Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.

Molecular regionalization of the developing amphioxus neural tube challenges major partitions of the vertebrate brain.

All vertebrate brains develop following a common Bauplan defined by anteroposterior (AP) and dorsoventral (DV) subdivisions, characterized by largely conserved differential expression of gene markers. However, it is still unclear how this Bauplan originated during evolution. We studied the relative expression of 48 genes with key roles in vertebrate neural patterning in a representative amphioxus embryonic stage. Unlike nonchordates, amphioxus develops its central nervous system (CNS) from a neural plate that is homologous to that of vertebrates, allowing direct topological comparisons. The resulting genoarchitectonic model revealed that the amphioxus incipient neural tube is unexpectedly complex, consisting of several AP and DV molecular partitions. Strikingly, comparison with vertebrates indicates that the vertebrate thalamus, pretectum, and midbrain domains jointly correspond to a single amphioxus region, which we termed Di-Mesencephalic primordium (DiMes). This suggests that these domains have a common developmental and evolutionary origin, as supported by functional experiments manipulating secondary organizers in zebrafish and mice.

The evolution of ependymin-related proteins.

BACKGROUND: Ependymins were originally defined as fish-specific secreted glycoproteins involved in central nervous system plasticity and memory formation. Subsequent research revealed that these proteins represent a fish-specific lineage of a larger ependymin-related protein family (EPDRs). EPDRs have now been identified in a number of bilaterian animals and have been implicated in diverse non-neural functions. The recent discoveries of putative EPDRs in unicellular holozoans and an expanded EPDR family with potential roles in conspecific communication in crown-of-thorns starfish suggest that the distribution and diversity of EPDRs is significantly broader than currently understood. RESULTS: We undertook a systematic survey to determine the distribution and evolution of EPDRs in eukaryotes. In addition to Bilateria, EPDR genes were identified in Cnidaria, Placozoa, Porifera, Choanoflagellatea, Filasterea, Apusozoa, Amoebozoa, Charophyta and Percolozoa, and tentatively in Cercozoa and the orphan group Malawimonadidae. EPDRs appear to be absent from prokaryotes and many eukaryote groups including ecdysozoans, fungi, stramenopiles, alveolates, haptistans and cryptistans. The EPDR family can be divided into two major clades and has undergone lineage-specific expansions in a number of metazoan lineages, including in poriferans, molluscs and cephalochordates. Variation in a core set of conserved residues in EPDRs reveals the presence of three distinct protein types; however, 3D modelling predicts overall protein structures to be similar. CONCLUSIONS: Our results reveal an early eukaryotic origin of the EPDR gene family and a dynamic pattern of gene duplication and gene loss in animals. This research provides a phylogenetic framework for the analysis of the functional evolution of this gene family.

Vertebrate-like regeneration in the invertebrate chordate amphioxus.

An important question in biology is why some animals are able to regenerate, whereas others are not. The basal chordate amphioxus is uniquely positioned to address the evolution of regeneration. We report here the high regeneration potential of the European amphioxus Branchiostoma lanceolatum. Adults regenerate both anterior and posterior structures, including neural tube, notochord, fin, and muscle. Development of a classifier based on tail regeneration profiles predicts the assignment of young and old adults to their own class with >94% accuracy. The process involves loss of differentiated characteristics, formation of an msx-expressing blastema, and neurogenesis. Moreover, regeneration is linked to the activation of satellite-like Pax3/7 progenitor cells, the extent of which declines with size and age. Our results provide a framework for understanding the evolution and diversity of regeneration mechanisms in vertebrates.

The evolutionary origins of chordate hematopoiesis and vertebrate endothelia.

The vertebrate circulatory system is the most complex vascular system among those of metazoans, with key innovations including a multi-chambered heart and highly specialized blood cells. Invertebrate vessels, on the other hand, consist of hemal spaces between the basal laminae of epithelia. How the evolutionary transition from an invertebrate-type system to the complex vertebrate one occurred is, however, poorly understood. We investigate here the development of the cardiovascular system of the cephalochordate amphioxus Branchiostoma lanceolatum in order to gain insight into the origin of the vertebrate cardiovascular system. The cardiac markers Hand, Csx (Nkx2-5) and Tbx4/5 reveal a broad cardiac-like domain in amphioxus; such a decentralized organization during development parallels that seen in the adult anatomy. Our data therefore support the hypothesis that amphioxus never possessed a proper heart, even transiently during development. We also define a putative hematopoietic domain, supported by the expression of the hematopoietic markers Scl and Pdvegfr. We show that this area is closed to the dorsal aorta anlages, partially linked to excretory tissues, and that its development is regulated by retinoic acid, thus recalling the aorta-gonads-mesonephros (AGM) area of vertebrates. This region probably produces Pdvegfr+ hemal cells, with an important role in amphioxus vessel formation, since treatments with an inhibitor of PDGFR/VEGFR lead to a decrease of Laminin in the basal laminae of developing vessels. Our results point to a chordate origin of hematopoiesis in an AGM-like area from where hemal Pdvegfr+ cells are produced. These Pdvegfr+ cells probably resemble the ancestral chordate blood cells from which the vertebrate endothelium later originated.

Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits.

FGF signaling is one of the few cell-cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

Stepwise assembly of the Nova-regulated alternative splicing network in the vertebrate brain.

Novel organismal structures in metazoans are often undergirded by complex gene regulatory networks; as such, understanding the emergence of new structures through evolution requires reconstructing the series of evolutionary steps leading to these underlying networks. Here, we reconstruct the step-by-step assembly of the vertebrate splicing network regulated by Nova, a splicing factor that modulates alternative splicing in the vertebrate central nervous system by binding to clusters of YCAY motifs on pre-RNA transcripts. Transfection of human HEK293T cells with Nova orthologs indicated vertebrate-like splicing regulatory activity in bilaterian invertebrates, thus Nova acquired the ability to bind YCAY clusters and perform vertebrate-like splicing modulation at least before the last common ancestor of bilaterians. In situ hybridization studies in several species showed that Nova expression became restricted to CNS later on, during chordate evolution. Finally, comparative genomics studies revealed a diverse history for Nova-regulated exons, with target exons arising through both de novo exon creation and acquisition of YCAY motifs by preexisting exons throughout chordate and vertebrate history. In addition, we find that tissue-specific Nova expression patterns emerged independently in other lineages, suggesting independent assembly of tissue-specific regulatory networks.

D-Aspartic acid is a novel endogenous neurotransmitter.

D-aspartic acid (D-Asp) is present in invertebrate and vertebrate neuroendocrine tissues, where it carries out important physiological functions and is implicated in nervous system development. We show here that D-Asp is a novel endogenous neurotransmitter in two distantly related animals, a mammal (Rattus norvegicus) and a mollusk (Loligo vulgaris). Our main findings demonstrate that D-Asp is present in high concentrations in the synaptic vesicles of axon terminals; synthesis for this amino acid occurs in neurons by conversion of L-Asp to D-Asp via D-aspartate racemase; depolarization of nerve endings with K(+) ions evokes an immediate release of D-Asp in a Ca(2+) dependent manner; specific receptors for D-Asp occur at the postsynaptic membrane, as demonstrated by binding assays and by the expansion of squid skin chromatophores; D-aspartate oxidase, the specific enzyme that oxidizes D-Asp, is present in the postsynaptic membranes; and stimulation of nerve endings with D-Asp triggers signal transduction by increasing the second messenger cAMP. Taken together, these data demonstrate that D-Asp fulfills all criteria necessary to be considered a novel endogenous neurotransmitter. Given its known role in neurogenesis, learning, and neuropathologies, our results have important implications for biomedical and clinical research.

Three-dimensional fine structure of fibroblasts and other mesodermally derived tissues in the dermis of adults of the Bahamas lancelet (Chordata, Cephalohordata), as seen by serial block-face scanning electron microscopy.

Tissues of adult cephalochordates include sparsely distributed fibroblasts. Previous work on these cells has left unsettled such questions as their developmental origin, range of functions, and even their overall shape. Here, we describe fibroblasts of a cephalochordate, the Bahamas lancelet, Asymmetron lucayanum, by serial block-face scanning electron microscopy to demonstrate their three-dimensional (3D) distribution and fine structure in a 0.56-mm length of the tail. The technique reveals in detail their position, abundance, and morphology. In the region studied, we found only 20 fibroblasts, well separated from one another. Each was strikingly stellate with long cytoplasmic processes rather similar to those of a vertebrate telocyte, a possibly fortuitous resemblance that is considered in the discussion section. In the cephalochordate dermis, the fibroblasts were never linked with one another, although they occasionally formed close associations of unknown significance with other cell types. The fibroblasts, in spite of their name, showed no signs of directly synthesizing fibrillar collagen. Instead, they appeared to be involved in the production of nonfibrous components of the extracellular matrix-both by the release of coarsely granular dense material and by secretion of more finely granular material by the local breakdown of their cytoplasmic processes. For context, the 3D structures of two other mesoderm-derived tissues (the midline mesoderm and the posteriormost somite) are also described for the region studied.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

Adult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long-lived, lineage-restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ-restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by 'stemness' gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ-cell markers, but often lack germ-line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole-body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the 'wobbling Penrose' landscape. Here, totipotent ASCs adopt ascending/descending courses of an 'Escherian stairwell', in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Tail regeneration in a cephalochordate, the Bahamas lancelet, Asymmetron lucayanum.

Lancelets (Phylum Chordata, subphylum Cephalochordata) readily regenerate a lost tail. Here, we use light microscopy and serial blockface scanning electron microscopy (SBSEM) to describe tail replacement in the Bahamas lancelet, Asymmetron lucayanum. One day after amputation, the monolayered epidermis has migrated over the wound surface. At 4 days, the regenerate is about 3% as long as the tail length removed. The re-growing nerve cord is a tubular outgrowth of ependymal cells, and the new part of the notochord consists of several degenerating lamellar cells anterior to numerous small vacuolated cells. The cut edges of the mesothelium project into the regenerate as tubular extensions. These tubes anastomose with each other and with midline mesodermal canals beneath the regenerating edges of the dorsal and ventral fins. SBSEM did not reveal a blastema-like aggregation of undifferentiated cells anywhere in the regenerate. At 6 days, the regenerate (10% of the amputated tail length) includes a notochord in which the small vacuolated cells mentioned above are differentiating into lamellar cells. At 10 days, the regenerate is 22% of the amputated tail length: myocytes have appeared in the walls of the myomeres, and sclerocoels have formed. By 14 days, the regenerate is 35% the length of the amputated tail, and the new tissues resemble smaller versions of those originally lost. The present results for A. lucayanum, a species regenerating quickly and with little inter-specimen variability, provide the morphological background for future cell-tracer, molecular genetic, and genomic studies of cephalochordate regeneration.

JNK Mediates Differentiation, Cell Polarity and Apoptosis During Amphioxus Development by Regulating Actin Cytoskeleton Dynamics and ERK Signalling.

c-Jun N-terminal kinase (JNK) is a multi-functional protein involved in a diverse array of context-dependent processes, including apoptosis, cell cycle regulation, adhesion, and differentiation. It is integral to several signalling cascades, notably downstream of non-canonical Wnt and mitogen activated protein kinase (MAPK) signalling pathways. As such, it is a key regulator of cellular behaviour and patterning during embryonic development across the animal kingdom. The cephalochordate amphioxus is an invertebrate chordate model system straddling the invertebrate to vertebrate transition and is thus ideally suited for comparative studies of morphogenesis. However, next to nothing is known about JNK signalling or cellular processes in this lineage. Pharmacological inhibition of JNK signalling using SP600125 during embryonic development arrests gastrula invagination and causes convergence extension-like defects in axial elongation, particularly of the notochord. Pharynx formation and anterior oral mesoderm derivatives like the preoral pit are also affected. This is accompanied by tissue-specific transcriptional changes, including reduced expression of six3/6 and wnt2 in the notochord, and ectopic wnt11 in neurulating embryos treated at late gastrula stages. Cellular delamination results in accumulation of cells in the gut cavity and a dorsal fin-like protrusion, followed by secondary Caspase-3-mediated apoptosis of polarity-deficient cells, a phenotype only partly rescued by co-culture with the pan-Caspase inhibitor Z-VAD-fmk. Ectopic activation of extracellular signal regulated kinase (ERK) signalling in the neighbours of extruded notochord and neural cells, possibly due to altered adhesive and tensile properties, as well as defects in cellular migration, may explain some phenotypes caused by JNK inhibition. Overall, this study supports conserved functions of JNK signalling in mediating the complex balance between cell survival, apoptosis, differentiation, and cell fate specification during cephalochordate morphogenesis.

Serial blockface SEM suggests that stem cells may participate in adult notochord growth in an invertebrate chordate, the Bahamas lancelet.

BACKGROUND: The cellular basis of adult growth in cephalochordates (lancelets or amphioxus) has received little attention. Lancelets and their constituent organs grow slowly but continuously during adult life. Here, we consider whether this slow organ growth involves tissue-specific stem cells. Specifically, we focus on the cell populations in the notochord of an adult lancelet and use serial blockface scanning electron microscopy (SBSEM) to reconstruct the three-dimensional fine structure of all the cells in a tissue volume considerably larger than normally imaged with this technique. RESULTS: In the notochordal region studied, we identified 10 cells with stem cell-like morphology at the posterior tip of the organ, 160 progenitor (Müller) cells arranged along its surface, and 385 highly differentiated lamellar cells constituting its core. Each cell type could clearly be distinguished on the basis of cytoplasmic density and overall cell shape. Moreover, because of the large sample size, transitions between cell types were obvious. CONCLUSIONS: For the notochord of adult lancelets, a reasonable interpretation of our data indicates growth of the organ is based on stem cells that self-renew and also give rise to progenitor cells that, in turn, differentiate into lamellar cells. Our discussion compares the cellular basis of adult notochord growth among chordates in general. In the vertebrates, several studies implied that proliferating cells (chordoblasts) in the cortex of the organ might be stem cells. However, we think it is more likely that such cells actually constitute a progenitor population downstream from and maintained by inconspicuous stem cells. We venture to suggest that careful searches should find stem cells in the adult notochords of many vertebrates, although possibly not in the notochordal vestiges (nucleus pulposus regions) of mammals, where the presence of endogenous proliferating cells remains controversial.

The sensory peripheral nervous system in the tail of a cephalochordate studied by serial blockface scanning electron microscopy.

Serial blockface scanning electron microscopy (SBSEM) is used to describe the sensory peripheral nervous system (PNS) in the tail of a cephalochordate, Asymmetron lucayanum. The reconstructed region extends from the tail tip to the origin of the most posterior peripheral nerves from the dorsal nerve cord. As peripheral nerves ramify within the dermis, all the nuclei along their course belong to glial cells. Invaginations in the glial cell cytoplasm house the neurites, an association reminiscent of the nonmyelinated Schwann cells of vertebrates. Peripheral nerves pass from the dermis to the epidermis via small fenestrae in the sub-epidermal collagen fibril layer; most nerves exit abruptly, but a few run obliquely within the collagen fibril layer for many micrometers before exiting. Within the epidermis, each nerve begins ramifying repeatedly, but the branches are too small to be followed to their tips with SBSEM at low magnification (previous studies on other cephalochordates indicate that the branches end freely or in association with epidermal sensory cells). In Asymmetron, two morphological kinds of sensory cells are scattered in the epidermis, usually singly, but sometimes in pairs, evidently the recent progeny of a single precursor cell. The discussion considers the evolution of the sensory PNS in the phylum Chordata. In cephalochordates, Retzius bipolar neurons with intramedullary perikarya likely correspond to the Rohon-Beard cells of vertebrates. However, extramedullary neurons originating from ventral epidermis in cephalochordates (and presumably in ancestral chordates) contrast with vertebrate sensory neurons, which arise from placodes and neural crest.

Beyond Adult Stem Cells: Dedifferentiation as a Unifying Mechanism Underlying Regeneration in Invertebrate Deuterostomes.

The diversity of regenerative phenomena seen in adult metazoans, as well as their underlying mechanistic bases, are still far from being comprehensively understood. Reviewing both ultrastructural and molecular data, the present work aims to showcase the increasing relevance of invertebrate deuterostomes, i.e., echinoderms, hemichordates, cephalochordates and tunicates, as invaluable models to study cellular aspects of adult regeneration. Our comparative approach suggests a fundamental contribution of local dedifferentiation -rather than mobilization of resident undifferentiated stem cells- as an important cellular mechanism contributing to regeneration in these groups. Thus, elucidating the cellular origins, recruitment and fate of cells, as well as the molecular signals underpinning tissue regrowth in regeneration-competent deuterostomes, will provide the foundation for future research in tackling the relatively limited regenerative abilities of vertebrates, with clear applications in regenerative medicine.

FGFRL1 is a neglected putative actor of the FGF signalling pathway present in all major metazoan phyla.

BACKGROUND: Fibroblast Growth Factors (FGF) and their receptors are well known for having major implications in cell signalling controlling embryonic development. Recently, a gene coding for a protein closely related to FGFRs (Fibroblast Growth Factor Receptors) called FGFR5 or FGFR-like 1 (FGFRL1), has been described in vertebrates. An orthologous gene was also found in the cephalochordate amphioxus, but no orthologous genes were found by the authors in other non-vertebrate species, even if a FGFRL1 gene was identified in the sea urchin genome, as well as a closely related gene, named nou-darake, in the planarian Dugesia japonica. These intriguing data of a deuterostome-specific gene that might be implicated in FGF signalling prompted us to search for putative FGFRL1 orthologues in the completely sequenced genomes of metazoans. RESULTS: We found FGFRL1 genes in the cnidarian Nematostella vectensis as well as in many bilaterian species. Our analysis also shows that FGFRL1 orthologous genes are linked in the genome with other members of the FGF signalling pathway from cnidarians to bilaterians (distance < 10 Mb). To better understand the implication of FGFRL1 genes in chordate embryonic development, we have analyzed expression patterns of the amphioxus and the mouse genes by whole mount in situ hybridization. We show that some homologous expression territories can be defined, and we propose that FGFRL1 and FGF8/17/18 were already co-expressed in the pharyngeal endoderm in the ancestor of chordates. CONCLUSION: Our work sheds light on the existence of a putative FGF signalling pathway actor present in the ancestor of probably all metazoans, the function of which has received little attention until now.

Light sheet microscopy with acoustic sample confinement.

Contactless sample confinement would enable a whole host of new studies in developmental biology and neuroscience, in particular, when combined with long-term, wide-field optical imaging. To achieve this goal, we demonstrate a contactless acoustic gradient force trap for sample confinement in light sheet microscopy. Our approach allows the integration of real-time environmentally controlled experiments with wide-field low photo-toxic imaging, which we demonstrate on a variety of marine animal embryos and larvae. To illustrate the key advantages of our approach, we provide quantitative data for the dynamic response of the heartbeat of zebrafish larvae to verapamil and norepinephrine, which are known to affect cardiovascular function. Optical flow analysis allows us to explore the cardiac cycle of the zebrafish and determine the changes in contractile volume within the heart. Overcoming the restrictions of sample immobilisation and mounting can open up a broad range of studies, with real-time drug-based assays and biomechanical analyses.

Evidence for stasis and not genetic piracy in developmental expression patterns of Branchiostoma lanceolatum and Branchiostoma floridae, two amphioxus species that have evolved independently over the course of 200 Myr.

Cephalochordates, the most basal extant group in the phylum Chordata, are represented chiefly by about 20 species of the genus Branchiostoma, commonly called amphioxus or lancelets. In recent years, insights into the evolutionary origin of the vertebrates have been gained from molecular genetic studies during the development of three of these amphioxus species (Branchiostoma floridae in North America, Branchiostoma lanceolatum in Europe, and Branchiostoma belcheri in East Asia). In spite of an estimated divergence time of 100-200 Myr among these species, all three are remarkably similar morphologically, and students of amphioxus have tacitly assumed that such resemblances arise during ontogeny from nearly identical networks of developmental genes. We felt that this assumption needed to be reexamined because instances are known--even in comparisons of closely related species--where characters seeming homologous on the basis of morphology actually develop under the control of conspicuously divergent genetic programs (a phenomenon termed "genetic piracy"). In the present work, we tested the hypothesis that morphological similarities reflect strict conservation of developmentally important genes' expression patterns in order to assess whether the developmental genetics of different amphioxus species show evidence of genetic piracy. To these ends, we cloned 18 genes implicated in different developmental functions in B. lanceolatum and compared their gene expression patterns with the known expression patterns of their orthologous genes in B. floridae. We show that, for the most part, conservation of gene expression parallels that of morphology in these two species. We also identified some differences in gene expression, likely reflecting experimental sensitivity, with the exception of Pax1/9, which may result from true developmental specificities in each amphioxus species. Our results demonstrate that morphological conservation reflects stasis in developmental gene expression patterns and find no evidence for genetic piracy. Thus, different species of amphioxus appear to be very similar, not only morphologically, but also in the genetic programs directing the development of their structural features. Moreover, we provide the first catalogue of gene expression data for the European species, B. lanceolatum.

Pax3/7 duplicated and diverged independently in amphioxus, the basal chordate lineage.

The Pax3/7 transcription factor family is integral to developmental gene networks contributing to important innovations in vertebrate evolution, including the neural crest. The basal chordate lineage of amphioxus is ideally placed to understand the dynamics of the gene regulatory network evolution that produced these novelties. We report here the discovery that the cephalochordate lineage possesses two Pax3/7 genes, Pax3/7a and Pax3/7b. The tandem duplication is ancestral to all extant amphioxus, occurring in both Asymmetron and Branchiostoma, but originated after the split from the lineage leading to vertebrates. The two paralogues are differentially expressed during embryonic development, particularly in neural and somitic tissues, suggesting distinct regulation. Our results have implications for the study of amphioxus regeneration, neural plate and crest evolution, and differential tandem paralogue evolution.

A Revised Spiralian Homeobox Gene Classification Incorporating New Polychaete Transcriptomes Reveals a Diverse TALE Class and a Divergent Hox Gene.

The diversity of mechanisms and capacity for regeneration across the Metazoa present an intriguing challenge in evolutionary biology, impacting on the burgeoning field of regenerative medicine. Broad taxonomic sampling is essential to improve our understanding of regeneration, and studies outside of the traditional model organisms have proved extremely informative. Within the historically understudied Spiralia, the Annelida have an impressive variety of tractable regenerative systems. The biomeralizing, blastema-less regeneration of the head appendage (operculum) of the serpulid polychaete keelworm Spirobranchus (formerly Pomatoceros) lamarcki is one such system. To profile potential regulatory mechanisms, we classified the homeobox gene content of opercular regeneration transcriptomes. As a result of retrieving several difficult-to-classify homeobox sequences, we performed an extensive search and phylogenetic analysis of the TALE and PRD-class homeobox gene content of a broad selection of lophotrochozoan genomes. These analyses contribute to our increasing understanding of the diversity, taxonomic extent, rapid evolution, and radical flexibility of these recently discovered homeobox gene radiations. Our expansion and integration of previous nomenclature systems helps to clarify their cryptic orthology. We also describe an unusual divergent S. lamarcki Antp gene, a previously unclassified lophotrochozoan orphan gene family (Lopx), and a number of novel Nk class orphan genes. The expression and potential involvement of many of these lineage- and clade-restricted homeobox genes in S. lamarcki operculum regeneration provides an example of diversity in regenerative mechanisms, as well as significantly improving our understanding of homeobox gene evolution.