Amphioxus (Branchiostoma lanceolatum) in the North Adriatic Sea: ecological observations and spawning behavior.

The European amphioxus (Branchiostoma lanceolatum) is a member of the chordate subphylum Cephalochordata, and, as such, a key model organism for providing insights into the origin and evolution of vertebrates. Despite its significance and global distribution, detailed characterizations of natural populations of cephalochordates are still very limited. This study investigates the abundance, habitat, and spawning behavior of amphioxus in the North Adriatic Sea. Across 32 sampled sites, adult amphioxus were consistently present, reaching densities exceeding 300 individuals m- 2. DNA barcoding confirmed the species as B. lanceolatum, and environmental analyses revealed an amphioxus preference for slightly gravelly sand with low silt content and a correlation between amphioxus density and the presence of specific macroinvertebrate taxa. Remarkably, the amphioxus population was breeding in early spring and possibly late fall, in contrast to the typical late spring/early summer spawning season described for other populations of European amphioxus. Amphioxus adults kept in captivity maintained the spawning seasonality of their place of origin, suggesting the possibility of extending the overall spawning season of European amphioxus in laboratory settings by exploiting populations from diverse geographic origins. This study thus expands our understanding of B. lanceolatum ecology and reproduction in the Mediterranean Sea, emphasizing the role of the North Adriatic Sea as a substantial reservoir.

Amphioxus as a model to study the evolution of development in chordates.

Cephalochordates and tunicates represent the only two groups of invertebrate chordates, and extant cephalochordates - commonly known as amphioxus or lancelets - are considered the best proxy for the chordate ancestor, from which they split around 520 million years ago. Amphioxus has been an important organism in the fields of zoology and embryology since the 18th century, and the morphological and genomic simplicity of cephalochordates (compared to vertebrates) makes amphioxus an attractive model for studying chordate biology at the cellular and molecular levels. Here we describe the life cycle of amphioxus, and discuss the natural histories and habitats of the different species of amphioxus. We also describe their use as laboratory animal models, and discuss the techniques that have been developed to study different aspects of amphioxus.

The long protostomic-type cytoplasmic intermediate filament (cIF) protein in Branchiostoma supports the phylogenetic transition between the protostomic- and the chordate-type cIFs.

We identified 23 and 20 cytoplasmic IF (cIF) genes in the two Branchiostoma belcheri and Branchiostoma lanceolatum cephalochordates, respectively. Combining these results with earlier data on the related Branchiostoma floridae, the following conclusions can be drawn. First, the Branchiostoma N4 protein with a long lamin-like coil 1B segment is the only protostomic-type cIF found so far in any analysed chordate or vertebrate organism. Second, Branchiostoma is the only organism known so far containing both the long protostomic- and the short chordate-prototypes of cIFs. This finding provides so far missing molecular evidence for the phylogenetic transition between the protostomic- and the chordate-type IF sequences at the base of the cephalochordates and vertebrates. Third, this finding also brings some support for another hypothesis, that the long protostomic-type cIF is subjected to evolutionary constraints in order to preclude inappropriate interactions with lamin and that the latter complexes might be prevented by a several heptad-long rod deletion, which released the selective constraints on it and promoted, at least in part, its expansion in nematodes, cephalochordates, and in vertebrates. Finally, here-presented data confirmed our previous results that cephalochordates do not have any vertebrate type III or type IV IF homolog.

Cranial cartilages: Players in the evolution of the cranium during evolution of the chordates in general and of the vertebrates in particular.

The present contribution is chiefly a review, augmented by some new results on amphioxus and lamprey anatomy, that draws on paleontological and developmental data to suggest a scenario for cranial cartilage evolution in the phylum chordata. Consideration is given to the cartilage-related tissues of invertebrate chordates (amphioxus and some fossil groups like vetulicolians) as well as in the two major divisions of the subphylum Vertebrata (namely, agnathans, and gnathostomes). In the invertebrate chordates, which can be considered plausible proxy ancestors of the vertebrates, only a viscerocranium is present, whereas a neurocranium is absent. For this situation, we examine how cartilage-related tissues of this head region prefigure the cellular cartilage types in the vertebrates. We then focus on the vertebrate neurocranium, where cyclostomes evidently lack neural-crest derived trabecular cartilage (although this point needs to be established more firmly). In the more complex gnathostome, several neural-crest derived cartilage types are present: namely, the trabecular cartilages of the prechordal region and the parachordal cartilage the chordal region. In sum, we present an evolutionary framework for cranial cartilage evolution in chordates and suggest aspects of the subject that should profit from additional study.

Retinoic Acid and POU Genes in Developing Amphioxus: A Focus on Neural Development.

POU genes are a family of evolutionarily conserved transcription factors with key functions in cell type specification and neurogenesis. In vitro experiments have indicated that the expression of some POU genes is controlled by the intercellular signaling molecule retinoic acid (RA). In this work, we aimed to characterize the roles of RA signaling in the regulation of POU genes in vivo. To do so, we studied POU genes during the development of the cephalochordate amphioxus, an animal model crucial for understanding the evolutionary origins of vertebrates. The expression patterns of amphioxus POU genes were assessed at different developmental stages by chromogenic in situ hybridization and hybridization chain reaction. Expression was further assessed in embryos subjected to pharmacological manipulation of endogenous RA signaling activity. In addition to a detailed description of the effects of these treatments on amphioxus POU gene expression, our survey included the first description of Pou2 and Pou6 expression in amphioxus embryos. We found that Pit-1, Pou2, Pou3l, and Pou6 expression are not affected by alterations of endogenous RA signaling levels. In contrast, our experiments indicated that Brn1/2/4 and Pou4 expression are regulated by RA signaling in the endoderm and the nerve cord, respectively. The effects of the treatments on Pou4 expression in the nerve cord revealed that, in developing amphioxus, RA signaling plays a dual role by (1) providing anteroposterior patterning information to neural cells and (2) specifying neural cell types. This finding is coherent with a terminal selector function of Pou4 for GABAergic neurons in amphioxus and represents the first description of RA-induced changes in POU gene expression in vivo.

Evolution of calcitonin/calcitonin gene-related peptide family in chordates: Identification of CT/CGRP family peptides in cartilaginous fish genome.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is a peptide gene family that is widely found in bilaterians. CT, CGRP, adrenomedullin (AM), amylin (AMY), and CT receptor-stimulating peptide (CRSP) are members of the CT/CGRP family. In mammals, CT is involved in calcium homeostasis, while CGRP and AM primarily function in vasodilation. AMY and CRSP are associated with anorectic effects. Diversification of the molecular features and physiological functions of the CT/CGRP family in vertebrate lineages have been extensively reported. However, the origin and diversification mechanisms of the vertebrate CT/CGRP family of peptides remain unclear. In this review, the molecular characteristics of CT/CGRP family peptides and their receptors, along with their major physiological functions in mammals and teleosts, are introduced. Furthermore, novel candidates of the CT/CGRP family in cartilaginous fish are presented based on genomic information. The CT/CGRP family peptides and receptors in urochordates and cephalochordates, which are closely related to vertebrates, are also described. Finally, a putative evolutionary scenario of the CT/CGRP family peptides and receptors in chordates is discussed.

FISH for All: A Fast and Efficient Fluorescent In situ Hybridization (FISH) Protocol for Marine Embryos and Larvae.

In situ hybridization is one the most commonly used techniques for developmental and evolutionary biology and has extensively contributed to the identification of distinct cell types and cell states, as well dissecting several molecular mechanisms involved in physiological processes. Moreover, it has been used as a tool to compare distinct gene expression patterns and, therefore, genetic programs across animal species. Nowadays, the predominance of transcriptomics in science has imposed the need to establish a reliable, fast and easy whole mount in situ hybridization protocol. Here we describe a fluorescent in situ hybridization protocol that is rapid, accurate and applicable in a great variety of marine species.

Is There a Prechordal Region and an Acroterminal Domain in Amphioxus?

This essay re-examines the singular case of the supposedly unique rostrally elongated notochord described classically in amphioxus. We start from our previous observations in hpf 21 larvae [Albuixech-Crespo et al.: PLoS Biol. 2017;15(4):e2001573] indicating that the brain vesicle has rostrally a rather standard hypothalamic molecular configuration. This correlates with the notochord across a possible rostromedian acroterminal hypothalamic domain. The notochord shows some molecular differences that specifically characterize its pre-acroterminal extension beyond its normal rostral end under the mamillary region. We explored an alternative interpretation that the putative extension of this notochord actually represents a variant form of the prechordal plate in amphioxus, some of whose cells would adopt the notochordal typology, but would lack notochordal patterning properties, and might have some (but not all) prechordal ones instead. We survey in detail the classic and recent literature on gastrulation, prechordal plate, and notochord formation in amphioxus, compare the observed patterns with those of some other vertebrates of interest, and re-examine the literature on differential gene expression patterns in this rostralmost area of the head. We noted that previous literature failed to identify the amphioxus prechordal primordia at appropriate stages. Under this interpretation, a consistent picture can be drawn for cephalochordates, tunicates, and vertebrates. Moreover, there is little evidence for an intrinsic capacity of the early notochord to grow rostralwards (it normally elongates caudalwards). Altogether, we conclude that the hypothesis of a prechordal nature of the elongated amphioxus notochord is consistent with the evidence presented.

Functional Conservation and Genetic Divergence of Chordate Glycinergic Neurotransmission: Insights from Amphioxus Glycine Transporters.

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and neurons, respectively. Only limited information is available on glycinergic neurotransmission in invertebrates, and the evolution of glycinergic neurotransmission is poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possess three glycine transporter genes. Two of these (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, whereas the third (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. GlyT2.2 expression is predominantly non-neural, whereas GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are differentially expressed, respectively, in neurons and glia. Vertebrate glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by a paralog-specific inversion of gene expression. Despite this genetic divergence between amphioxus and vertebrates, we found strong evidence for conservation in the role glycinergic neurotransmission plays during larval swimming, the implication being that the neural networks controlling the rhythmic movement of chordate bodies may be homologous.

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.

The Ontology of the Amphioxus Anatomy and Life Cycle (AMPHX).

An ontology is a computable representation of the different parts of an organism and its different developmental stages as well as the relationships between them. The ontology of model organisms is therefore a fundamental tool for a multitude of bioinformatics and comparative analyses. The cephalochordate amphioxus is a marine animal representing the earliest diverging evolutionary lineage of chordates. Furthermore, its morphology, its anatomy and its genome can be considered as prototypes of the chordate phylum. For these reasons, amphioxus is a very important animal model for evolutionary developmental biology studies aimed at understanding the origin and diversification of vertebrates. Here, we have constructed an amphioxus ontology (AMPHX) which combines anatomical and developmental terms and includes the relationships between these terms. AMPHX will be used to annotate amphioxus gene expression patterns as well as phenotypes. We encourage the scientific community to adopt this amphioxus ontology and send recommendations for future updates and improvements.

Amphioxus neuroglia: Molecular characterization and evidence for early compartmentalization of the developing nerve cord.

Glial cells play important roles in the development and homeostasis of metazoan nervous systems. However, while their involvement in the development and function in the central nervous system (CNS) of vertebrates is increasingly well understood, much less is known about invertebrate glia and the evolutionary history of glial cells more generally. An investigation into amphioxus glia is therefore timely, as this organism is the best living proxy for the last common ancestor of all chordates, and hence provides a window into the role of glial cell development and function at the transition of invertebrates and vertebrates. We report here our findings on amphioxus glia as characterized by molecular probes correlated with anatomical data at the transmission electron microscopy (TEM) level. The results show that amphioxus glial lineages express genes typical of vertebrate astroglia and radial glia, and that they segregate early in development, forming what appears to be a spatially separate cell proliferation zone positioned laterally, between the dorsal and ventral zones of neural cell proliferation. Our study provides strong evidence for the presence of vertebrate-type glial cells in amphioxus, while highlighting the role played by segregated progenitor cell pools in CNS development. There are implications also for our understanding of glial cells in a broader evolutionary context, and insights into patterns of precursor cell deployment in the chordate nerve cord.

My Favorite Animal, Amphioxus: Unparalleled for Studying Early Vertebrate Evolution.

Amphioxus represents the most basally divergent group in chordates and probably the best extant proxy to the ancestor of all chordates including vertebrates. The amphioxus, or lancelets, are benthic filter feeding marine animals and their interest as a model in research is due to their phylogenetic position and their anatomical and genetic stasis throughout their evolutionary history. From the first works in the 19th century to the present day, enormous progress is made mainly favored by technical development at different levels, from spawning induction and husbandry techniques, through techniques for studies of gene function or of the role of different signalling pathways through embryonic development, to functional genomics techniques. Together, these advances foretell a plethora of interesting developments in the world of research with the amphioxus model. Here, the discovery and development of amphioxus as a superb model organism in evolutionary and evolutionary-developmental biology are reviewed.

Genome-wide comparison of the protein-coding repertoire reveals fast evolution of immune-related genes in cephalochordates and Osteichthyes superclass.

Amphioxus is used to investigate the origin and evolution of vertebrates. To better understand the characteristics of genome evolution from cephalochordates to Osteichthyes, we conducted a genome-wide pairwise comparison of protein-coding genes within amphioxus (a comparable group) and parallel analyses within Osteichthyes (two comparable groups). A batch of fast-evolving genes in each comparable group was identified. Of these genes, the most fast-evolving genes (top 20) were scrutinized, most of which were involved in immune system. An analysis of the fast-evolving genes showed that they were enriched into gene ontology (GO) terms and pathways primarily involved in immune-related functions. Similarly, this phenomenon was detected within Osteichthyes, and more well-known and abundant GO terms and pathways involving innate immunity were found in Osteichthyes than in cephalochordates. Next, we measured the expression responses of four genes belonging to metabolism or energy production-related pathways to lipopolysaccharide challenge in the muscle, intestine or skin of B. belcheri; three of these genes (HMGCL, CYBS and MDH2) showed innate immune responses. Additionally, some genes involved in adaptive immunity showed fast evolution in Osteichthyes, such as those involving "intestinal immune network for IgA production" or "T-cell receptor signaling pathway". In this study, the fast evolution of immune-related genes in amphioxus and Osteichthyes was determined, providing insights into the evolution of immune-related genes in chordates.

Molecular signatures that are distinctive characteristics of the vertebrates and chordates and supporting a grouping of vertebrates with the tunicates.

Members of the phylum Chordata and the subphylum Vertebrata are presently distinguished solely on the basis of morphological characteristics. The relationship of the vertebrates to the two non-vertebrate chordate subphyla is also a subject of debate. Analyses of protein sequences have identified multiple conserved signature indels (CSIs) that are specific for Chordata or for Vertebrata. Five CSIs in 4 important proteins are specific for the Vertebrata, whereas two other CSIs are uniquely found in all sequenced chordate species including Ciona intestinalis and Oikapleura dioica (Tunicates) as well as Branchiostoma floridae (Cephalochordates). The shared presence of these molecular signatures by all vertebrates/chordate species, but in no other animal taxa, strongly indicates that the genetic changes represented by the identified CSIs diagnose monophyletic groups. Two other discovered CSIs are uniquely shared by different vertebrate species and by either one (Ciona intestinalis) or both tunicate (Ciona and Oikapleura) species, but they are not found in Branchiostoma or other animal species. Specific presence of these CSIs in different vertebrates and either one or both tunicate species provides strong independent evidence that the vertebrate species are more closely related to the urochordates (tunicates) than to the cephalochordates.

Development, metamorphosis, morphology, and diversity: The evolution of chordate muscles and the origin of vertebrates.

Recent findings that urochordates are the closest sister-group of vertebrates have dramatically changed our understanding of chordate evolution and vertebrate origins. To continue to deepen our understanding of chordate evolution and diversity, in particular the morphological and taxonomical diversity of the vertebrate clade, one must explore the origin, development, and comparative anatomy of not only hard tissues, but also soft tissues such as muscles. Building on a recent overview of the discovery of a cardiopharyngeal field in urochordates and the profound implications for reconstructing the origin and early evolution of vertebrates, in this study we focus on the broader comparative and developmental anatomy of chordate cephalic muscles and their relation to life history, and to developmental, morphological and taxonomical diversity. We combine our recent findings on cephalochordates, urochordates, and vertebrates with a literature review and suggest that developmental changes related to metamorphosis and/or heterochrony (e.g., peramorphosis) played a crucial role in the early evolution of chordates and vertebrates. Recent studies reviewed here supported de Beer's "law of diversity" that peramorphic animals (e.g., ascidians, lampreys) are taxonomically and morphologically less diverse than nonperamorphic animals (e.g., gnathostomes), probably because their "too specialized" development and adult anatomy constrain further developmental and evolutionary innovations. Developmental Dynamics 244:1046-1057, 2015. © 2014 Wiley Periodicals, Inc.

Neuronal Organization of the Brain in the Adult Amphioxus (Branchiostoma lanceolatum): A Study With Acetylated Tubulin Immunohistochemistry.

Amphioxus (Cephalochordata) belongs to the most basal extant chordates, and knowledge of their brain organization appears to be key to deciphering the early stages of evolution of vertebrate brains. Most comprehensive studies of the organization of the central nervous system of adult amphioxus have investigated the spinal cord. Some brain populations have been characterized via neurochemistry and electron microscopy, and the overall cytoarchitecture of the brain was studied by Ekhart et al. (2003; J. Comp. Neurol. 466:319-330) with general staining methods and retrograde transport from the spinal cord. Here, the cytoarchitecture of the brain of adult amphioxus Branchiostoma lanceolatum was reinvestigated by using acetylated tubulin immunohistochemistry, which specifically stains neurons and fibers, in combination with some ancillary methods. This method allowed reproducible staining and mapping of types of neuron, mostly in brain regions caudal to the entrance level of nerve 2, and its comparison with spinal cord populations. The brain populations studied and discussed in detail were the Retzius bipolar cells, lamellate cells, Joseph cells, various types of translumenal cells, somatic motoneurons, Rohde nucleus cells, small ventral multipolar neurons, and Edinger cells. These observations expand our knowledge of the distribution of cell types and provide additional data on the number of cells and the axonal tracts and commissural regions of the adult amphioxus brain. The results of this comprehensive study provide a framework for comparison of complex adult populations with the early brain neuronal populations revealed in developmental studies of the amphioxus.

The cephalochordate Branchiostoma genome contains 26 intermediate filament (IF) genes: implications for evolution of chordate IF proteins.

We analyzed the draft genome of the cephalochordate Branchiostoma floridae (B. floridae) for genes encoding intermediate filament (IF) proteins. From 26 identified IF genes 13 were not reported before. Four of the new IF genes belong to the previously established Branchiostoma IF group A, four to the Branchiostoma IF group B, one is homologous to the type II keratin E2 while the remaining four new IF sequences N1 to N4 could not be readily classified in any of the previously established Branchiostoma IF groups. All eleven identified A and B2-type IF genes are located on the same genomic scaffold and arose due to multiple cephalochordate-specific duplications. Another IF gene cluster, identified in the B. floridae genome, contains three keratins (E1, Y1, D1), two keratin-like IF genes (C2, X1), one new IF gene (N1) and one IF unrelated gene, but does not show any similarities to the well defined vertebrate type I or type II keratin gene clusters. In addition, some type III sequence features were documented in the new IF protein N2, which, however, seems to share a common ancestry with the Branchiostoma keratins D1 and two keratin-related genes C. Thus, a few type I and type II keratin genes existed in a common ancestor of cephalochordates and vertebrates, which after separation of these two lineages gave rise to the known complexities of the vertebrate cytoplasmic type I-IV IF proteins, as well as to the multiple keratin and related IF genes in cephalochordates, due to multiple gene duplications, deletions and sequence divergences.