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Evolutionary history of deuterostomes has remained unsolved and is intimately related to how chordates originated. Within the clade of deuterostomes, hemichordates and echinoderms (together called Ambulacraria) are sister groups of chordates; comparative studies among the three are insightful for understanding deuterostome evolution. Indirect-developing hemichordates, such as Ptychodera flava, develop into planktonic larvae, which are morphologically similar to echinoderm larvae, before metamorphosing into the adult body plan that retains an anteroposterior polarity homologous to that of chordates. Therefore, deciphering developmental programmes of indirect-developing hemichordates may provide a key for understanding the evolution of deuterostomes and chordate origins. Here, we characterise transcriptomes and chromatin accessibility across multiple developmental stages of P. flava and uncover biphasic developmental programmes controlled by different sets of transcription factors and their corresponding cis-regulatory elements. Transcriptome age and network analyses reveal that the gastrula transcriptome is relatively ancient and highly connected. By comparing developmental transcriptomes of hemichordate, sea urchin and amphioxus, a high conservation of gene expression during gastrulation is identified and extended to neurula stages of amphioxus, in addition to the highly similar larval transcriptomes among the three species. Moreover, P. flava possesses conserved interactions of transcription factors required for the development of echinoderm endomesoderm and chordate axial mesoderm. These results suggest a deuterostome phylotypic stage during gastrulation (corresponding to amphioxus gastrulation and neurulation) controlled by gene regulatory networks with conserved cis-regulatory interactions, and support the hypothesis that the deuterostome ancestor is an indirect-developer.

Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation [A. Pires-daSilva, R. J. Sommer, Nat. Rev. Genet. 4, 39-49 (2003)], although only a few of these pathways operate during this period [J. J. Sanz-Ezquerro, A. E. Munsterberg, € S. Stricker, Front. Cell Dev. Biol. 5, 76 (2017)]. Moreover, most of them have been largely conserved during metazoan evolution [L. S. Babonis, M. Q. Martindale, Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20150477 (2017)]. How evolution has generated a vast diversity of animal morphologies with such a limited number of tools is still largely unknown. Here, we show that gain of interconnectivity between signaling pathways and the GRNs they control may have critically contributed to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF, and Nodal signaling pathways during gastrulation in the invertebrate chordate amphioxus and zebrafish and compared the effects on gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through vertebrate-specific CREs. Moreover, in contrast to amphioxus, many of these CREs responded to multiple pathways in zebrafish, which reflects their high interconnectivity. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than in tissues with more ancient origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and to the formation of new cell types and morphological novelties in this lineage., This project has received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (Grant agreement no. 740041) and the Spanish Ministerio de Economía y Competitividad (Grants BFU2016-74961-P and PID2019-103921GB-I00 to J.L.G.-S. and J.J.T.). This work was also supported by the institutional grant Unidad de Excelencia María de Maeztu (Grant MDM-2016-0687 to the Department of Gene regulation and morphogenesis of Centro Andaluz de Biología del Desarrollo). M.F. was funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement [#800396]. M.S., H.E., and S.B. were supported by the CNRS, and H.E. and S.B. additionally by Agence Nationale de la Recherche (ANR) CHORELAND (Grant ANR-16-CE12-0008-01) and the Institut Universitaire de France. Y.-H.S. and J.-K.Y. are supported by intramural funds from Academia Sinica and grants from Ministry of Science and Technology, Taiwan (Grants 110-2326-B-001-006 to Y.-H.S. and 110-2621-B-001-001-MY3 to J.-K.Y.).

Most sea urchin species produce planktonic feeding larvae with distinct dorsal-ventral polarity. Such morphological indicators of polarity arise after gastrulation, when several morphogenesis and cell differentiation events occur differentially along the dorsal-ventral axis. For instance, the gut bends toward the ventral side where the mouth will form, skeletogenesis occurs initially near the ventral side with the forming skeleton extending dorsally, and pigment cells differentiate and embed in the dorsal ectoderm. The patterning mechanisms and gene regulatory networks underlying these events have been extensively studied. Two opposing TGF-β signaling pathways, Nodal and BMP, play key roles in all three germ layers to respectively pattern the sea urchin ventral and dorsal sides. In this chapter, I describe our current understanding of sea urchin dorsal-ventral patterning mechanisms. Additionally, differences in the patterning mechanisms observed in lecithotrophic sea urchins (nonfeeding larvae) and in cidaroid sea urchins are also discussed, along with evolutionary insights gained from comparative analyses.

Spinal motor neurons (MNs) integrate sensory stimuli and brain commands to generate motor movements in vertebrates. Distinct MN populations and their diversity has long been hypothesized to co-evolve with motor circuit to provide the neural basis from undulatory to ambulatory locomotion during aquatic-to-terrestrial transition of vertebrates. However, how these subtypes are evolved remains largely enigmatic. Using single-cell transcriptomics, we investigate heterogeneity in mouse MNs and discover novel segment-specific subtypes. Among limb-innervating MNs, we reveal a diverse neuropeptide code for delineating putative motor pool identities. We further uncovered that axial MNs are subdivided by three conserved and molecularly distinct subpopulations, defined by Satb2, Nr2f2 or Bcl11b expression. Although axial MNs are conserved from cephalochordates to humans, subtype diversity becomes prominent in land animals and appears to continue evolving in humans. Overall, our study provides a unified classification system for spinal MNs and paves the way towards deciphering how neuronal subtypes are evolved.

Sea urchin larval skeletons are produced by skeletogenic primary mesenchyme cells (PMCs), which migrate to form two ventrolateral clusters (VLCs) at the sites where biomineralization is initiated. Both PMC migration and biomineralization are controlled by VEGF signals emitted from lateral ectodermal cells. In mammals, VEGF signaling can be activated by hypoxia-inducible factor alpha (HIFα), an oxygen-sensitive transcription factor. Our previous study showed that the sea urchin maternal HIFα is involved in regulating gene expression along the dorsoventral axis. In this study, we discovered that zygotic hifα is expressed in PMCs, and at the late gastrula stage, hifα transcripts display a graded pattern, with stronger signal in the ventral PMCs than in the dorsal PMCs. We further showed that PMCs are hypoxic, which is a condition typically required for HIFα function. In embryos injected with a splice-blocking morpholino against hifα, elongation of the skeleton was impaired, and expression of vegfr-10-Ig (encodes VEGF receptor; VEGFR) was significantly reduced. This morpholino-caused defect could be partially rescued by injection of vegfr-10-Ig mRNA. Expression patterns of transcription factor and biomineralization genes, such as alx1, tbr, msp130, and the sm30 family, were affected when HIFα was knocked down or when VEGF signaling was inhibited. These results suggest that zygotic HIFα acts upstream or in parallel with VEGF signaling to regulate skeletogenic gene expression and participate in spicule elongation. Our study therefore links HIFα with the known role of VEGF signaling in sea urchin biomineralization.

Primordial germ cells (PGCs) are specified during development by either one of two major mechanisms, the preformation mode or the inductive mode. Because the inductive mode is widely employed by many bilaterians and early branching metazoan lineages, it has been postulated as an ancestral mechanism. However, among the deuterostome species that have been studied, invertebrate chordates use the preformation mode, while many vertebrate and echinoderm species are known to utilize an inductive mechanism, thus leaving the evolutionary history of PGC specification in the deuterostome lineage unclear. Hemichordates are the sister phylum of echinoderms, and together they form a clade called Ambulacraria that represents the closest group to the chordates. Thus, research in hemichordates is highly informative for resolving this issue. In this study, we investigate the developmental process of PGCs in an indirect-developing hemichordate, Ptychodera flava. We show that maternal transcripts of the conserved germline markers vasa, nanos, and piwi1 are ubiquitously distributed in early P. flava embryos, and these genes are coexpressed specifically in the dorsal hindgut starting from the gastrula stage. Immunostaining revealed that Vasa protein is concentrated toward the vegetal pole in early P. flava embryos, and it is restricted to cells in the dorsal hindgut of gastrulae and newly hatched larvae. The Vasa-positive cells later contribute to the developing trunk coeloms of the larvae and eventually reside in the adult gonads. We further show that bone morphogenetic protein (BMP) signaling is required to activate expression of the germline determinants in the gastrula hindgut, suggesting that PGC specification is induced by BMP signaling in P. flava. Our data support the hypothesis that the inductive mode is a conserved mechanism in Ambulacraria, which might even trace back to the common ancestor of Deuterostomes.

Extensive adenosine-to-inosine (A-to-I) editing of nuclear-transcribed RNAs is the hallmark of metazoan transcriptional regulation, and is fundamental to numerous biochemical processes. Here we explore the origin and evolution of this regulatory innovation, by quantifying its prevalence in 22 species that represent all major transitions in metazoan evolution. We provide substantial evidence that extensive RNA editing emerged in the common ancestor of extant metazoans. We find the frequency of RNA editing varies across taxa in a manner independent of metazoan complexity. Nevertheless, cis-acting features that guide A-to-I editing are under strong constraint across all metazoans. RNA editing seems to preserve an ancient mechanism for suppressing the more recently evolved repetitive elements, and is generally nonadaptive in protein-coding regions across metazoans, except forDrosophilaand cephalopods. Interestingly, RNA editing preferentially target genes involved in neurotransmission, cellular communication and cytoskeleton, and recodes identical amino acid positions in several conserved genes across diverse taxa, emphasizing broad roles of RNA editing in cellular functions during metazoan evolution that have been previously underappreciated.

Studies on the gene regulatory networks (GRNs) of sea urchin embryos have provided a basic understanding of the molecular mechanisms controlling animal development. The causal links in GRNs have been verified experimentally through perturbation of gene functions. Microinjection of antisense morpholino oligonucleotides (MOs) into the egg is the most widely used approach for gene knockdown in sea urchin embryos. The modification of MOs into a membrane-permeable form (vivo-MOs) has allowed gene knockdown at later developmental stages. Recent advances in genome editing tools, such as zinc-finger nucleases, transcription activator-like effector-based nucleases and the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) system, have provided methods for gene knockout in sea urchins. Here, we review the use of vivo-MOs and genome editing tools in sea urchin studies since the publication of its genome in 2006. Various applications of the CRISPR/Cas9 system and their potential in studying sea urchin development are also discussed. These new tools will provide more sophisticated experimental methods for studying sea urchin development.

A defining feature of chordates is the unique presence of a dorsal hollow neural tube that forms by internalization of the ectodermal neural plate specified via inhibition of BMP signaling during gastrulation. While BMP controls dorsoventral (DV) patterning across diverse bilaterians, the BMP-active side is ventral in chordates and dorsal in many other bilaterians. How this phylum-specific DV inversion occurs and whether it is coupled to the emergence of the dorsal neural plate are unknown. Here we explore these questions by investigating an indirect-developing enteropneust from the hemichordate phylum, which together with echinoderms form a sister group of the chordates. We found that in the hemichordate larva, BMP signaling is required for DV patterning and is sufficient to repress neurogenesis. We also found that transient overactivation of BMP signaling during gastrulation concomitantly blocked mouth formation and centralized the nervous system to the ventral ectoderm in both hemichordate and sea urchin larvae. Moreover, this mouthless, neurogenic ventral ectoderm displayed a medial-to-lateral organization similar to that of the chordate neural plate. Thus, indirect-developing deuterostomes use BMP signaling in DV and neural patterning, and an elevated BMP level during gastrulation drives pronounced morphological changes reminiscent of a DV inversion. These findings provide a mechanistic basis to support the hypothesis that an inverse chordate body plan emerged from an indirect-developing ancestor by tinkering with BMP signaling.

Summary Certain vertebrates such as salamanders and zebrafish are able to regenerate complex tissues (e.g., limbs and fins) with remarkable fidelity. However, how positional information of the missing structure is recalled by appendage stump cells has puzzled researchers for centuries. Here, we report that sizing information for adult zebrafish tailfins is encoded within proliferating blastema cells during a critical period of regeneration. Using a chemical mutagenesis screen, we identified a temperature-sensitive allele of the gene encoding DNA polymerase alpha subunit 2 (pola2) that disrupts fin regeneration in zebrafish. Temperature shift assays revealed a 48-h window of regeneration, during which positional identities could be disrupted in pola2 mutants, leading to regeneration of miniaturized appendages. These fins retained memory of the new size in subsequent rounds of amputation and regeneration. Similar effects were observed upon transient genetic or pharmacological disruption of progenitor cell proliferation after plucking of zebrafish scales or head or tail amputation in amphioxus and annelids. Our results provide evidence that positional information in regenerating tissues is not hardwired but malleable, based on regulatory mechanisms that appear to be evolutionarily conserved across distantly related phyla.

Oxygen is essential to contemporary life, providing the major electron sink underlying cellular energy metabolism. In addition to providing energy, largely involving redox reactions within mitochondria, oxidative metabolism produces reactive byproducts that are damaging to cellular components. Eukaryotic organisms have evolved multiple physiological mechanisms and signaling pathways to deal with fluctuating levels of oxygen and reactive oxygen species (ROS), and many of these are used in animals to regulate developmental processes. Here we review recent findings showing how mitochondria, ROS and hypoxia signaling contribute to the regulation of early axial patterning in embryos, to nervous system development, and to the regulation of cell proliferation and differentiation during development and regeneration.

Small micromeres of the sea urchin are believed to be primordial germ cells (PGCs), fated to give rise to sperm or eggs in the adult. Sea urchin PGCs are formed at the fifth cleavage, undergo one additional division during blastulation, and migrate to the coelomic pouches of the pluteus larva. The goal of this chapter is to detail classical and modern techniques used to analyze primordial germ cell specification, gene expression programs, and cell behaviors in fixed and live embryos. The transparency of the sea urchin embryo enables both live imaging techniques and in situ RNA hybridization and immunolabeling for a detailed molecular characterization of these cells. Four approaches are presented to highlight small micromeres with fluorescent molecules for analysis by live and fixed cell microscopy: (1) small molecule dye accumulation during cleavage and blastula stages, (2) primordial germ cell targeted RNA expression using the Nanos untranslated regions, (3) fusing genes of interest with a Nanos2 targeting peptide, and (4) EdU and BrdU labeling. Applications of the live labeling techniques are discussed, including sorting by fluorescence-activated cell sorting for transcriptomic analysis, and, methods to image small micromere behavior in whole and dissociated embryos by live confocal microscopy. Finally, summary table of antibody and RNA probes as well as small molecule dyes to label small micromeres at a variety of developmental stages is provided.

Background Mesoderm is generally considered to be a germ layer that is unique to Bilateria, and it develops into diverse tissues, including muscle, and in the case of vertebrates, the skeleton and notochord. Studies on various deuterostome animals have demonstrated that fibroblast growth factor (FGF) signaling is required for the formation of many mesodermal structures, such as vertebrate somites, from which muscles are differentiated, and muscles in sea urchin embryos, suggesting an ancient role of FGF signaling in muscle development. However, the formation of trunk muscles in invertebrate chordates is FGF-independent, leading to ambiguity about this ancient role in deuterostomes. To further understand the role of FGF signaling during deuterostome evolution, we investigated the development of mesodermal structures during embryogenesis and metamorphosis in Ptychodera flava, an indirect-developing hemichordate that has larval morphology similar to echinoderms and adult body features that are similar to chordates. Results Here we show that genes encoding FGF ligands, FGF receptors and transcription factors that are known to be involved in mesoderm formation and myogenesis are expressed dynamically during embryogenesis and metamorphosis. FGF signaling at the early gastrula stage is required for the specification of the mesodermal cell fate in P. flava. The mesoderm cells are then differentiated stepwise into the hydroporic canal, the pharyngeal muscle and the muscle string; formation of the last two muscular structures are controlled by FGF signaling. Moreover, augmentation of FGF signaling during metamorphosis accelerated the process, facilitating the transformation from cilia-driven swimming larvae into muscle-driven worm-like juveniles. Conclusions Our data show that FGF signaling is required for mesoderm induction and myogenesis in the P. flava embryo, and it is reiteratively used for the morphological transition during metamorphosis. The dependence of muscle development on FGF signaling in both planktonic larvae and sand-burrowing worms supports its ancestral role in deuterostomes. Electronic supplementary material The online version of this article (10.1186/s12862-018-1235-9) contains supplementary material, which is available to authorized users.

Severe injury to the central nervous system of chordates often results in permanent and irreversible mental and physical challenges. While some chordates are able to repair and/or regenerate portions of their nervous system, no chordate has been shown to be able to regenerate all regions of its central nervous system after catastrophic injury or amputation. Some hemichordates, on the other hand, are able to efficiently regenerate all neural structures, including their dorsal, hollow neural tube after complete ablation. Solitary hemichordates are marine acorn worms and a sister group to the echinoderms. The hemichordate Ptychodera flava progresses from a pelagic, feeding tornaria larva to a tripartite benthic worm with an anterior proboscis, a middle collar region, and a long posterior trunk. The adult worm regenerates all body parts when bisected in the trunk, but it was unknown whether the regeneration process was present in tornaria larvae. Now, we show that P. flava larvae are capable of robust regeneration after bisection through the sagittal, coronal, and axial planes. We also use antibody staining to show that the apical sensory organ regenerates a rich, serotonin-positive complex of cells within two weeks after amputation. Cells labeled with 5-ethynyl-2'-deoxyuridine confirm that regeneration is occurring through epimorphic processes as new cells are added at the cut site and throughout the regenerating tissue. This study verifies that P. flava larvae can be used for future functional studies aimed at identifying the genetic and morphological mechanisms controlling central nervous system regeneration in a stem deuterostome.

Table S1. Accession numbers for genes/proteins used in this study. Table S2. Primers used for cloning. (DOCX 25 kb)

Supplementary methods. (DOCX 77 kb)

The indirect-developing enteropneust acorn worm Ptychodera flava has been used as a hemichordate model system for studying the developmental evolution of deuterostome body plans and the origins of chordate characteristics. However, research progress has been hindered by the limited accessibility of its embryonic materials and metamorphosing larvae. In this study, we identified an abundant population of P. flava in Penghu, Taiwan, and examined the feasibility of using this animal for developmental studies. Through histological examination, we established that the reproductive season of this population is between September and December, with a peak breeding period in October and November. In addition, we have developed new procedures that can induce P. flava spawning at any time of the day during the breeding season, with a higher successful rate than that achieved using a previously published method. Moreover, the culturing system we developed enables rearing of P. flava larvae through various planktonic stages and eventual metamorphosis into benthic juveniles, all under laboratory conditions. We anticipate that the animal resources and new technical procedures reported here will further facilitate the use of P. flava as a model organism for evolutionary and developmental biology research.

Fibroblast growth factors (FGFs) are a group of ligands that play multiple roles during development by transducing signals through FGF receptors (FGFRs) to downstream factors. At least 22 FGF ligands and 4 receptors have been identified in vertebrates, while six to eight FGF ligands and a single FGFR are present in invertebrate chordates, such as tunicates and amphioxus. The chordate FGFs can be categorized into at least seven subfamilies, and the members of which expanded during the evolution of early vertebrates. In contrast, only one FGF and two FGFRs have been found in sea urchins. Thus, it is unclear whether the FGF subfamilies duplicated in the lineage leading to the chordates, or sea urchins lost several fgf genes. Analyses of the FGF signaling repertoire in hemichordates, which together with echinoderms form the closest group to the chordates, may provide insights into the evolution of FGF signaling in deuterostomes. In this study, we identified five FGFs and three FGFRs from Ptychodera flava, an indirect-developing hemichordate acorn worm. Phylogenetic analyses revealed that hemichordates possess a conserved FGF8/17/18 in addition to several putative hemichordate-specific FGFs. Analyses of sequence similarity and protein domain organizations suggested that the sea urchin and hemichordate FGFRs arose from independent lineage-specific duplications. Furthermore, the acorn worm fgf and fgfr genes were demonstrated to be expressed during P. flava embryogenesis. These results set the foundations for further functional studies of FGF signaling in hemichordates and provided insights into the evolutionary history of the FGF repertoire.

Acorn worms, also known as enteropneust (literally, 'gut-breathing') hemichordates, are marine invertebrates that share features with echinoderms and chordates. Together, these three phyla comprise the deuterostomes. Here we report the draft genome sequences of two acorn worms, Saccoglossus kowalevskii and Ptychodera flava. By comparing them with diverse bilaterian genomes, we identify shared traits that were probably inherited from the last common deuterostome ancestor, and then explore evolutionary trajectories leading from this ancestor to hemichordates, echinoderms and chordates. The hemichordate genomes exhibit extensive conserved synteny with amphioxus and other bilaterians, and deeply conserved non-coding sequences that are candidates for conserved gene-regulatory elements. Notably, hemichordates possess a deuterostome-specific genomic cluster of four ordered transcription factor genes, the expression of which is associated with the development of pharyngeal 'gill' slits, the foremost morphological innovation of early deuterostomes, and is probably central to their filter-feeding lifestyle. Comparative analysis reveals numerous deuterostome-specific gene novelties, including genes found in deuterostomes and marine microbes, but not other animals. The putative functions of these genes can be linked to physiological, metabolic and developmental specializations of the filter-feeding ancestor.

Quantum Hall ferromagnetic transitions are typically achieved by increasing the Zeeman energy through in-situ sample rotation, while transitions in systems with pseudo-spin indices can be induced by gate control. We report here a gate-controlled quantum Hall ferromagnetic transition between two real spin states in a conventional two-dimensional system without any in-plane magnetic field. We show that the ratio of the Zeeman splitting to the cyclotron gap in a Ge two-dimensional hole system increases with decreasing density owing to inter-carrier interactions. Below a critical density of ~2.4 × 1010 cm−2, this ratio grows greater than 1, resulting in a ferromagnetic ground state at filling factor ν = 2. At the critical density, a resistance peak due to the formation of microscopic domains of opposite spin orientations is observed. Such gate-controlled spin-polarizations in the quantum Hall regime opens the door to realizing Majorana modes using two-dimensional systems in conventional, low-spin-orbit-coupling semiconductors.

Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs the hypoxic acclimation and results in various diseases, including cancers. The role of the hypoxia pathway in early embryogenesis remains unclear. Here, we show that in the sea urchin Strongylocentrotus purpuratus blastula, hypoxia-inducible factor α (HIFα), the downstream transcription factor of this pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricted the expression of nodal to the ventral side and activated several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results present an unprecedented role of the hypoxia pathway in animal development.

As a group closely related to chordates, hemichordate acorn worms are in a key phylogenic position for addressing hypotheses of chordate origins. The stomochord of acorn worms is an anterior outgrowth of the pharynx endoderm into the proboscis. In 1886 Bateson proposed homology of this organ to the chordate notochord, crowning this animal group "hemichordates." Although this proposal has been debated for over a century, the question still remains unresolved. Here we review recent progress related to this question. First, the developmental mode of the stomochord completely differs from that of the notochord. Second, comparison of expression profiles of genes including Brachyury, a key regulator of notochord formation in chordates, does not support the stomochord/notochord homology. Third, FoxE that is expressed in the stomochord-forming region in acorn worm juveniles is expressed in the club-shaped gland and in the endostyle of amphioxus, in the endostyle of ascidians, and in the thyroid gland of vertebrates. Based on these findings, together with the anterior endodermal location of the stomochord, we propose that the stomochord has evolutionary relatedness to chordate organs deriving from the anterior pharynx rather than to the notochord.

Symbiotic copepods compose one-third of the known copepod species and are associated with a wide range of animal groups. Two parasitic copepods endoparasitic in acorn worms (Hemichordata), Ive balanoglossi and Ubius hilli, collected in the Mediterranean Sea and Australian waters, respectively, were described a century ago. Here we report a new parasitic copepod species, Ive ptychoderae sp. nov., found in Ptychodera flava, a widespread acorn worm in the Indo-Pacific Ocean and an emerging organism for developmental and evolutionary studies. The female of I. ptychoderae is characterized by having a reduced maxilliped and five pairs of annular swellings along the body that are morphologically similar but distinguishable from those in the two previously described parasitic copepods in acorn worms. Phylogenetic analysis based on the 18S rDNA sequence shows that I. ptychoderae may belong to Poecilostomatoida but represent a new family, which we name Iveidae fam. nov. Ive ptychoderae is commonly found in the acorn worm population with an average prevalence of 42% during the collecting period. The infection of the parasite induces the formation of cysts and causes localized lesions of the host tissues, suggesting that it may have negative effects on its host. Interestingly, most cysts contain a single female with one or multiple male copepods, suggesting that their sex determination may be controlled by environmental conditions. The relationships between the parasitic copepods and acorn worms thus provide a platform for understanding physiological and ecological influences and coevolution between parasites and hosts.

Summary Left–right asymmetry of internal organs is a common feature in bilateria. It is generally considered that three steps are required to establish the left–right axis. These include the initial symmetry-breaking step that leads to asymmetric gene expression and ultimately results in side-specific organogenesis along the left–right axis. During the development of indirect-developing sea urchins, the adult rudiments consistently form on the left side of the feeding larvae. Recent studies have revealed that several signaling molecules are required to regulate the initial right-sided nodal expression in the archenteron. Right-sided Nodal signaling then restricts BMP signaling to the left side. These two TGFβ signals are important for asymmetric gene expression, cell behavior, and morphogenesis along the left–right axis. Although considerable progress has been made regarding sea urchin left–right asymmetry, many questions remain unanswered. This review attempts to synthesize what is known about the left–right asymmetric controls in sea urchins. Additionally, questions raised from previous findings and possible mechanisms concerning symmetry breaking are also discussed. genesis 52:269–278. © 2014 Wiley Periodicals, Inc.

Understanding the evolutionary history of deuterostomes requires elucidating the phylogenetic interrelationships amongst the constituent taxa. Although the monophyly and interrelationships among the three principal groups—the chordates, the echinoderms, and the hemichordates—are well established, as are the internal relationships among the echinoderm and chordate taxa, the interrelationships among the principal groups of hemichordates—the harrimaniid enteropneusts, the ptychoderid enteropneusts, and the pterobranchs—remain unresolved. Depending on the study some find enteropneusts paraphyletic with pterobranchs (e.g., Cephalodiscus) more closely related to the harrimaniid enteropneusts (e.g., Saccoglossus) than either are to the ptychoderid enteropneusts (e.g., Ptychodera), whereas other studies support a monophyletic Enteropneusta. To try and resolve between these two competing hypotheses, we turned to microRNAs, small ∼22 nt non-coding RNA genes that have been shown to shed insight into particularly difficult phylogenetic questions. Using deep sequencing we characterized the small RNA repertoires of two hemichordate species, Cephalodiscus hodgsoni and Ptychodera flava, and the crinoid echinoderm Antedon mediterranea, and combined our results with the described complements of the hemichordate Saccoglossus kowalevskii, the sea urchin Strongylocentrotus purpuratus, and the starfish Patiria miniata. Our data unambiguously support the monophyly of Enteropneusts as S. kowalevskii shares 12 miRNA sequences with P. flava that are not present in the C. hodgsoni or A. mediterranea libraries, and have never been reported from another metazoan taxon. Thus, these data resolve the phylogenetic position of pterobranchs, ultimately allowing for a better understanding of body plan evolution throughout the deuterostomes. J. Exp. Zool. (Mol. Dev. Evol.) 320B: 368–374, 2013. © 2013 Wiley Periodicals, Inc.

Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin

The origin of germline cells was a crucial step in animal evolution. Therefore, in both developmental biology and evolutionary biology, the mechanisms of germline specification have been extensively studied over the past two centuries. However, in many animals, the process of germline specification remains unclear. Here, we show that in the cephalochordate amphioxus Branchiostoma floridae, the germ cell-specific molecular markers Vasa and Nanos become localized to the vegetal pole cytoplasm during oogenesis and are inherited asymmetrically by a single blastomere during cleavage. After gastrulation, this founder cell gives rise to a cluster of progeny that display typical characters of primordial germ cells (PGCs). Blastomeres separated at the two-cell stage grow into twin embryos, but one of the twins fails to develop this Vasa-positive cell population, suggesting that the vegetal pole cytoplasm is required for the formation of putative PGCs in amphioxus embryos. Contrary to the hypothesis that cephalochordates may form their PGCs by epigenesis, our data strongly support a preformation mode of germ cell specification in amphioxus. In addition to the early localization of their maternal transcripts in the putative PGCs, amphioxus Vasa and Nanos are also expressed zygotically in the tail bud, which is the posterior growth zone of amphioxus. Thus, in addition to PGC specification, amphioxus Vasa and Nanos may also function in highly proliferating somatic stem cells.

The temporal and spatial expression patterns of regulatory genes are required for building a gene regulatory network (GRN). The current ectoderm GRN model for the sea urchin embryo includes pregastrular specification functions in the oral (OE) and aboral ectoderm (AE). Unlike the OE, which is resolved into several subdomains, the AE is considered a simpler territory due to the lack of detailed gene expression studies in this territory. Here, we perform temporal and spatial gene expression studies on the eight transcription factor genes constituting the AE GRN. Based on the differential gene expression patterns, we conclude that the AE contains at least three subdomains at the mesenchyme blastula stage. We also performed immunostaining for pSmad1/5/8 to monitor the activation of the BMP signaling pathway. The dynamic changes in the expression patterns of these transcription factor genes and the nuclearization of pSmad1/5/8 may provide a foundation for resolving the AE GRN. Developmental Dynamics, 2011. © 2010 Wiley-Liss, Inc.

The current gene regulatory network (GRN) for the sea urchin embryo pertains to pregastrular specification functions in the endomesodermal territories. Here we extend gene regulatory network analysis to the adjacent oral and aboral ectoderm territories over the same period. A large fraction of the regulatory genes predicted by the sea urchin genome project and shown in ancillary studies to be expressed in either oral or aboral ectoderm by 24 h are included, though universally expressed and pan-ectodermal regulatory genes are in general not. The loci of expression of these genes have been determined by whole mount in situ hybridization. We have carried out a global perturbation analysis in which expression of each gene was interrupted by introduction of morpholino antisense oligonucleotide, and the effects on all other genes were measured quantitatively, both by QPCR and by a new instrumental technology (NanoString Technologies nCounter Analysis System). At its current stage the network model, built in BioTapestry, includes 22 genes encoding transcription factors, 4 genes encoding known signaling ligands, and 3 genes that are yet unknown but are predicted to perform specific roles. Evidence emerged from the analysis pointing to distinctive subcircuit features observed earlier in other parts of the GRN, including a double negative transcriptional regulatory gate, and dynamic state lockdowns by feedback interactions. While much of the regulatory apparatus is downstream of Nodal signaling, as expected from previous observations, there are also cohorts of independently activated oral and aboral ectoderm regulatory genes, and we predict yet unidentified signaling interactions between oral and aboral territories.

The spatially opposed expression of Antidorsalizing morphogenetic protein (Admp) and BMP signals controls dorsoventral (DV) polarity across Bilateria and hence represents an ancient regulatory circuit. Here, we show that in addition to the conserved admp1 that constitutes the ancient circuit, a second admp gene (admp2) is present in Ambulacraria (Echinodermata+Hemichordata) and two marine worms belonging to Xenoturbellida and Acoelomorpha. The phylogenetic distribution implies that the two admp genes were duplicated in the Bilaterian common ancestor and admp2 was subsequently lost in chordates and protostomes. We show that the ambulacrarian admp1 and admp2 are under opposite transcriptional control by BMP signals and knockdown of Admps in sea urchins impaired their DV polarity. Over-expression of either Admps reinforced BMP signaling but resulted in different phenotypes in the sea urchin embryo. Our study provides an excellent example of signaling circuit rewiring and protein functional changes after gene duplications.

Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60–80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67–100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos.

Evolutionary origin of muscle is a central question when discussing mesoderm evolution. Developmental mechanisms underlying somatic muscle development have mostly been studied in vertebrates and fly where multiple signals and hierarchic genetic regulatory cascades selectively specify myoblasts from a pool of naive mesodermal progenitors. However, due to the increased organismic complexity and distant phylogenetic position of the two systems, a general mechanistic understanding of myogenesis is still lacking. In this study, we propose a gene regulatory network (GRN) model that promotes myogenesis in the sea urchin embryo, an early branching deuterostome. A fibroblast growth factor signaling and four Forkhead transcription factors consist the central part of our model and appear to orchestrate the myogenic process. The topological properties of the network reveal dense gene interwiring and a multilevel transcriptional regulation of conserved and novel myogenic genes. Finally, the comparison of the myogenic network architecture among different animal groups highlights the evolutionary plasticity of developmental GRNs. DOI: http://dx.doi.org/10.7554/eLife.07343.001, eLife digest Muscles, bones, and blood vessels all develop from a tissue called the mesoderm, which forms early on in the development of an embryo. Networks of genes control which parts of the mesoderm transform into different cell types. The gene networks that control the development of muscle cells from the mesoderm have so far been investigated in flies and several species of animals with backbones. However, these species are complex, which makes it difficult to work out the general principles that control muscle cell development. Sea urchins are often studied in developmental biology as they have many of the same genes as more complex animals, but are much simpler and easier to study in the laboratory. Andrikou et al. therefore investigated the ‘gene regulatory network’ that controls muscle development in sea urchins. This revealed that proteins called Forkhead transcription factors and a process called FGF signaling are crucial for controlling muscle development in sea urchins. These are also important factors for developing muscles in other animals. Andrikou et al. then produced models that show the interactions between the genes that control muscle formation at three different stages of embryonic development. These models reveal several important features of the muscle development gene regulatory network. For example, the network is robust: if one gene fails, the network is connected in a way that allows it to still make muscle. This also allows the network to adapt and evolve without losing the ability to perform any of its existing roles. Comparing the gene regulatory network that controls muscle development in sea urchins with the networks found in other animals showed that many of the same genes are used across different species, but are connected into different network structures. Investigating the similarities and differences of the regulatory networks in different species could help us to understand how muscles have evolved and could ultimately lead to a better understanding of the causes of developmental diseases. DOI: http://dx.doi.org/10.7554/eLife.07343.002

The indirect-developing enteropneust acorn worm Ptychodera flava has been used as a hemichordate model system for studying the developmental evolution of deuterostome body plans and the origins of chordate characteristics. However, research progress has been hindered by the limited accessibility of its embryonic materials and metamorphosing larvae. In this study, we identified an abundant population of P. flava in Penghu, Taiwan, and examined the feasibility of using this animal for developmental studies. Through histological examination, we established that the reproductive season of this population is between September and December, with a peak breeding period in October and November. In addition, we have developed new procedures that can induce P. flava spawning at any time of the day during the breeding season, with a higher successful rate than that achieved using a previously published method. Moreover, the culturing system we developed enables rearing of P. flava larvae through various planktonic stages and eventual metamorphosis into benthic juveniles, all under laboratory conditions. We anticipate that the animal resources and new technical procedures reported here will further facilitate the use of P. flava as a model organism for evolutionary and developmental biology research.

The stomachs of most vertebrates operate at an acidic pH of 2 generated by the gastric H+/K+-ATPase located in parietal cells. The acidic pH in stomachs of vertebrates is believed to aid digestion and to protect against environmental pathogens. Little attention has been placed on whether acidic gastric pH regulation is a vertebrate character or a deuterostome ancestral trait. Here, we report alkaline conditions up to pH 10.5 in the larval digestive systems of ambulacraria (echinoderm + hemichordate), the closest relative of the chordate. Microelectrode measurements in combination with specific inhibitors for acid-base transporters and ion pumps demonstrated that the gastric alkalization machinery in sea urchin larvae is mainly based on direct H+ secretion from the stomach lumen and involves a conserved set of ion pumps and transporters. Hemichordate larvae additionally utilized HCO3− transport pathways to generate even more alkaline digestive conditions. Molecular analyses in combination with acidification experiments supported these findings and identified genes coding for ion pumps energizing gastric alkalization. Given that insect larval guts were also reported to be alkaline, our discovery raises the hypothesis that the bilaterian ancestor utilized alkaline digestive system while the vertebrate lineage has evolved a strategy to strongly acidify their stomachs.

The metabolism, flagellar beating, and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. As is true of most cells, swimming sperm maintain intracellular Ca 2+ concentrations at submicromolar levels. Here we describe a K + -dependent Na + /Ca 2+ exchanger (suNCKX) from sea urchin sperm. The suNCKX is phylogenetically related to other NCKXs, which use high relative intracellular K + , and high relative extracellular Na + , to couple the efflux of 1 Ca 2+ and 1 K + to the influx of 4 Na + . The 652-aa suNCKX shares structural topology with other NCKX proteins, and has two protein kinase A sites and a His-rich region in its cytoplasmic loop. The suNCKX is encoded by a single gene, which is highly expressed in testes. The suNCKX activity of whole sperm shows Na + and K + dependence, and like other NCKXs can run in reverse exchange mode. An inhibitor blocks the suNCKX activity and sperm motility. suNCKX localizes to the plasma membrane over the sperm flagellum. The suNCKX may play a major role in keeping Ca 2+ low in swimming sperm.

The unusual rate and extent of environmental changes due to human activities may exceed the capacity of marine organisms to deal with this phenomenon. The identification of physiological systems that set the tolerance limits and their potential for phenotypic buffering in the most vulnerable ontogenetic stages become increasingly important to make large-scale projections. Here, we demonstrate that the differential sensitivity of non-calcifying Ambulacraria (echinoderms and hemichordates) larvae towards simulated ocean acidification is dictated by the physiology of their digestive systems. Gastric pH regulation upon experimental ocean acidification was compared in six species of the superphylum Ambulacraria. We observed a strong correlation between sensitivity to ocean acidification and the ability to regulate gut pH. Surprisingly, species with tightly regulated gastric pH were more sensitive to ocean acidification. This study provides evidence that strict maintenance of highly alkaline conditions in the larval gut of Ambulacraria early life stages may dictate their sensitivity to decreases in seawater pH. These findings highlight the importance of identifying and understanding pH regulatory systems in marine larval stages that may contribute to substantial energetic challenges under near-future ocean acidification scenarios.