the stimulus of adhesion. Our results provide a better understanding of the mechanisms underlying the regulation of the timing of ascidian metamorphosis.

Differentiation of endostyle cells by Nkx2-1 and FoxE in the ascidian Ciona intestinalis type A: insights into shared gene regulation in glandular- and thyroid-equivalent elements of the chordate endostyle.

Due to similarities in iodine concentrations and peroxidase activities, the thyroid in vertebrates is considered to originate from the endostyle of invertebrate chordates even though it is a glandular (mucus-producing) organ for aquatic suspension feeding. Among chordates with an endostyle, urochordates are useful evolutionary research models for the study of vertebrate traits. The ascidian Ciona intestinalis forms an endostyle with specific components of glandular- and thyroid-related elements, and molecular markers have been identified for these components. Since we previously examined a simple endostyle in the larvacean Oikopleura dioica, the expression of the thyroid-related transcription factor genes, Ciona Nkx2-1 and FoxE, was perturbed by TALEN-mediated gene knockout in the present study to elucidate the shared and/or divergent features of a complex ascidian endostyle. The knockout of Ciona Nkx2-1 and FoxE exerted different effects on the morphology of the developing endostyle. The knockout of Nkx2-1 eliminated the expression of both glandular and thyroidal differentiation marker genes, e.g., vWFL1, vWFL2, CiEnds1, TPO, and Duox, while that of FoxE eliminated the expression of the differentiation marker genes, TPO and CiEnds1. The supporting element-related expression of Pax2/5/8a, Pax2/5/8b, FoxQ1, and ß-tubulin persisted in the hypoplastic endostyles of Nkx2-1- and FoxE-knockout juveniles. Although the gene regulation of ascidian-specific CiEnds1 remains unclear, these results provide insights into the evolution of the vertebrate thyroid as well as the urochordate endostyle.

Polyzoa is back: The effect of complete gene sets on the placement of Ectoprocta and Entoprocta.

The phylogenomic approach has largely resolved metazoan phylogeny and improved our knowledge of animal evolution based on morphology, paleontology, and embryology. Nevertheless, the placement of two major lophotrochozoan phyla, Entoprocta (Kamptozoa) and Ectoprocta (Bryozoa), remains highly controversial: Originally considered as a single group named Polyzoa (Bryozoa), they were separated on the basis of morphology. So far, each new study of lophotrochozoan evolution has still consistently proposed different phylogenetic positions for these groups. Here, we reinvestigated the placement of Entoprocta and Ectoprocta using highly complete datasets with rigorous contamination removal. Our results from maximum likelihood, Bayesian, and coalescent analyses strongly support the topology in which Entoprocta and Bryozoa form a distinct clade, placed as a sister group to all other lophotrochozoan clades: Annelida, Mollusca, Brachiopoda, Phoronida, and Nemertea. Our study favors the evolutionary scenario where Entoprocta, Cycliophora, and Bryozoa constitute one of the earliest branches among Lophotrochozoa and thus supports the Polyzoa hypothesis.

d-Serine controls epidermal vesicle release via NMDA receptor, allowing tissue migration during the metamorphosis of the chordate Ciona.

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.

Neuronal identities derived by misexpression of the POU IV sensory determinant in a protovertebrate.

The protovertebrate Ciona intestinalis type A (sometimes called Ciona robusta) contains a series of sensory cell types distributed across the head-tail axis of swimming tadpoles. They arise from lateral regions of the neural plate that exhibit properties of vertebrate placodes and neural crest. The sensory determinant POU IV/Brn3 is known to work in concert with regional determinants, such as Foxg and Neurogenin, to produce palp sensory cells (PSCs) and bipolar tail neurons (BTNs), in head and tail regions, respectively. A combination of single-cell RNA-sequencing (scRNA-seq) assays, computational analysis, and experimental manipulations suggests that misexpression of POU IV results in variable transformations of epidermal cells into hybrid sensory cell types, including those exhibiting properties of both PSCs and BTNs. Hybrid properties are due to coexpression of Foxg and Neurogenin that is triggered by an unexpected POU IV feedback loop. Hybrid cells were also found to express a synthetic gene battery that is not coexpressed in any known cell type. We discuss these results with respect to the opportunities and challenges of reprogramming cell types through the targeted misexpression of cellular determinants.

Gene regulation of adult skeletogenesis in starfish and modifications during gene network co-option.

The larval skeleton of the echinoderm is believed to have been acquired through co-option of a pre-existing gene regulatory network (GRN); that is, the mechanism for adult skeleton formation in the echinoderm was deployed in early embryogenesis during echinoderm diversification. To explore the evolutionary changes that occurred during co-option, we examined the mechanism for adult skeletogenesis using the starfish Patiria pectinifera. Expression patterns of skeletogenesis-related genes (vegf, vegfr, ets1/2, erg, alx1, ca1, and clect) suggest that adult skeletogenic cells develop from the posterior coelom after the start of feeding. Treatment with inhibitors and gene knockout using transcription activator-like effector nucleases (TALENs) suggest that the feeding-nutrient sensing pathway activates Vegf signaling via target of rapamycin (TOR) activity, leading to the activation of skeletogenic regulatory genes in starfish. In the larval skeletogenesis of sea urchins, the homeobox gene pmar1 activates skeletogenic regulatory genes, but in starfish, localized expression of the pmar1-related genes phbA and phbB was not detected during the adult skeleton formation stage. Based on these data, we provide a model for the adult skeletogenic GRN in the echinoderm and propose that the upstream regulatory system changed from the feeding-TOR-Vegf pathway to a homeobox gene-system during co-option of the skeletogenic GRN.

Nkx2-1 and FoxE regionalize glandular (mucus-producing) and thyroid-equivalent traits in the endostyle of the chordate Oikopleura dioica.

The endostyle is a ventral pharyngeal organ used for internal filter feeding of basal chordates and is considered homologous to the follicular thyroid of vertebrates. It contains mucus-producing (glandular) and thyroid-equivalent regions organized along the dorsoventral (DV) axis. Although thyroid-related genes (Nkx2-1, FoxE, and thyroid peroxidase (TPO)) are known to be expressed in the endostyle, their roles in establishing regionalization within the organ have not been demonstrated. We report that Nkx2-1 and FoxE are essential for establishing DV axial identity in the endostyle of Oikopleura dioica. Genome and expression analyses showed von Willebrand factor-like (vWFL) and TPO/dual oxidase (Duox)/Nkx2-1/FoxE as orthologs of glandular and thyroid-related genes, respectively. Knockdown experiments showed that Nkx2-1 is necessary for the expression of glandular and thyroid-related genes, whereas FoxE is necessary only for thyroid-related genes. Moreover, Nkx2-1 expression is necessary for FoxE expression in larvae during organogenesis. The results demonstrate the essential roles of Nkx2-1 and FoxE in establishing regionalization in the endostyle, including (1) the Nkx2-1-dependent glandular region, and (2) the Nkx2-1/FoxE-dependent thyroid-equivalent region. DV axial regionalization may be responsible for organizing glandular and thyroid-equivalent traits of the pharynx along the DV axis.

Vasopressin Promoter Transgenic and Vasopressin Gene-Edited Ascidian, Ciona intestinalis Type A (Ciona robusta): Innervation, Gene Expression Profiles, and Phenotypes.

Oxytocin (OT) and vasopressin (VP) superfamily neuropeptides are distributed in not only vertebrates but also diverse invertebrates. However, no VPergic innervation of invertebrates has ever been documented. In the ascidian, Ciona intestinalis Type A (Ciona robusta), an OT/VP superfamily peptide was identified, and the Ciona vasopressin (CiVP) induces oocyte maturation and ovulation. In the present study, we characterize the innervation and phenotypes of genetically modified Ciona: CiVP promoter-Venus transgenic and CiVP mutants. CiVP promoter-Venus transgenic Ciona demonstrated that CiVP gene was highly expressed in the cerebral ganglion and several nerves. Fluorescence was also detected in the ovary of young CiVP promoter-Venus transgenic ascidians, suggesting that the CiVP gene is also expressed temporarily in the ovary of young ascidians. Furthermore, a marked decrease of post-vitellogenic (stage III) follicles was observed in the ovary of CiVP mutants, whereas pre-vitellogenic (stage I) and vitellogenic (stage II) follicles were increased in the mutant ovary, compared with that of wildtype Ciona. Gene expression profiles showed that the expression of various genes, including genes related to ovarian follicle growth, was altered in the ovary of CiVP mutants. Altogether, these results indicated that CiVP, mainly as a neuropeptide, plays pivotal roles in diverse biological functions, including growth of early-stage ovarian follicles via regulation of the expression of a wide variety of genes. This is the first report describing a VP gene promoter-transgenic and VP gene-edited invertebrate and also on its gene expression profiles and phenotypes.