Cephalochordate Hemocytes: First Demonstration for Asymmetron lucayanum (Bahamas Lancelet) Plus Augmented Description for Branchiostoma floridae (Florida Amphioxus).

AbstractWithin phylum Chordata, the subphylum Cephalochordata (amphioxus and lancelets) has figured large in considerations of the evolutionary origin of the vertebrates. To date, these discussions have been predominantly based on knowledge of a single cephalochordate genus (Branchiostoma), almost to the exclusion of the other two genera (Asymmetron and Epigonichthys). This uneven pattern is illustrated by cephalochordate hematology, until now known entirely from work done on Branchiostoma. The main part of the present study is to describe hemocytes in the dorsal aorta of a species of Asymmetron by serial block-face scanning electron microscopy. This technique, which demonstrates three-dimensional fine structure, showed that the hemocytes have a relatively uniform morphology characterized by an oval shape and scanty cytoplasm. Ancillary information is also included for Branchiostoma hemocytes, known from previous studies to have relatively abundant cytoplasm; our serial block-face scanning electron microscopy provides more comprehensive views of the highly variable shapes of these cells, which typically extend one or several pseudopodium-like protrusions. The marked difference in hemocyte morphology found between Asymmetron and Branchiostoma was unexpected and directs attention to investigating comparable cells in the genus Epigonichthys. A broader knowledge of the hemocytes in all three cephalochordate genera would provide more balanced insights into the evolution of vertebrate hematopoiesis.

Evolutionary Adaptation of the Thyroid Hormone Signaling Toolkit in Chordates.

The specification of the endostyle in non-vertebrate chordates and of the thyroid gland in vertebrates are fundamental steps in the evolution of the thyroid hormone (TH) signaling to coordinate development and body physiology in response to a range of environmental signals. The physiology and biology of TH signaling in vertebrates have been studied in the past, but a complete understanding of such a complex system is still lacking. Non-model species from non-vertebrate chordates may greatly improve our understanding of the evolution of this complex endocrine pathway. Adaptation of already existing proteins in order to perform new roles is a common feature observed during the course of evolution. Through sequence similarity approaches, we investigated the presence of bona fide thyroid peroxidase (TPO), iodothyronine deiodinase (DIO), and thyroid hormone receptors (THRs) in non-vertebrate and vertebrate chordates. Additionally, we determined both the conservation and divergence degrees of functional domains at the protein level. This study supports the hypothesis that non-vertebrate chordates have a functional thyroid hormone signaling system and provides additional information about its possible evolutionary adaptation.

MeDAS: a Metazoan Developmental Alternative Splicing database.

Alternative splicing is widespread throughout eukaryotic genomes and greatly increases transcriptomic diversity. Many alternative isoforms have functional roles in developmental processes and are precisely temporally regulated. To facilitate the study of alternative splicing in a developmental context, we created MeDAS, a Metazoan Developmental Alternative Splicing database. MeDAS is an added-value resource that re-analyses publicly archived RNA-seq libraries to provide quantitative data on alternative splicing events as they vary across the time course of development. It has broad temporal and taxonomic scope and is intended to assist the user in identifying trends in alternative splicing throughout development. To create MeDAS, we re-analysed a curated set of 2232 Illumina polyA+ RNA-seq libraries that chart detailed time courses of embryonic and post-natal development across 18 species with a taxonomic range spanning the major metazoan lineages from Caenorhabditis elegans to human. MeDAS is freely available at https://das.chenlulab.com both as raw data tables and as an interactive browser allowing searches by species, tissue, or genomic feature (gene, transcript or exon ID and sequence). Results will provide details on alternative splicing events identified for the queried feature and can be visualised at the gene-, transcript- and exon-level as time courses of expression and inclusion levels, respectively.

Is the Gill Skeleton of Acorn Worms (Enteropneusta) Similar to the Gill Skeleton of Amphioxus (Cephalochordata)?

The gill skeleton of the enteropneust Saccoglossus mereschkowskii consists of a series of tridents. The central prong of each trident bifurcates in its ventral end. The most anterior gill skeletal element has a simple horseshoe shape. Homologues of the elements of the enteropneust gill apparatus were found in the structure of the gill apparatus of Cephalochordata. The organization of the gill skeleton of Enteropneusta and Cephalochordata can be derived from the metameric horseshoe-shaped elements. The similarity of the structure of the gill skeleton of Enteropneusta and Cephalochordata contradicts a common "upside-down theory" of the origin of Chordata.

Asymmetron lucayanum: How many species are valid?

The cephalochordates amphioxus or lancelets are benthic marine animals representing the earliest divergent evolutionary lineage within chordates. Although amphioxus are present in most of the world's tropical and temperate oceans, only about thirty different species grouped into three different genera, Branchiostoma, Epigonichthys and Asymmetron have been described. In the genus Asymmetron, only two species have been characterized, although for one of them, A. lucayanum, several cryptic lineages exist. In this work we have sequenced and analyzed the mitogenome of an A. lucayanum population previously described in the Red Sea. The phylogenetic study using this complete mitogenome as well as the analysis of COI gene sequences of several individuals of this Red Sea population show that the Red Sea population is a new cryptic species. We propose to call this new species Asymmetron rubrum.

ANISEED 2019: 4D exploration of genetic data for an extended range of tunicates.

ANISEED (https://www.aniseed.cnrs.fr) is the main model organism database for the worldwide community of scientists working on tunicates, the vertebrate sister-group. Information provided for each species includes functionally-annotated gene and transcript models with orthology relationships within tunicates, and with echinoderms, cephalochordates and vertebrates. Beyond genes the system describes other genetic elements, including repeated elements and cis-regulatory modules. Gene expression profiles for several thousand genes are formalized in both wild-type and experimentally-manipulated conditions, using formal anatomical ontologies. These data can be explored through three complementary types of browsers, each offering a different view-point. A developmental browser summarizes the information in a gene- or territory-centric manner. Advanced genomic browsers integrate the genetic features surrounding genes or gene sets within a species. A Genomicus synteny browser explores the conservation of local gene order across deuterostome. This new release covers an extended taxonomic range of 14 species, including for the first time a non-ascidian species, the appendicularian Oikopleura dioica. Functional annotations, provided for each species, were enhanced through a combination of manual curation of gene models and the development of an improved orthology detection pipeline. Finally, gene expression profiles and anatomical territories can be explored in 4D online through the newly developed Morphonet morphogenetic browser.

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.

Evolution of thyroid hormone signaling in animals: Non-genomic and genomic modes of action.

Much research has focused on vertebrate thyroid hormone (TH) synthesis and their function in development and metabolism. While important differences in TH synthesis and signaling exist, comparative studies between vertebrates fail to explain the evolutionary origins of this important regulatory axis. For that, one needs to make sense out of the diverse TH effects which have been described in invertebrate phyla but for which a mechanistic understanding is largely missing. Almost every major group of non-vertebrate animals possesses the capability to synthesize and metabolize thyroid hormones and there is evidence for a nuclear thyroid hormone receptor mediated mechanism in the bilateria, especially in molluscs, echinoderms, cephalochordates and ascidians. Still, genomic pathways cannot fully explain many observed effects of thyroid hormones in groups such as cnidarians, molluscs, and echinoderms and it is therefore possible that TH may signal via other mechanisms, such as non-genomic signaling systems via membrane bound or cytoplasmic receptors. Here we provide a brief review of TH actions in selected invertebrate species and discuss the hypothesis that non-genomic TH action may have played a critical role in TH signaling throughout animal evolution.

Metamerism in cephalochordates and the problem of the vertebrate head.

The vertebrate head characteristically exhibits a complex pattern with sense organs, brain, paired eyes and jaw muscles, and the brain case is not found in other chordates. How the extant vertebrate head has evolved remains enigmatic. Historically, there have been two conflicting views on the origin of the vertebrate head, segmental and non-segmental views. According to the segmentalists, the vertebrate head is organized as a metameric structure composed of segments equivalent to those in the trunk; a metamere in the vertebrate head was assumed to consist of a somite, a branchial arch and a set of cranial nerves, considering that the head evolved from rostral segments of amphioxus-like ancestral vertebrates. Non-segmentalists, however, considered that the vertebrate head was not segmental. In that case, the ancestral state of the vertebrate head may be non-segmented, and rostral segments in amphioxus might have been secondarily gained, or extant vertebrates might have evolved through radical modifications of amphioxus-like ancestral vertebrate head. Comparative studies of mesodermal development in amphioxus and vertebrate gastrula embryos have revealed that mesodermal gene expressions become segregated into two domains anteroposteriorly to specify the head mesoderm and trunk mesoderm only in vertebrates; in this segregation, key genes such as delta and hairy, involved in segment formation, are expressed in the trunk mesoderm, but not in the head mesoderm, strongly suggesting that the head mesoderm of extant vertebrates is not segmented. Taken together, the above finding possibly adds a new insight into the origin of the vertebrate head; the vertebrate head mesoderm would have evolved through an anteroposterior polarization of the paraxial mesoderm if the ancestral vertebrate had been amphioxus-like.

Amphioxus regeneration: evolutionary and biomedical implications.

Regeneration is a variable trait in chordates, with some species capable of impressive abilities, and others of only wound healing with scarring. Regenerative capacity has been reported in the literature for 5 species from two cephalochordate genera, Branchiostoma and Asymmetron. Its cellular and molecular bases have been studied in some detail in only two species: tail regeneration in the European amphioxus B. lanceolatum; and oral cirrus regeneration in the Asian species B. japonicum. Gene expression analyses of germline formation and posterior elongation in cephalochordate embryos provide some insight into regulation of progenitor and stem cell function. When combined with functional studies of gene function, including overexpression and knockdown, these will open the door to amphioxus as a good model not only for understanding the evolution of regeneration, but also for biomedical purposes.

Developmental cell-cell communication pathways in the cephalochordate amphioxus: actors and functions.

During embryonic development, cells of metazoan embryos need to communicate in order to construct the correct bodyplan. To do so, they use several signals that usually act through interactions between ligands and receptors. Interestingly, only a few pathways are known to be fundamental during animal development, and they are usually found in all the major metazoan clades, raising the following question: how have evolution of the actors and of the functions of these pathways participated in the appearance of the current diversity of animal morphologies? The chordate lineage comprises vertebrates, their sister group the urochordates, and the cephalochordates (i.e. amphioxus). Urochordates are quite derived relative to the chordate ancestor, whereas cephalochordates and vertebrates share many morphological traits. Thus, comparing embryonic development between vertebrates and cephalochordates should give us some insight into the ancestral characters present in chordates and into the morphological evolution in this clade. However, while much is known about the function of different signalling pathways in vertebrates, data are still scarce in the literature for cephalochordates. In this review, we summarize the current state of the field concerning the expression of actors and the function of the major cell-cell communication pathways, including Hedgehog (Hh), Notch, Nuclear Receptor (NR), Receptor Tyrosine Kinase (RTK), Transforming Growth Factor-ß (TGF-ß) and Wingless/Int (Wnt), in amphioxus.

Amphioxus Sp5 is a member of a conserved Specificity Protein complement and is modulated by Wnt/ß-catenin signalling.

A cluster of three Specificity Protein (Sp) genes (Sp1-4, Sp5 and Sp6-9) is thought to be ancestral in both chordates and the wider Eumetazoa. Sp5 and Sp6-9 gene groups are associated with embryonic growth zones, such as tailbuds, and are both Wnt/ß-catenin signalling pathway members and targets. Currently, there are conflicting reports as to the number and identity of Sp genes in the cephalochordates, the sister group to the vertebrates and urochordates. We confirm the SP complement of Branchiostoma belcheri and Branchiostoma lanceolatum, as well as their genomic arrangement, protein domain structure and residue frequency. We assay Sp5 expression in B. lanceolatum embryos, and determine its response to pharmacologically increased ß-catenin signalling. Branchiostoma possesses three Sp genes, located on the same genomic scaffold. Phylogenetic and domain structure analyses are consistent with their identification as SP1-4, SP5 and SP6-9, although SP1-4 contains a novel glutamine-rich N-terminal region. SP5 is expressed in axial mesoderm and neurectoderm, and marks the cerebral vesicle and presumptive pharynx. Early exposure to increased ß-catenin caused ubiquitous SP5 expression in late gastrula, while later treatment at gastrula stages reduced SP5 expression in the posterior growth zone during axis elongation. Amphioxus possess a typical invertebrate eumetazoan SP complement, and SP5 expression in embryos is well conserved with vertebrate homologues. Its expression in the tailbud, a posterior growth zone, is consistent with expression seen in other bilaterians. Branchiostoma SP5 shows a dynamic response to Wnt/ß-catenin signalling.

Conserved Noncoding Elements in the Most Distant Genera of Cephalochordates: The Goldilocks Principle.

Cephalochordates, the sister group of vertebrates + tunicates, are evolving particularly slowly. Therefore, genome comparisons between two congeners of Branchiostoma revealed so many conserved noncoding elements (CNEs), that it was not clear how many are functional regulatory elements. To more effectively identify CNEs with potential regulatory functions, we compared noncoding sequences of genomes of the most phylogenetically distant cephalochordate genera, Asymmetron and Branchiostoma, which diverged approximately 120-160 million years ago. We found 113,070 noncoding elements conserved between the two species, amounting to 3.3% of the genome. The genomic distribution, target gene ontology, and enriched motifs of these CNEs all suggest that many of them are probably cis-regulatory elements. More than 90% of previously verified amphioxus regulatory elements were re-captured in this study. A search of the cephalochordate CNEs around 50 developmental genes in several vertebrate genomes revealed eight CNEs conserved between cephalochordates and vertebrates, indicating sequence conservation over >500 million years of divergence. The function of five CNEs was tested in reporter assays in zebrafish, and one was also tested in amphioxus. All five CNEs proved to be tissue-specific enhancers. Taken together, these findings indicate that even though Branchiostoma and Asymmetron are distantly related, as they are evolving slowly, comparisons between them are likely optimal for identifying most of their tissue-specific cis-regulatory elements laying the foundation for functional characterizations and a better understanding of the evolution of developmental regulation in cephalochordates.

The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus).

Green Fluorescent Protein (GFP) was originally found in cnidarians, and later in copepods and cephalochordates (amphioxus) (Branchiostoma spp). Here, we looked for GFP-encoding genes in Asymmetron, an early-diverged cephalochordate lineage, and found two such genes closely related to some of the Branchiostoma GFPs. Dim fluorescence was found throughout the body in adults of Asymmetron lucayanum, and, as in Branchiostoma floridae, was especially intense in the ripe ovaries. Spectra of the fluorescence were similar between Asymmetron and Branchiostoma. Lineage-specific expansion of GFP-encoding genes in the genus Branchiostoma was observed, largely driven by tandem duplications. Despite such expansion, purifying selection has strongly shaped the evolution of GFP-encoding genes in cephalochordates, with apparent relaxation for highly duplicated clades. All cephalochordate GFP-encoding genes are quite different from those of copepods and cnidarians. Thus, the ancestral cephalochordates probably had GFP, but since GFP appears to be lacking in more early-diverged deuterostomes (echinoderms, hemichordates), it is uncertain whether the ancestral cephalochordates (i.e. the common ancestor of Asymmetron and Branchiostoma) acquired GFP by horizontal gene transfer (HGT) from copepods or cnidarians or inherited it from the common ancestor of copepods and deuterostomes, i.e. the ancestral bilaterians.

Functional Pairing of Class B1 Ligand-GPCR in Cephalochordate Provides Evidence of the Origin of PTH and PACAP/Glucagon Receptor Family.

Several hypotheses have been proposed regarding the origin and evolution of the secretin family of peptides and receptors. However, identification of homologous ligand-receptor pairs in invertebrates and vertebrates is difficult because of the low levels of sequence identity between orthologs of distant species. In this study, five receptors structurally related to the vertebrate class B1 G protein-coupled receptor (GPCR) family were characterized from amphioxus (Branchiostoma floridae). Phylogenetic analysis showed that they clustered with vertebrate parathyroid hormone receptors (PTHR) and pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon receptors. These PTHR-like receptors shared synteny with several PTH and PACAP/glucagon receptors identified in spotted gar, Xenopus, and human, indicating that amphioxus preserves the ancestral chordate genomic organization of these receptor subfamilies. According to recent data by Mirabeau and Joly, amphioxus also expresses putative peptide ligands including homologs of PTH (bfPTH1 and 2) and PACAP/GLUC-like peptides (bfPACAP/GLUCs) that may interact with these receptors. Functional analyses showed that bfPTH1 and bfPTH2 activated one of the amphioxus receptors (bf98C) whereas bfPACAP/GLUCs strongly interacted with bf95. In summary, our data confirm the presence of PTH and PACAP/GLUC ligand-receptor pairs in amphioxus, demonstrating that functional homologs of vertebrate PTH and PACAP/glucagon GPCR subfamilies arose before the cephalochordate divergence from the ancestor of tunicates and vertebrates.

Identification, evolution and expression of an insulin-like peptide in the cephalochordate Branchiostoma lanceolatum.

Insulin is one of the most studied proteins since it is central to the regulation of carbohydrate and fat metabolism in vertebrates and its expression and release are disturbed in diabetes, the most frequent human metabolic disease worldwide. However, the evolution of the function of the insulin protein family is still unclear. In this study, we present a phylogenetic and developmental analysis of the Insulin Like Peptide (ILP) in the cephalochordate amphioxus. We identified an ILP in the European amphioxus Branchiostoma lanceolatum that displays structural characteristics of both vertebrate insulin and Insulin-like Growth Factors (IGFs). Our phylogenetic analysis revealed that amphioxus ILP represents the sister group of both vertebrate insulin and IGF proteins. We also characterized both temporal and spatial expression of ILP in amphioxus. We show that ilp is highly expressed in endoderm and paraxial mesoderm during development, and mainly expressed in the gut of both the developing embryo and adult. We hypothesize that ILP has critical implications in both developmental processes and metabolism and could display IGF- and insulin-like functions in amphioxus supporting the idea of a common ancestral protein.

Evolution of endothelin receptors in vertebrates.

Endothelin receptors are G protein coupled receptors (GPCRs) of the ß-group of rhodopsin receptors that bind to endothelin ligands, which are 21 amino acid long peptides derived from longer prepro-endothelin precursors. The most basal Ednr-like GPCR is found outside vertebrates in the cephalochordate amphioxus, but endothelin ligands are only present among vertebrates, including the lineages of jawless vertebrates (lampreys and hagfishes), cartilaginous vertebrates (sharks, rays, and chimaeras), and bony vertebrates (ray-finned fishes and lobe-finned vertebrates including tetrapods). A bona fide endothelin system is thus a vertebrate-specific innovation with important roles for regulating the cardiovascular system, renal and pulmonary processes, as well as for the development of the vertebrate-specific neural crest cell population and its derivatives. Expectedly, dysregulation of endothelin receptors and the endothelin system leads to a multitude of human diseases. Despite the importance of different types of endothelin receptors for vertebrate development and physiology, current knowledge on endothelin ligand-receptor interactions, on the expression of endothelin receptors and their ligands, and on the functional roles of the endothelin system for embryonic development and in adult vertebrates is very much biased towards amniote vertebrates. Recent analyses from a variety of vertebrate lineages, however, have shown that the endothelin system in lineages such as teleost fish and lampreys is more diverse and is divergent from the mammalian endothelin system. This diversity is mainly based on differential evolution of numerous endothelin system components among vertebrate lineages generated by two rounds of whole genome duplication (three in teleosts) during vertebrate evolution. Here we review current understanding of the evolutionary history of the endothelin receptor family in vertebrates supplemented with surveys on the endothelin receptor gene complement of newly available genome assemblies from phylogenetically informative taxa. Our assessment further highlights the diversity of the vertebrate endothelin system and calls for detailed functional and pharmacological analyses of the endothelin system beyond tetrapods.

Deuterostome brains: synopsis and commentary.

The living deuterostomes comprise six monophyletic groups: (1) echinoderms + hemichordates, (2) tunicates, (3) cephalochordates, (4) myxinoids, (5) petromyzontoids, and (6) gnathostomes. The morphotype of the craniote (myxinoids + petromyzontoids + gnathostomes) central nervous system (CNS) comprises a fixed number of histogenetic units, formed by the intersection of transversely oriented neuromeres and longitudinally arranged zones. A well-developed built-in, natural coordinate system adds the third dimension to this morphotype. The classical subdivisions of the craniote CNS: prosencephalon (P), mesencephalon (M), rhombencephalon (R), and spinal cord (S) are each composed of a number of neuromeres. Chordates (larval tunicates + cephalochordates + craniotes) share a highly characteristic axial complex, encompassing a dorsal tubular CNS, a notochord and bilateral series of segmental muscles. In all chordates the CNS can be divided into a rostral (P-like + M-like), an intermediate (R-like) and a caudal (S-like) sector, and sets of homologous developmental genes play a role in this tripartitioning. There are no indications for the presence of olfactory or other telencephalic regions in the brain of non-craniote chordates. Convincing evidence that parts of the chordate CNS are homologous to parts of the larval or adult CNS of non-chordate deuterostomes (echinoderms + hemichordates) is lacking. The dorsal tubular CNS is most probably a chordate autapomorphy.