Transcriptomic evidence for Brachyury expression in the caudal tip region of adult Ptychodera flava (Hemichordata).

Most metazoans have a single copy of the T-box transcription factor gene Brachyury. This gene is expressed in cells of the blastopore of late blastulae and the archenteron invagination region of gastrulae. It appears to be crucial for gastrulation and mesoderm differentiation of embryos. Although this expression pattern is shared by most deuterostomes, Brachyury expression has not been reported in adult stages. Here we show that Brachyury of an indirect developer, the hemichordate acorn worm Ptychodera flava, is expressed not only in embryonic cells, but also in cells of the caudal tip (anus) region of adults. This spatially restricted expression, shown by whole-mount in situ hybridization, was confirmed by Iso-Seq RNA sequencing and single-cell RNA-seq (scRNA-seq) analysis. Iso-Seq analysis showed that gene expression occurs only in the caudal region of adults, but not in anterior regions, including the stomochord. scRNA-seq analysis showed a cluster that contained Brachyury-expressing cells comprising epidermis- and mesoderm-related cells, but which is unlikely to be associated with the nervous system or muscle. Although further investigation is required to examine the roles of Brachyury in adults, this study provides important clues for extending studies on Brachyury expression involved in development of the most posterior region of deuterostomes.

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.

I-SceI Meganuclease-mediated transgenesis in the acorn worm, Saccoglossus kowalevskii.

Hemichordates are a phylum of marine invertebrate deuterostomes that are closely related to chordates, and represent one of the most promising models to provide insights into early deuterostome evolution. The genome of the hemichordate, Saccoglossus kowalevskii, reveals an extensive set of non-coding elements conserved across all three deuterostome phyla. Functional characterization and cross-phyla comparisons of these putative regulatory elements will enable a better understanding of enhancer evolution, and subsequently how changes in gene regulation give rise to morphological innovation. Here, we describe an efficient method of transgenesis for the characterization of non-coding elements in S. kowalevskii. We first test the capacity of an I-SceI transgenesis system to drive ubiquitous or regionalized gene expression, and to label specific cell types. Finally, we identified a minimal promoter that can be used to test the capacity of putative enhancers in S. kowalevskii. This work demonstrates that this I-SceI transgenesis technique, when coupled with an understanding of chromatin accessibility, can be a powerful tool for studying how evolutionary changes in gene regulatory mechanisms contributed to the diversification of body plans in deuterostomes.

Regeneration of the acorn worm pygochord with the implication for its convergent evolution with the notochord.

The origin of the notochord is a central issue in chordate evolution. This study examined the development of the acorn worm pygochord, a putative homologue of the notochord. Because the pygochord differentiates only after metamorphosis, the developmental was followed process by inducing regeneration after artificial amputation in Ptychodera flava. It was found that although the regeneration of the posterior part of the body did not proceed via formation of an obvious regeneration bud, pygochord regeneration was observed within a few weeks, possibly via trans-differentiation of endoderm cells. The expression of the fibrillary collagen gene (Fcol) and elav in the pygochord during regeneration was detected. This indicates that pygochord cells are not part of gut epithelial cells, but that they differentiated as a distinct cell type. Our gene expression analyses do not provide supporting evidence for the homology between the pygochord and notochord, but rather favored the convergent evolution between them.

Anteroposterior molecular registries in ectoderm of the echinus rudiment.

BACKGROUND: Echinoderms and hemichordates are sister taxa that both have larvae with tripartite coeloms. Hemichordates inherit the coelom plan and ectoderm from larvae, whereas echinoderms form the adult rudiment comprising rearranged coeloms and a vestibule that then develops into adult oral ectoderm. Molecular networks that control patterns of the ectoderm and the central nervous system along the anteroposterior (AP) axis are highly conserved between hemichordates and chordates, respectively. In echinoderms, however, little is known about the AP registry in the ectoderm. RESULTS: We isolated ectodermal AP map genes from the sand dollar Peronella japonica and examined their expression. Comparative expression analyses showed that (1) P. japonica orthologs of hemichordate anterior markers are expressed in the larval apical plate, which degenerates during metamorphosis; (2) P. japonica orthologs of the medial markers are expressed in the ambulacral ectoderm of the rudiment; and (3) few P. japonica orthologs of the posterior markers are expressed in ectoderm. CONCLUSIONS: We suggest that echinoids only inherit the ambulacral ectoderm from a common ambulacrarian ancestor, which largely corresponds to the collar ectoderm in hemichordates. The ectodermal AP registry provides insights into the AP axis and evolutionary processes of echinoderms from a common ambulacrarian ancestor. Developmental Dynamics 247:1297-1307, 2018. © 2018 Wiley Periodicals, Inc.

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.

Regeneration in the enteropneust hemichordate, Ptychodera flava, and its evolutionary implications.

Hemichordates are marine invertebrates that are closely related to chordates, but while their body plans are comparable to those of chordates, they possess a remarkable capacity for regeneration, even as adults. A small fragment is sufficient to form a complete individual. Unlike echinoderms, their larvae transform directly into adults; therefore, hemichordate systems offer clear morphological and molecular parallels between regeneration and development. Morphological events in regeneration are generally similar to organogenesis in juveniles. Nonetheless, comparative analysis of gene expression in these two morphological phenomena suggests that hemichordate regeneration is regulated by regeneration-specific mechanisms, as well as by developmental mechanisms. Dependency upon resident pluripotent/multipotent stem cells is a significant difference in metazoan regeneration, and such stem cells are essential for regeneration in many lineages. Based on the present gene expression study, regeneration in acorn worms is more closely related to that in vertebrates, because it employs endogenous stem cell-independent transdifferentiation.

Head regeneration in hemichordates is not a strict recapitulation of development.

BACKGROUND: Head or anterior body part regeneration is commonly associated with protostome, but not deuterostome invertebrates. However, it has been shown that the solitary hemichordate Ptychodera flava possesses the remarkable capacity to regenerate their entire nervous system, including their dorsal neural tube and their anterior head-like structure, or proboscis. Hemichordates, also known as acorn worms, are marine invertebrate deuterostomes that have retained chordate traits that were likely present in the deuterostome ancestor, placing these animals in a vital position to study regeneration and chordate evolution. All acorn worms have a tripartite body plan, with an anterior proboscis, middle collar region, and a posterior trunk. The collar houses a hollow, dorsal neural tube in ptychoderid hemichordates and numerous chordate genes involved in brain and spinal cord development are expressed in a similar anterior-posterior spatial arrangement along the body axis. RESULTS: We have examined anterior regeneration in the hemichordate Ptychodera flava and report the spatial and temporal morphological changes that occur. Additionally, we have sequenced, assembled, and analyzed the transcriptome for eight stages of regenerating P. flava, revealing significant differential gene expression between regenerating and control animals. CONCLUSIONS: Importantly, we have uncovered developmental steps that are regeneration-specific and do not strictly follow the embryonic program. Developmental Dynamics 245:1159-1175, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Spatiotemporal development of the embryonic nervous system of Saccoglossus kowalevskii.

Defining the organization and temporal onset of key steps in neurogenesis in invertebrate deuterostomes is critical to understand the evolution of the bilaterian and deuterostome nervous systems. Although recent studies have revealed the organization of the nervous system in adult hemichordates, little attention has been paid to neurogenesis during embryonic development in this third major phylum of deuterostomes. We examine the early events of neural development in the enteropneust hemichordate Saccoglossus kowalevskii by analyzing the expression of 11 orthologs of key genes associated with neurogenesis in an expansive range of bilaterians. Using in situ hybridization (ISH) and RT-PCR, we follow the course of neural development to track the transition of the early embryonic diffuse nervous system to the more regionalized midline nervous system of the adult. We show that in Saccoglossus, neural progenitor markers are expressed maternally and broadly encircle the developing embryo. An increase in their expression and the onset of pan neural markers, indicate that neural specification occurs in late blastulae - early gastrulae. By mid-gastrulation, punctate expression of markers of differentiating neurons encircling the embryo indicate the presence of immature neurons, and at the end of gastrulation when the embryo begins to elongate, markers of mature neurons are expressed. At this stage, expression of a subset of neuronal markers is concentrated along the trunk ventral and dorsal midlines. These data indicate that the diffuse embryonic nervous system of Saccoglossus is transient and quickly reorganizes before hatching to resemble the adult regionalized, centralized nervous system. This regionalization occurs at a much earlier developmental stage than anticipated indicating that centralization is not linked in S. kowalevskii to a lifestyle change of a swimming larva metamorphosing to a crawling worm-like adult.

Molecular phylogeny of four homeobox genes from the purple sea star Pisaster ochraceus.

Homeobox genes cloned from the purple sea star Pisaster ochraceus (Phylum Echinodermata/Class Asteroidea) were used along with related sequences available from members of other representative animal phyla to generate molecular phylogenies for Distal-less/Dlx, Hox5, Hox7, and Hox9/10 homeobox genes. Phylogenetic relationships were inferred based on the predicted 60 amino acid homeodomain, using amino acid (AA) and nucleotide (NT) models as well as the recently developed codon substitution models of sequence evolution. The resulting phylogenetic trees were mostly congruent with the consensus species-tree, grouping these newly identified genes with those isolated from other Asteroidea. This analysis also allowed a preliminary comparison of the performance of codon models with that of NT and AA evolutionary models in the inference of homeobox phylogeny. We found that, overall, the NT models displayed low reliability in recovering major clades at the Superphylum/Phylum level, and that codon models were slightly more dependable than AA models. Remarkably, in the majority of cases, codon substitution models seemed to outperform both AA and NT models at both the Class level and homeobox paralogy-group level of classification.

Evolution of basal deuterostome nervous systems.

Understanding the evolution of deuterostome nervous systems has been complicated by the by the ambiguous phylogenetic position of the Xenocoelomorpha (Xenoturbellids, acoel flat worms, nemertodermatids), which has been placed either as basal bilaterians, basal deuterostomes or as a sister group to the hemichordate/echinoderm clade (Ambulacraria), which is a sister group of the Chordata. None of these groups has a single longitudinal nerve cord and a brain. A further complication is that echinoderm nerve cords are not likely to be evolutionarily related to the chordate central nervous system. For hemichordates, opinion is divided as to whether either one or none of the two nerve cords is homologous to the chordate nerve cord. In chordates, opposition by two secreted signaling proteins, bone morphogenetic protein (BMP) and Nodal, regulates partitioning of the ectoderm into central and peripheral nervous systems. Similarly, in echinoderm larvae, opposition between BMP and Nodal positions the ciliary band and regulates its extent. The apparent loss of this opposition in hemichordates is, therefore, compatible with the scenario, suggested by Dawydoff over 65 years ago, that a true centralized nervous system was lost in hemichordates.

Hedgehog Expression During Development and Regeneration in the Hemichordate, Ptychodera flava.

Hedgehog is a toolkit gene conserved in metazoans. However, its function differs among taxa, and it shows versatile expression patterns in morphogenesis. We analyzed the expression pattern of hedgehog in the indirect development of the hemichordate, Ptychodera flava, during development and regeneration. Pf-Hh showed distinct enteropneust-specific expression at the anterior tip of the larvae, as well as deuterostome-conserved expression in the pharyngeal endoderm. In contrast, the gene is expressed only in the pharyngeal region during anterior regeneration, but not in the anterior tip of the proboscis. These data suggest that anterior regeneration is driven not only by conserved developmental mechanisms, but also by some regeneration-specific mechanism(s).

A cDNA resource for gene expression studies of a hemichordate, Ptychodera flava.

Recent investigations into the evolution of deuterostomes and the origin of chordates have paid considerable attention to hemichordates (acorn worms), as hemichordates and echinoderms are the closest chordate relatives. The present study prepared cDNA libraries from Ptychodera flava, to study expression and function of genes involved in development of the hemichordate body plan. Expressed sequence tag (EST) analyses of nine cDNA libraries yielded 18,832 cloned genes expressed in eggs, 18,739 in blastulae, 18,539 in gastrulae, 18,811 in larvae, 18,978 in juveniles, 11,802 in adult proboscis, 17,259 in stomochord, 11,886 in gills, and 11,580 in liver, respectively. A set of 34,159 uni-gene clones of P. flava was obtained. This cDNA resource will be valuable for studying temporal and spatial expression of acorn worm genes during development.

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.

Anterior-posterior Wnt signaling network conservation between indirect developing sea urchin and hemichordate embryos.

How animal body plans evolved and diversified is a major question in evolutionary developmental biology. To address this question, it is important to characterize the exact molecular mechanisms that establish the major embryonic axes which give rise to the adult animal body plan. The anterior-posterior (AP) axis is the first axis to be established in most animal embryos, and in echinoderm sea urchin embryos its formation is governed by an integrated network of three different Wnt signaling pathways: Wnt/ß-catenin, Wnt/JNK, and Wnt/PKC pathway. The extent to which this embryonic patterning mechanism is conserved among deuterostomes, or more broadly in metazoans, is an important open question whose answers could lead to a deeper appreciation of the evolution of the AP axis. Because Ambulacrarians (echinoderms and hemichordates) reside in a key phylogenetic position as the sister group to chordates, studies in these animals can help inform on how chordate body plans may have evolved. Here, we assayed the spatiotemporal gene expression of a subset of sea urchin AP Wnt patterning gene orthologs in the hemichordate, Schizocardium californicum. Our results show that positioning of the anterior neuroectoderm (ANE) to a territory around the anterior pole during early AP formation is spatially and temporally similar between indirect developing hemichordates and sea urchins. Furthermore, we show that the expression of wnt8 and frizzled5/8, two known drivers of ANE patterning in sea urchins, is similar in hemichordate embryos. Lastly, our results highlight divergence in embryonic expression of several early expressed Wnt genes (wnt1, wnt2 and wnt4). These results suggest that expression of the sea urchin AP Wnt signaling network is largely conserved in indirect developing hemichordates setting the foundation for future functional studies in S. californicum.

A Silurian pseudocolonial pterobranch.

Pterobranchs, a major group of the phylum Hemichordata, first appear in the fossil record during the Cambrian,1 and there are more than 600 fossil genera dominated by the mainly planktic graptolites of the Paleozoic, which are widely used as zone fossils for correlating sedimentary rock sequences.2 Pterobranchs are rare today; they are sessile marine forms represented by Rhabdopleura, which is considered the only living graptolite, and Cephalodiscus. Unlike their sister taxon, the colonial graptolites, cephalodiscids are pseudocolonial.3,4 Here, we describe a problematic fossil from the Silurian (Pridoli) Bertie Group of Ontario (420 mya), a sequence of near-shore sediments well known for its remarkably preserved diversity of eurypterids (sea scorpions).5 The fossil, Rotaciurca superbus, a new genus and species, was familiarly known as Ezekiel's Wheel,5 with reference to the unusual circular arrangement of the tubes that compose it. The structure and arrangement of the tubes identify Rotaciurca as a pterobranch, and phylogenetic analysis groups it with the cephalodiscids. We place it in a new family Rotaciurcidae to distinguish it from Cephalodiscidae. A large structure associated with the tubes is interpreted as a float, which would distinguish Rotaciurca as the only known planktic cephalodiscid-thus cephalodiscids, like the graptolites, invaded the water column. This mode of life reflects the rarity of pseudocolonial macroinvertebrates in planktic ocean communities, a role occupied by the tunicates (Chordata) known as salps today. Our estimates of divergence times, the first using relaxed total-evidence clocks, date the origins of both hemichordates and pterobranchs to the earliest Cambrian (Fortunian).

Peripheral and central employment of acid-sensing ion channels during early bilaterian evolution.

Nervous systems are endowed with rapid chemosensation and intercellular signaling by ligand-gated ion channels (LGICs). While a complex, bilaterally symmetrical nervous system is a major innovation of bilaterian animals, the employment of specific LGICs during early bilaterian evolution is poorly understood. We therefore questioned bilaterian animals' employment of acid-sensing ion channels (ASICs), LGICs that mediate fast excitatory responses to decreases in extracellular pH in vertebrate neurons. Our phylogenetic analysis identified an earlier emergence of ASICs from the overarching DEG/ENaC (degenerin/epithelial sodium channel) superfamily than previously thought and suggests that ASICs were a bilaterian innovation. Our broad examination of ASIC gene expression and biophysical function in each major bilaterian lineage of Xenacoelomorpha, Protostomia, and Deuterostomia suggests that the earliest bilaterian ASICs were probably expressed in the periphery, before being incorporated into the brain as it emerged independently in certain deuterostomes and xenacoelomorphs. The loss of certain peripheral cells from Ecdysozoa after they separated from other protostomes likely explains their loss of ASICs, and thus the absence of ASICs from model organisms Drosophila and Caenorhabditis elegans. Thus, our use of diverse bilaterians in the investigation of LGIC expression and function offers a unique hypothesis on the employment of LGICs in early bilaterian evolution.

Most animals on Earth, from worms to chimpanzees, belong to a group known as the bilaterians. Despite their rich variety of shapes and lifestyles, all these creatures share similarities ­ in particular, a complex nervous system where neurons can quickly relay electric signals. This is made possible by a class of proteins, known as ligand-gated ion channels, which are studded through the membrane of cells. There, they help neurons efficiently communicate with each other by converting external chemical information into internal electrical signals. Yet despite their importance, how and when these proteins have evolved remains poorly understood. Marti-Solans et al. decided to explore this question by focusing on acid-sensing ion channels, a family which often forms the linchpin of bilaterian neural networks. They examined when these proteins first evolved (that is, in which putative ancestral animals) and where in the body. To do so, they combed through genetic data from all major bilaterian lineages as well as from non-biletarian groups; this included previously unexplored datasets that give insight into the type of cells in which a particular gene is active. The analyses revealed that the channels are specific to bilaterians, but that they appeared earlier than previously thought, being present in the very first members of this group. However, at this stage, the proteins were mainly located in cells at the periphery of the body rather than in those from emerging neural circuits. This suggests that the channels were co-opted by nerve cells later on, when the nervous systems became more complex. The proteins being initially located in cells at the outer edge of the body could also explain why they are absent in bilaterian creatures such as fruit flies and nematode worms; these animals all belong to a lineage where growth takes place by shedding their external layers. Acid-sensing ion channels are an important group of potential drug targets, often being implicated in pain and diseases of the nervous system. The work of Marti-Solans et al. offers an insight into the diversity of roles these proteins can play in the body, demonstrating once again how evolution can repurpose the same biophysical functions to serve a range of needs inside an organism.

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum.

BACKGROUND: There are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79-88, 2012) to answer these questions. RESULTS: We identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis. CONCLUSIONS: Cell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.

Saccoglossus kowalevskii: Evo-devo insights from the mud.

Hemichordates have long been recognized as a critical group for addressing hypotheses of chordate origins. Historically this was due to anatomical traits that resembled those of chordates, most strikingly the dorsolateral gill slits. As molecular data and phylogenetic analyses were found to support a close phylogenetic relationship between hemichordates and chordates within the deuterostomes, interest was revived in hemichordates. In particular, Saccoglossus kowalevskii has been developed as a molecular model to represent hemichordate developmental biology. Herein, we highlight the considerations when choosing a particular species to study and the challenges we encountered when developing S. kowalevskii. We discuss our findings and how method and tool development enabled them, and how we envision expanding our repertoire of molecular tools in the future. Establishing a new model organism comes with many obstacles-from identifying a reliable season to collect animals, to developing modern molecular techniques. The Saccoglossus research community has benefited greatly from the collaborations and teamwork established over the years. As a result, Saccoglossus is well positioned to contribute to a new century of evolutionary developmental (evo-devo) research.

Molecular insights into deuterostome evolution from hemichordate developmental biology.

Hemichordates, along with echinoderms and chordates, belong to the lineage of bilaterians called the deuterostomes. Their phylogenetic position as an outgroup to chordates provides an opportunity to investigate the evolutionary origins of the chordate body plan and reconstruct ancestral deuterostome characters. The body plans of the hemichordates and chordates are organizationally divergent making anatomical comparisons very challenging. The developmental underpinnings of animal body plans are often more conservative than the body plans they regulate, and offer a novel data set for making comparisons between morphologically divergent body architectures. Here I review the hemichordate developmental data generated over the past 20 years that further test hypotheses of proposed morphological affinities between the two taxa, but also compare the conserved anteroposterior, dorsoventral axial patterning programs and germ layer specification programs. These data provide an opportunity to determine which developmental programs are ancestral deuterostome or bilaterian innovations, and which ones occurred in stem chordates or vertebrates representing developmental novelties of the chordate body plan.