Chromosome-level genome assemblies of 2 hemichordates provide new insights into deuterostome origin and chromosome evolution.

Deuterostomes are a monophyletic group of animals that includes Hemichordata, Echinodermata (together called Ambulacraria), and Chordata. The diversity of deuterostome body plans has made it challenging to reconstruct their ancestral condition and to decipher the genetic changes that drove the diversification of deuterostome lineages. Here, we generate chromosome-level genome assemblies of 2 hemichordate species, Ptychodera flava and Schizocardium californicum, and use comparative genomic approaches to infer the chromosomal architecture of the deuterostome common ancestor and delineate lineage-specific chromosomal modifications. We show that hemichordate chromosomes (1N = 23) exhibit remarkable chromosome-scale macrosynteny when compared to other deuterostomes and can be derived from 24 deuterostome ancestral linkage groups (ALGs). These deuterostome ALGs in turn match previously inferred bilaterian ALGs, consistent with a relatively short transition from the last common bilaterian ancestor to the origin of deuterostomes. Based on this deuterostome ALG complement, we deduced chromosomal rearrangement events that occurred in different lineages. For example, a fusion-with-mixing event produced an Ambulacraria-specific ALG that subsequently split into 2 chromosomes in extant hemichordates, while this homologous ALG further fused with another chromosome in sea urchins. Orthologous genes distributed in these rearranged chromosomes are enriched for functions in various developmental processes. We found that the deeply conserved Hox clusters are located in highly rearranged chromosomes and that maintenance of the clusters are likely due to lower densities of transposable elements within the clusters. We also provide evidence that the deuterostome-specific pharyngeal gene cluster was established via the combination of 3 pre-assembled microsyntenic blocks. We suggest that since chromosomal rearrangement events and formation of new gene clusters may change the regulatory controls of developmental genes, these events may have contributed to the evolution of diverse body plans among deuterostomes.

Gain of gene regulatory network interconnectivity at the origin of vertebrates.

SignificanceIn this manuscript, we address an essential question in developmental and evolutionary biology: How have changes in gene regulatory networks contributed to the invertebrate-to-vertebrate transition? To address this issue, we perturbed four signaling pathways critical for body plan formation in the cephalochordate amphioxus and in zebrafish and compared the effects of such perturbations on gene expression and gene regulation in both species. Our data reveal that many developmental genes have gained response to these signaling pathways in the vertebrate lineage. Moreover, we show that the interconnectivity between these pathways is much higher in zebrafish than in amphioxus. We conclude that this increased signaling pathway complexity likely contributed to vertebrate morphological novelties during evolution.

Insights into RAG Evolution from the Identification of "Missing Link" Family A RAGL Transposons.

A series of "molecular domestication" events are thought to have converted an invertebrate RAG-like (RAGL) transposase into the RAG1-RAG2 (RAG) recombinase, a critical enzyme for adaptive immunity in jawed vertebrates. The timing and order of these events are not well understood, in part because of a dearth of information regarding the invertebrate RAGL-A transposon family. In contrast to the abundant and divergent RAGL-B transposon family, RAGL-A most closely resembles RAG and is represented by a single orphan RAG1-like (RAG1L) gene in the genome of the hemichordate Ptychodera flava (PflRAG1L-A). Here, we provide evidence for the existence of complete RAGL-A transposons in the genomes of P. flava and several echinoderms. The predicted RAG1L-A and RAG2L-A proteins encoded by these transposons intermingle sequence features of jawed vertebrate RAG and RAGL-B transposases, leading to a prediction of DNA binding, catalytic, and transposition activities that are a hybrid of RAG and RAGL-B. Similarly, the terminal inverted repeats (TIRs) of the RAGL-A transposons combine features of both RAGL-B transposon TIRs and RAG recombination signal sequences. Unlike all previously described RAG2L proteins, RAG2L-A proteins contain an acidic hinge region, which we demonstrate is capable of efficiently inhibiting RAG-mediated transposition. Our findings provide evidence for a critical intermediate in RAG evolution and argue that certain adaptations thought to be specific to jawed vertebrates (e.g. the RAG2 acidic hinge) actually arose in invertebrates, thereby focusing attention on other adaptations as the pivotal steps in the completion of RAG domestication in jawed vertebrates.

Zygotic hypoxia-inducible factor alpha regulates spicule elongation in the sea urchin embryo.

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.

Molecular asymmetry in the cephalochordate embryo revealed by single-blastomere transcriptome profiling.

Studies in various animals have shown that asymmetrically localized maternal transcripts play important roles in axial patterning and cell fate specification in early embryos. However, comprehensive analyses of the maternal transcriptomes with spatial information are scarce and limited to a handful of model organisms. In cephalochordates (amphioxus), an early branching chordate group, maternal transcripts of germline determinants form a compact granule that is inherited by a single blastomere during cleavage stages. Further blastomere separation experiments suggest that other transcripts associated with the granule are likely responsible for organizing the posterior structure in amphioxus; however, the identities of these determinants remain unknown. In this study, we used high-throughput RNA sequencing of separated blastomeres to examine asymmetrically localized transcripts in two-cell and eight-cell stage embryos of the amphioxus Branchiostoma floridae. We identified 111 and 391 differentially enriched transcripts at the 2-cell stage and the 8-cell stage, respectively, and used in situ hybridization to validate the spatial distribution patterns for a subset of these transcripts. The identified transcripts could be categorized into two major groups: (1) vegetal tier/germ granule-enriched and (2) animal tier/anterior-enriched transcripts. Using zebrafish as a surrogate model system, we showed that overexpression of one animal tier/anterior-localized amphioxus transcript, zfp665, causes a dorsalization/anteriorization phenotype in zebrafish embryos by downregulating the expression of the ventral gene, eve1, suggesting a potential function of zfp665 in early axial patterning. Our results provide a global transcriptomic blueprint for early-stage amphioxus embryos. This dataset represents a rich platform to guide future characterization of molecular players in early amphioxus development and to elucidate conservation and divergence of developmental programs during chordate evolution.

BMP controls dorsoventral and neural patterning in indirect-developing hemichordates providing insight into a possible origin of chordates.

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.

Evidence for BMP-mediated specification of primordial germ cells in an indirect-developing hemichordate.

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.

Hemichordate genomes and deuterostome origins.

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.

Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

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 Strongylocentrotus purpuratus, hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia 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 restricts the expression of nodal to the ventral side and activates 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 reveal an unprecedented role of the hypoxia pathway in animal development.

Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava.

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.

Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.

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.

Regulatory circuit rewiring and functional divergence of the duplicate admp genes in dorsoventral axial patterning.

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.

Variability in larval gut pH regulation defines sensitivity to ocean acidification in six species of the Ambulacraria superphylum.

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.

Reproductive periodicity, spawning induction, and larval metamorphosis of the hemichordate acorn worm Ptychodera flava.

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.

Opposing nodal and BMP signals regulate left-right asymmetry in the sea urchin larva.

Nodal and BMP signals are important for establishing left-right (LR) asymmetry in vertebrates. In sea urchins, Nodal signaling prevents the formation of the rudiment on the right side. However, the opposing pathway to Nodal signaling during LR axis establishment is not clear. Here, we revealed that BMP signaling is activated in the left coelomic pouch, specifically in the veg2 lineage, but not in the small micromeres. By perturbing BMP activities, we demonstrated that BMP signaling is required for activating the expression of the left-sided genes and the formation of the left-sided structures. On the other hand, Nodal signals on the right side inhibit BMP signaling and control LR asymmetric separation and apoptosis of the small micromeres. Our findings show that BMP signaling is the positive signal for left-sided development in sea urchins, suggesting that the opposing roles of Nodal and BMP signals in establishing LR asymmetry are conserved in deuterostomes.

Telling left from right: left-right asymmetric controls in sea urchins.

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 TGFß 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.

On a possible evolutionary link of the stomochord of hemichordates to pharyngeal organs of chordates.

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.

Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs, and cell fate lockdown.

The regulation of oral-aboral ectoderm specification in the sea urchin embryo has been extensively studied in recent years. The oral-aboral polarity is initially imposed downstream of a redox gradient induced by asymmetric maternal distribution of mitochondria. Two TGF-ß signaling pathways, Nodal and BMP, are then respectively utilized in the generation of oral and aboral regulatory states. However, a causal understanding of the regulation of aboral ectoderm specification has been lacking. In this work control of aboral ectoderm regulatory state specification was revealed by combining detailed regulatory gene expression studies, perturbation and cis-regulatory analyses. Our analysis illuminates a dynamic system where different factors dominate at different developmental times. We found that the initial activation of aboral genes depends directly on the redox sensitive transcription factor, hypoxia inducible factor 1α (HIF-1α). Two BMP ligands, BMP2/4 and BMP5/8, then significantly enhance aboral regulatory gene transcription. Ultimately, encoded feedback wiring lockdown the aboral ectoderm regulatory state. Our study elucidates the different regulatory mechanisms that sequentially dominate the spatial localization of aboral regulatory states.

Tracing the evolutionary origin of chordate somites in the hemichordate Ptychodera flava.

Metameric somites are a novel character of chordates with unclear evolutionary origins. In the early branching chordate amphioxus, anterior somites are derived from the paraxial mesodermal cells that bud off the archenteron (i.e., enterocoely) at the end of gastrulation. Development of the anterior somites requires FGF signaling, and distinct somite compartments express orthologs of vertebrate non-axial mesodermal markers. Thus, it has been proposed that the amphioxus anterior somites are homologous to the vertebrate head mesoderm, paraxial mesoderm and lateral plate mesoderm. To trace the evolutionary origin of somites, it is essential to study the chordates' closest sister group, Ambulacraria, which includes hemichordates and echinoderms. The anterior coeloms of hemichordate and sea urchin embryos (respectively called protocoel and coelomic pouches) are also formed by enterocoely and require FGF signals for specification and/or differentiation. In this study, we applied RNA-seq to comprehensively screen for regulatory genes associated with the mesoderm-derived protocoel of the hemichordate Ptychodera flava. We also used a candidate gene approach to identify P. flava orthologs of chordate somite markers. In situ hybridization results showed that many of these candidate genes are expressed in distinct or overlapping regions of the protocoel, which indicates that molecular compartments exist in the hemichordate anterior coelom. Given that the hemichordate protocoel and amphioxus anterior somites share a similar ontogenic process (enterocoely), induction signal (FGF), and characteristic expression of orthologous genes, we propose that these two anterior coeloms are indeed homologous. In the lineage leading to the emergence of chordates, somites likely evolved from enterocoelic, FGF-dependent, and molecularly compartmentalized anterior coeloms of the deuterostome last common ancestor.