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.

Early expression onset of tissue-specific effector genes during the specification process in sea urchin embryos.

BACKGROUND: In the course of animal developmental processes, various tissues are differentiated through complex interactions within the gene regulatory network. As a general concept, differentiation has been considered to be the endpoint of specification processes. Previous works followed this view and provided a genetic control scheme of differentiation in sea urchin embryos: early specification genes generate distinct regulatory territories in an embryo to express a small set of differentiation driver genes; these genes eventually stimulate the expression of tissue-specific effector genes, which provide biological identity to differentiated cells, in each region. However, some tissue-specific effector genes begin to be expressed in parallel with the expression onset of early specification genes, raising questions about the simplistic regulatory scheme of tissue-specific effector gene expression and the current concept of differentiation itself. RESULTS: Here, we examined the dynamics of effector gene expression patterns during sea urchin embryogenesis. Our transcriptome-based analysis indicated that many tissue-specific effector genes begin to be expressed and accumulated along with the advancing specification GRN in the distinct cell lineages of embryos. Moreover, we found that the expression of some of the tissue-specific effector genes commences before cell lineage segregation occurs. CONCLUSIONS: Based on this finding, we propose that the expression onset of tissue-specific effector genes is controlled more dynamically than suggested in the previously proposed simplistic regulation scheme. Thus, we suggest that differentiation should be conceptualized as a seamless process of accumulation of effector expression along with the advancing specification GRN. This pattern of effector gene expression may have interesting implications for the evolution of novel cell types.

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.

Perspectives on divergence of early developmental regulatory pathways: Insight from the evolution of echinoderm double negative gate.

Evolution of gene regulatory networks (GRN) that orchestrate the highly coordinated course of development, is made possible by the network's robust nature for incorporating change without detrimental developmental outcome. It can be considered that the upstream network regulating early development, has immense influence over succeeding pathways thus may be less subjected to evolutionary modification. However, recent studies show incorporation of novel genes in such early developmental pathways such as the echinoderm pmar1 as evidence for drastic change occurring high in the GRN hierarchy. Here we discuss the mechanisms that underlie divergence of early developmental pathways utilizing promising insights from the evolution of echinoderm early mesoderm specification pathway of Pmar1-HesC double negative gate found solely in the euechinoid sea urchin lineage, as well as examples from other groups such as Spiralia and Drosophila.

Machinery and Developmental Role of Retinoic Acid Signaling in Echinoderms.

Although a recent genomic survey revealed its ancient evolutionary origin in the animal kingdom, retinoic acid (RA) signaling was previously thought to be unique to chordates. Echinoderms are of critical interest in researching the evolutionary history of RA signaling, as they represent a basal group of deuterostomes. Furthermore, our previous works have suggested that echinoderms may possess the ancestral function of RA signaling for metamorphosis regulation. In this paper, to facilitate future studies of RA signaling in echinoderms, we provide an overview of RA machinery in echinoderms, identify its signaling components, and discuss its developmental role.

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.

Retinoic Acid Signaling Regulates the Metamorphosis of Feather Stars (Crinoidea, Echinodermata): Insight into the Evolution of the Animal Life Cycle.

Many marine invertebrates have a life cycle with planktonic larvae, although the evolution of this type of life cycle remains enigmatic. We recently proposed that the regulatory mechanism of life cycle transition is conserved between jellyfish (Cnidaria) and starfish (Echinoderm); retinoic acid (RA) signaling regulates strobilation and metamorphosis, respectively. However, the function of RA signaling in other animal groups is poorly understood in this context. Here, to determine the ancestral function of RA signaling in echinoderms, we investigated the role of RA signaling during the metamorphosis of the feather star, Antedon serrata (Crinoidea, Echinodermata). Although feather stars have different larval forms from starfish, we found that exogenous RA treatment on doliolaria larvae induced metamorphosis, like in starfish. Furthermore, blocking RA synthesis or binding to the RA receptor suppressed metamorphosis. These results suggested that RA signaling functions as a regulator of metamorphosis in the ancestor of echinoderms. Our data provides insight into the evolution of the animal life cycle from the viewpoint of RA signaling.

The role of retinoic acid signaling in starfish metamorphosis.

BACKGROUND: Although retinoic acid (RA) signaling plays a crucial role in the body patterning of chordates, its function in non-chordate invertebrates, other than its mediation of environmental cues triggering metamorphosis in cnidarians, is largely unknown. We investigated the role of RA signaling in the metamorphosis of starfish (Echinodermata). RESULTS: We found that exogenous RA treatment induced metamorphosis in starfish larvae. In contrast, inhibitors of RA synthesis and RA receptors suppressed metamorphosis triggered by attachment to a substrate. Gene expressions of the RA signaling component were detected in competent larvae. CONCLUSIONS: This study provides insight into the ancestral function of RA signaling, which is conserved in the metamorphosis of cnidarians and starfish.