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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.

With the emergence of antifungal resistance, systematic infections with Aspergillus are becoming the major cause of the clinical morbidity. The development of novel antifungal agents with high efficacy, low drug tolerance, and few side effects is urgent. In response to that need, we have identified NP20. Here we demonstrate clearly that NP20 has antifungal activity, capable of killing the spores of Aspergillus niger and Aspergillus fumigatus as well as causing direct damage to the surface, membrane, cytoplasm, organelle, and nucleus of the fungal spores. Interestingly, NP20 is active under temperature stress and a wide range of pH. Subsequently, MTT assay, assay for binding of NP20 to fungal cell wall components, membrane depolarization assay, confocal microscopy, ROS assay, DNA replication, and protein synthesis assay are performed to clarify the mechanisms underlying NP20 against Aspergillus. The results show that NP20 can bind with and pass through the fungal cell wall, and then interfere with the lipid membrane. Moreover, NP20 can induce intracellular ROS production, DNA fragmentation, and protein synthesis inhibition of the fungal cells. These together indicate that NP20 is a novel antifungal peptide, which has considerable potential for future development as novel peptide antibiotics against Aspergillus.

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.

Previous studies show that some ribosomal proteins perform immune effector functions via killing bacteria directly. However, it remains largely unknown about other effector functions of ribosomal proteins during a bacterial infection. In this study, we expressed and purified four ribosomal proteins of the amphioxus Branchiostoma japonicum, termed rBjRPS15, rBjRPS18, rBjRPS19 and rBjRPS30-precursor (rBjRPS30P). They all exhibited bactericidal activity against Gram-positive Staphylococcus aureus, and with the exception of rBjRPS19 and rBjRPS30P, were capable of killing Gram-negative Escherichia coli. Importantly, rBjRPS15, rBjRPS19 and rBjRPS30P were able to agglutinate S. aureus in the presence of Mg2+, but none of them could agglutinate E. coli even in the presence of Mg2+ or Ca2+. Moreover, the S. aureus agglutination was achieved by the binding of these three proteins to the peptidoglycan component of the bacterial cell wall. This is the first report showing that some ribosomal proteins possess bacterial agglutinating activity, and these data provide a new angle to the roles of ribosomal proteins in immune defense.

[Background]: Amphioxus are non-vertebrate chordates characterized by a slow morphological and molecular evolution. They share the basic chordate body-plan and genome organization with vertebrates but lack their 2R whole-genome duplications and their developmental complexity. For these reasons, amphioxus are frequently used as an outgroup to study vertebrate genome evolution and Evo-Devo. Aside from whole-genome duplications, genes continuously duplicate on a smaller scale. Small-scale duplicated genes can be found in both amphioxus and vertebrate genomes, while only the vertebrate genomes have duplicated genes product of their 2R whole-genome duplications. Here, we explore the history of small-scale gene duplications in the amphioxus lineage and compare it to small- and large-scale gene duplication history in vertebrates. [Results]: We present a study of the European amphioxus (Branchiostoma lanceolatum) gene duplications thanks to a new, high-quality genome reference. We find that, despite its overall slow molecular evolution, the amphioxus lineage has had a history of small-scale duplications similar to the one observed in vertebrates. We find parallel gene duplication profiles between amphioxus and vertebrates and conserved functional constraints in gene duplication. Moreover, amphioxus gene duplicates show levels of expression and patterns of functional specialization similar to the ones observed in vertebrate duplicated genes. We also find strong conservation of gene synteny between two distant amphioxus species, B. lanceolatum and B. floridae, with two major chromosomal rearrangements. [Conclusions]: In contrast to their slower molecular and morphological evolution, amphioxus’ small-scale gene duplication history resembles that of the vertebrate lineage both in quantitative and in functional terms., RR acknowledges support from the Swiss National Science Foundation grant 31003A/173048, and from the Etat de Vaud. FMar acknowledges support from the Royal Society grant URF\R1\191161. MI acknowledges support from European Research Council ERC-StG-LS2-637591. HE acknowledges support from the Centre National de la Recherche Scientifique and Agence Nationale de la Recherche (ANR-16-CE12-0008-01 and ANR-19-CE13-0011). PP acknowledges support from the French Government under the “Investissements d’avenir” program managed by the Agence Nationale de la Recherche (Méditerranée Infection 10-IAHU-03). IM acknowledges support from the Spanish Ministry of Science and Innovation and the European Union (RYC-2016-20089 and PGC2018-099392-A-I00). JJT acknowledges support from the ERC (Grant Agreement No. 740041) and the Spanish Ministerio de Ciencia e Innovación (PID2019-103921GB-I00).

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

The core molecular mechanisms of dorsal organizer formation during gastrulation are highly conserved within the chordate lineage. One of the key characteristics is that Nodal signaling is required for the organizer-specific gene expression. This feature appears to be ancestral, as evidenced by the presence in the most basally divergent chordate amphioxus. To provide a better understanding of the evolution of organizer-specific gene regulation in chordates, we analyzed the cis-regulatory sequence of amphioxus Chordin in the context of the vertebrate embryo. First, we generated stable zebrafish transgenic lines, and by using light-sheet fluorescent microscopy, characterized in detail the expression pattern of GFP driven by the cis-regulatory sequences of amphioxus Chordin. Next, we performed a 5'deletion analysis and identified an enhancer sufficient to drive the expression of the reporter gene into a chordate dorsal organizer. Finally, we found that the identified enhancer element strongly depends on Nodal signaling, which is consistent with the well-established role of this pathway in the regulation of the expression of dorsal organizer-specific genes across chordates. The enhancer identified in our study may represent a suitable simple system to study the interplay of the evolutionarily conserved regulatory mechanisms operating during early chordate development.

Photoconvertible proteins are powerful tools widely used in cellular biology to study cell dynamics and organelles. Over the past decade, photoconvertible proteins have also been used for developmental biology applications to analyze cell lineage and cell fate during embryonic development. One of these photoconvertible proteins called Kaede, from the stony coral Trachyphyllia geoffroyi, undergoes irreversible photoconversion from green to red fluorescence when illuminated with UV light. Undertaking a cell tracing approach using photoconvertible proteins can be challenging when using unconventional animal models. In this protocol, we describe the use of Kaede to track specific cells during embryogenesis of the cephalochordate Branchiostoma lanceolatum. This protocol can be adapted to other unconventional models, especially marine animals.

Growing inputs of microplastics into marine sediment have increased significantly the needs for assessment of their potential risks to the marine benthos. A knowledge gap remains with regard to the effect of microplastics on benthos, such as cephalochordates. By employing amphioxus as a model benthic chordate, here we show that exposure to microplastics for 96 h at doses of 1 mg/L and 100 mg/L results in evident accumulation of the polyethylene microplastics. The accumulated microplastics are as much as 0.027% of body weight upon high-dose exposure, causing an abnormal body-bending phenotype that limits the locomotion capability of amphioxus. Mechanistic insight reveals that microplastics can bring about histological damages in gill, intestine and hepatic cecum; In-depth assay of relevant biomarkers including superoxide dismutase, catalase, glutathione, pyruvic acid and total cholesterol indicates the occurrence of oxidative damage and metabolic disorder; Further, microplastics exposure depresses the activity of acetylcholinesterase while allowing the level of acetylcholine to rise in muscle, suggesting the emergence of neurotoxicity. These consequences eventually contribute to the muscle dysfunction of amphioxus. This study rationalizes the abnormal response of the vulnerable notochord to microplastics, signifying the dilemma suffered by the ancient lineage under the emerging threat. Given the enrichment of microplastics through marine food chains, this study also raises significant concerns on the impact of microplastics to other marine organisms, and eventually human beings.

Background Amphioxus is a model organism for vertebrate evolutionary research. The significant contrast between morphological phenotypic similarity and high-level genetic polymorphism among amphioxus populations has aroused scientists' attention. Here we resequenced 21 amphioxus genomes to over 100X depth and mapped them to a haploid reference. Results More than 11.5 million common SNPs were detected in the amphioxus population, which mainly affect genes enriched in ion transport, signal transduction and cell adhesion, while protein structure analysis via AlphaFold2 revealed that these SNPs fail to bring effective structural variants. Conclusions Our work provides explanation for “amphioxus polymorphism paradox” in a micro view, and generates an enhanced genomic dataset for amphioxus research.

The growth hormone/insulin-like growth factor (GH/IGF) system, also called the pituitary-liver axis, has a somatotrophic role in the body. Although the GH/IGF system has always been regarded as a vertebrate-specific endocrine system, its actual origin remained unknown for a long time. The basal chordate, amphioxus, occupies an evolutionary position between vertebrates and invertebrates. Impressively, most of the members of the GH/IGF system are present in the amphioxus. The GH-like molecule in the amphioxus is mainly expressed in Hatschek’s pit. It functions similarly to vertebrate GH and has a GH receptor-like binding partner. The amphioxus IGF-like peptide shows mitogenic activity and an expression pattern resembling that of vertebrate IGF-I. The receptor of IGF-like peptide and IGF binding protein (IGFBP) have also been demonstrated to exist in the amphioxus. These results reveal the origin of the gene families in the GH/IGF system, providing strong evidence that this system emerged in the amphioxus.

Cephalochordate (Amphioxus), situated at a key phylogenetic position in the phylum Chordata, serves as a model organism for studying the origin and evolution of the vertebrate innate immune. In this study, five members of precursor miR-92 family (miR-92a-1, miR-92a-2, miR-92b, miR-92c and miR-92d) are identified in Branchiostoma belcheri, and their evolutionary conservation and potential molecular functions in innate immunity are analyzed. Among them, miR-92b-5p was validated in HEK293 cells to target the coding region but not classic 3'UTR of IKK (inhibitor of nuclear factor kappa-B kinase) mRNA, one integral component of MAPK and TLR4 immune signaling. Furthermore, the spatiotemporal expression patterns of miR-92b-5p and IKK were examined in different tissues or different time points (2 h, 4 h, 8 h, 12 h, 24 h and 48 h) post LPS stimulation at RNA and protein level in vivo. The seemingly inverse expression pattern between miR-92b-5p and IKK supports the involvement of miR-92b-5p in Branchiostoma belcheri innate immune response. In conclusion, our work not only illustrates the evolutionary pattern of Branchiostoma belcheri miR-92 family across chordates, but also reveals that miR-92b-5p could target IKK expression to regulate innate immune response.

Conventional role of ribosomal proteins is ribosome assembly and protein translation, but some ribosomal proteins also show antimicrobial peptide (AMP) activity, though their mode of action remains ill-defined. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933-55, Aquificae RPS1933-55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial surface via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67 were toxic to mammalian cells, making them promising lead molecules for the design of novel AMPs against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.

Amphioxus (cepholochordates) have long been used to infer how the vertebrates evolved from their invertebrate ancestors. However, some of the body part homologies between amphioxus and vertebrates have been controversial. This is not surprising as the amphioxus and vertebrate lineages separated half a billion years ago-plenty of time for independent loss and independent gain of features. The development of new techniques in the late 20th and early 21st centuries including transmission electron microscopy and serial blockface scanning electron microscopy in combination with in situ hybridization and immunocytochemistry to reveal spatio-temporal patterns of gene expression and gene products have greatly strengthened inference of some homologies (like those between regions of the central nervous system), although others (like nephridia) still need further support. These major advances in establishing homologies between amphioxus and vertebrates, together with strong support from comparative genomics, have firmly established amphioxus as a stand-in or model for the ancestral vertebrate.

Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation [A. Pires-daSilva, R. J. Sommer, Nat. Rev. Genet. 4, 39-49 (2003)], although only a few of these pathways operate during this period [J. J. Sanz-Ezquerro, A. E. Munsterberg, € S. Stricker, Front. Cell Dev. Biol. 5, 76 (2017)]. Moreover, most of them have been largely conserved during metazoan evolution [L. S. Babonis, M. Q. Martindale, Philos. Trans. R. Soc. Lond. B Biol. Sci. 372, 20150477 (2017)]. How evolution has generated a vast diversity of animal morphologies with such a limited number of tools is still largely unknown. Here, we show that gain of interconnectivity between signaling pathways and the GRNs they control may have critically contributed to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF, and Nodal signaling pathways during gastrulation in the invertebrate chordate amphioxus and zebrafish and compared the effects on gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through vertebrate-specific CREs. Moreover, in contrast to amphioxus, many of these CREs responded to multiple pathways in zebrafish, which reflects their high interconnectivity. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than in tissues with more ancient origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and to the formation of new cell types and morphological novelties in this lineage., This project has received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program (Grant agreement no. 740041) and the Spanish Ministerio de Economía y Competitividad (Grants BFU2016-74961-P and PID2019-103921GB-I00 to J.L.G.-S. and J.J.T.). This work was also supported by the institutional grant Unidad de Excelencia María de Maeztu (Grant MDM-2016-0687 to the Department of Gene regulation and morphogenesis of Centro Andaluz de Biología del Desarrollo). M.F. was funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement [#800396]. M.S., H.E., and S.B. were supported by the CNRS, and H.E. and S.B. additionally by Agence Nationale de la Recherche (ANR) CHORELAND (Grant ANR-16-CE12-0008-01) and the Institut Universitaire de France. Y.-H.S. and J.-K.Y. are supported by intramural funds from Academia Sinica and grants from Ministry of Science and Technology, Taiwan (Grants 110-2326-B-001-006 to Y.-H.S. and 110-2621-B-001-001-MY3 to J.-K.Y.).

Madeleine E. Aase-Remedios and Clara Coll-Lladó were supported by funding from the University of St Andrews, School of Biology and additional support from St Leonards College (MEAR), the CORBEL grant European Research Infrastructure cluster project and European Assemble Plus (H2020-INFRAIA-1-2016-2017; grant no.730984). Transcriptome sequencing was done with an award under the BBSRC TGAC Capacity and Capability Challenge. Background: The formation and functioning of muscles are fundamental aspects of animal biology, and the evolution of ‘muscle genes’ is central to our understanding of this tissue. Feeding-fasting-refeeding experiments have been widely used to assess muscle cellular and metabolic responses to nutrition. Though these studies have focused on vertebrate models and only a few invertebrate systems, they have found similar processes are involved in muscle degradation and maintenance. Motivation for these studies stems from interest in diseases whose pathologies involve muscle atrophy, a symptom also triggered by fasting, as well as commercial interest in the muscle mass of animals kept for consumption. Experimentally modelling atrophy by manipulating nutritional state causes muscle mass to be depleted during starvation and replenished with refeeding so that the genetic mechanisms controlling muscle growth and degradation can be understood. Results: Using amphioxus, the earliest branching chordate lineage, we address the gap in previous work stemming from comparisons between distantly related vertebrate and invertebrate models. Our amphioxus feeding-fasting-refeeding muscle transcriptomes reveal a highly conserved myogenic program and that the pro-orthologues of many vertebrate myoblast fusion genes were present in the ancestral chordate, despite these invertebrate chordates having unfused mononucleate myocytes. We found that genes differentially expressed between fed and fasted amphioxus were orthologous to the genes that respond to nutritional state in vertebrates. This response is driven in a large part by the highly conserved IGF/Akt/FOXO pathway, where depleted nutrient levels result in activation of FOXO, a transcription factor with many autophagy-related gene targets. Conclusion: Reconstruction of these gene networks and pathways in amphioxus muscle provides a key point of comparison between the distantly related groups assessed thus far, significantly refining the reconstruction of the ancestral state for chordate myoblast fusion genes and identifying the extensive role of duplicated genes in the IGF/Akt/FOXO pathway across animals. Our study elucidates the evolutionary trajectory of muscle genes as they relate to the increased complexity of vertebrate muscles and muscle development. Publisher PDF

The Hatschek’s pit in the cephalochordate amphioxus, an invertebrate deuterostome basal to chordates is suggested to be the functional homolog structure of the vertebrate adenohypophysis based on anatomy and expression of homologous neuroendocrine genes. However, the endocrine potential of the cephalochordate Hatschek’s pit remains to be demonstrated as well as the physiological actions of the secreted neuropeptides. In this study, we have explored the distribution and characterize the potential function of the amphioxus PACAP/GCG precursor, which is the ortholog of the hypothalamic PACAP neuropeptide in vertebrates. In amphioxi, two PACAP/GCG transcripts PACAP/GCGa and PACAP/GCGbc that are alternative isoforms of a single gene with different peptide coding potentials were isolated. Immunofluorescence staining detected their expression around the nucleus of Rohde, supporting that this structure may be homologous of the neurosecretory cells of the vertebrate hypothalamus where abundant PACAP is found. PACAP/GCGa was also detected in the infundibulum‐like downgrowth approaching the Hatschek’s pit, indicating diffusion of PACAP/GCGa from the CNS to the pit via the infundibulum‐like downgrowth. Under a high salinity challenge, PACAP/GCGa was upregulated in amphioxi head and PACAP/GCGa treatment increased expression of GHl in Hatschek’s pit in a dose-dependent manner, suggesting that PACAP/GCGa may be involved in the regulation of GHl via hypothalamic-pituitary (HP)-like axis similar as in the vertebrates. Our results support that the amphioxus Hatschek’s pit is likely to be the functional homolog of pituitary gland in vertebrates.

Background Vertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates. Results In order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates. Conclusions Our work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates.

The discovery of ancestral RAG transposons in early deuterostomia reveals the origin of vertebrate V(D)J recombination. Here, we analyze the functional regulation of a RAG transposon, ProtoRAG, in lancelet. We find that a specific interaction between the cis-acting element within the TIR sequences of ProtoRAG and a trans-acting factor, lancelet YY1-like (bbYY1), is important for the transcriptional regulation of lancelet RAG-like genes (bbRAG1L and bbRAG2L). Mechanistically, bbYY1 suppresses the transposition of ProtoRAG; meanwhile, bbYY1 promotes host DNA rejoins (HDJ) and TIR-TIR joints (TTJ) after TIR-dependent excision by facilitating the binding of bbRAG1L/2 L to TIR-containing DNA, and by interacting with the bbRAG1L/2 L complex. Our data thus suggest that bbYY1 has dual functions in fine-tuning the activity of ProtoRAG and maintaining the genome stability of the host., Lancelet expresses an ancestral RAG transposon, ProtoRAG, which predates human RAGgenes that are responsible for V(D)J recombination and adaptive immunity repertoire generation. Here the authors show that ProtoRAG is functionally regulated by a trans-acting factor, bbYY1, for tuning transposon activity and maintaining genome stability.

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.

Nitric oxide synthase is ubiquitously present in metazoans and is involved in a wide range of biological processes. Three distinct Nos genes have been so far identified in vertebrates exhibiting a complex expression pattern and transcriptional regulation. Nevertheless, although independent events of Nos duplication have been observed in several taxa, only few studies described the regulatory mechanisms responsible for their activation in non-vertebrate animals. To shed light on the mechanisms underlying neuronal-type Nos expression, we focused on two non-vertebrate chordates: the cephalochordate Branchiostoma lanceolatum and the tunicate Ciona robusta. Here, throughout transphyletic and transgenic approaches, we identified genomic regions in both species acting as Nos functional enhancers during development. In vivo analyses of Nos genomic fragments revealed their ability to recapitulate the endogenous expression territories. Therefore, our results suggest the existence of evolutionary conserved mechanisms responsible for neuronal-type Nos regulation in non-vertebrate chordates. In conclusion, this study paves the way for future characterization of conserved transcriptional logic underlying the expression of neuronal-type Nos genes in chordates.

Earliest craniates possess a newly enlarged, elaborated forebrain with new cell types and neuronal networks. A key question in vertebrate evolution is when and how this cerebral expansion took place. The exon-junction complex (EJC) plays an essential role in mRNA processing of all Eukarya. Recently, it has been proposed that the EJC represses recursive RNA splicing in Deuterostomes, with implication in human brain diseases like microcephaly and depression. However, the EJC or EJC subunit contribution to brain development in non-vertebrate Deuterostomes remained unknown. Being interested in the evolution of chordate characters, we focused on the model species, Branchiostoma lanceolatum (Cephalochordata) and Ciona robusta (Tunicata), with the aim to investigate the ancestral and the derived expression state of Magoh orthologous genes. This study identifies that Magoh is part of a conserved syntenic group exclusively in vertebrates and suggests that Magoh has experienced duplication and loss events in mammals. During early development in amphioxus and ascidian, maternal contribution and zygotic expression of Magoh genes in various types of progenitor cells and tissues are consistent with the condition observed in other Bilateria. Later in development, we also show expression of Magoh in the brain of cephalochordate and ascidian larvae. Collectively, these results provide a basis to further define what functional role(s) Magoh exerted during nervous system development and evolution.

Funding: Madeleine Aase-Remedios and Dr Clara Coll-Lladówere supported by funding from the University of St Andrews, School of Biology and additional support from the CORBEL grant European Research Infrastructure cluster project. The evolutionary transition from invertebrates to vertebrates involved extensive gene duplication, but understanding precisely how such duplications contributed to this transition requires more detailed knowledge of specific cases of genes and gene families. MyoD (Myogenic differentiation) has long been recognized as a master developmental control gene and member of the MyoD family of bHLH transcription factors (Myogenic regulatory factors, MRFs) that drive myogenesis across the bilaterians. Phylogenetic reconstructions within this gene family are complicated by multiple instances of gene duplication and loss in several lineages. Following two rounds of whole genome duplication (2R WGD) at the origin of the vertebrates the ancestral function of MRFs is thought to have become partitioned amongst the daughter genes, so that MyoD and Myf5 act early in myogenic determination while Myog and Myf6 are expressed later, in differentiating myoblasts. Comparing chordate MRFs, we find an independent expansion of MRFs in the invertebrate chordate amphioxus, with evidence for a parallel instance of subfunctionalisation relative to that of vertebrates. Conserved synteny between chordate MRF loci supports the 2R WGD events as a major force in shaping the evolution of vertebrate MRFs. We also resolve vertebrate MRF complements and organization, finding a new type of vertebrate MRF gene in the process, which allowed us to infer an ancestral two-gene state in the vertebrates corresponding to the early- and late-acting types of MRFs. This necessitates a revision of previous conclusions about the simple one-to-four origin of vertebrate MRFs. Publisher PDF

Sex determination is remarkably variable among animals with examples of environmental sex determination, male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) chromosomal sex determination, and other genetic mechanisms. The cephalochordate amphioxus occupies a key phylogenetic position as a basal chordate and outgroup to vertebrates, but its sex determination mechanism is unknown. During the course of generating Nodal mutants with transcription activator-like effector nucleases (TALENs) in amphioxus Branchiostoma floridae, serendipitously, we generated three mutant strains that reveal the sex determination mechanism of this animal. In one mutant strain, all heterozygous mutant offspring over three generations were female and all wild-type descendants were male. This pattern suggests the Nodal allele targeted is on a female-specific W chromosome. A second mutant showed the same W-linked inheritance pattern, with a female heterozygote passing the mutation only to daughters. In a third mutant strain, both male and female offspring could be heterozygous, but a female heterozygote passed the mutation only to sons. This pattern is consistent with the targeted allele being on a Z chromosome. We found an indel polymorphism linked to a Nodal allele present in most females, but no males in our cultured population. Together, these results indicate that Nodal is sex chromosome-linked in B. floridae, and that B. floridae has a ZZ/ZW sex chromosome system.

As the extant representatives of the basal chordate lineage, amphioxi (including the genera Branchiostoma, Asymmetron and Epigonichthys) play important roles in tracing the state of chordate ancestry. Previous studies have reported that members of the Branchiostoma species have similar morphological phenotypic characteristics, but in contrast, there are high levels of genetic polymorphisms in the populations. Here, we resequenced 20 Branchiostomabelcheri genomes to an average depth of approximately 12.5X using the Illumina HiSeq 2000 platform. In this study, over 52 million variations (~12% of the total genome) were detected in the B. belcheri population, and an average of 12.8 million variations (~3% of the total genome) were detected in each individual, confirming that Branchiostoma is one of the most genetically diverse species sequenced to date. Demographic inference analysis highlighted the role of historical global temperature in the long-term population dynamics of Branchiostoma, and revealed a population expansion at the Greenlandian stage of the current geological epoch. We detected 594 Single nucleotide polymorphism and 148 Indels in the Branchiostoma mitochondrial genome, and further analyzed their genetic mutations. A recent study found that the epithelial cells of the digestive tract in Branchiostoma can directly phagocytize food particles and convert them into absorbable nontoxic nutrients using powerful digestive and immune gene groups. In this study, we predicted all potential mutations in intracellular digestion-associated genes. The results showed that most “probably damaging” mutations were related to rare variants (MAF Branchiostoma. Due to the extremely high number of polymorphisms in the Branchiostoma genome, our analysis with a depth of approximately 12.5X can only be considered a preliminary analysis. However, the novel variant dataset provided here is a valuable resource for further investigation of phagocytic intracellular digestion in Branchiostoma and determination of the phenotypic and genotypic features of Branchiostoma.

Amphioxus belongs to the subphylum cephalochordata, an extant representative of the most basal chordates, whose regulation of endocrine system remains ambiguous. Here we clearly demonstrated the existence of a functional GRP neuropeptide in amphioxus, which was able to interact with GRP receptor, activate both PKC and PKA pathways, increase gh, igf, and vegf expression. We also showed that the transcription level of amphioxus grp was affected by temperature and light, indicating the role of this gene in the regulation of energy balance and circadian rhythms. In addition, the expression of the amphioxus grp was detected in cerebral vesicle that has been proposed to be the homologous organ of vertebrate brain. These data collectively suggested that a functional GRP neuropeptide had already emerged in amphioxus, which provided insights into the evolutionary origin of GRP in chordate and the functional homology between the cerebral vesicle and vertebrate brain.

Inflammatory response is an innate host defense mechanism, and its regulation is essential for the host to get rid of harm by the excessive reactions. We first utilized proteomics approach to identify amphioxus humoral fluid proteins in response to LPS-induced inflammation. A total of 26 differentially expressed proteins, mainly involved in energy metabolism and cytoskeleton rearrangement processes, were identified between LPS-treated and control animals. Furthermore, we found a single uncharacterized protein (termed BjIM1) out of the most up-regulated ones, and examined its role in the regulation of immune and inflammatory responses. BjIM1 is predominantly expressed in the hepatic caecum, and its promoter sequence includes many binding sites for immune-relevant transcription factors. Importantly, recombinant BjIM1 (rBjIM1) is able to inhibit LPS-induced up-regulation of TLR pathway genes, such as MyD88, IKK, NF-κB1, Rel, p38, JNK and AP-1, indicating that BjIM1 may negatively regulate the TLR signaling pathway in amphioxus. Moreover, rBjIM1 also modulates the expression of genes involved in the interaction network of inflammation, energy metabolism and cytoskeleton rearrangement, including SIRT1, Rac1 and NOX2, in the LPS-induced inflammatory response in amphioxus. Collectively, our studies suggest that BjIM1 is an uncharacterized protein functioning as a modulator of inflammatory networks in amphioxus.

The organizer is an essential signaling center required for axial formation during vertebrate embryonic development. In the basal chordate amphioxus, the dorsal blastopore lip of the gastrula has been proposed to be homologous to the vertebrate organizer. Lefty is one of the first genes to be expressed in the organizer. The present results show that Lefty overexpression abolishes the organizer; the embryos were severely ventralized and posteriorized, and failed to develop anterior and dorsal structures. In Lefty knockouts the organizer is enlarged, and anterior and dorsal structures are expanded. Different from Lefty morphants in vertebrates, amphioxus Lefty mutants also exhibited left-right defects. Inhibition of Nodal with SB505124 partially rescued the effects of Lefty loss-of-function on morphology. In addition, while SB505124 treatment blocked Lefty expression in the cleavage stages of amphioxus embryos, activation of Nodal signaling with Activin protein induced ectopic Lefty expression at these stages. These results show that the interplay between Lefty and Nodal signaling plays an essential role in the specification of the amphioxus organizer and axes.

Previous studies have shown that heat shock proteins (Hsps) are broadly associated in immune responses in a variety of animals. However, it remains largely unknown about the direct roles of Hsps during a bacterial infection. In this study, we have cloned and characterized the cDNAs of two Hsp genes in the amphioxus Branchiostoma japonicum, termed Bjhsp5 and Bjhsp90α, the first ones in this evolutionarily important animal. Both Bjhsp5 and Bjhsp90α showed distinct tissue expression patterns, and were inducible by challenge with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting they may be involved in anti-infectious responses. We also showed that both BjHsp5 and BjHsp90α displayed lectin-like property with affinity to both the Gram-negative and -positive bacteria as well as their signature molecules LPS and LTA, hinting they may both act as a pattern recognition receptor, capable of identifying pathogens. In addition, we found that BjHsp5 and BjHsp90α were both able to agglutinate the Gram-negative and -positive bacteria in the presence of Ca2+, suggesting they may be able to trap the invading pathogens together in vivo, avoiding them moving around and thereby protecting the host from pathogenic attack. These data provide a new angle to the roles of Hsps in immune defense.

Transforming growth factor-β activated kinase-1 (TAK1) is an important member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, which plays an important role in animal innate immune response. However, the TAK1 gene has yet not been reported in amphioxus to date. Here, we have identified and characterized a TAK1 gene from amphioxus (Branchiostoma belcheri) (named as AmphiTAK1) with the full-length cDNA of 3479 bp, including an ORF sequence of 1905 bp, a 5' UTR of 394 bp and a 3' UTR of 1180 bp. Moreover, the predicted AmphiTAK1 protein contains STKc_TAK1 domain, TAB1 and TAB2/3 binding domain which are conserved among chordate, and phylogenetic analysis also shows that the AmphiTAK1 is located at the bottom of the chordate, revealing AmphiTAK1 as a new member of the TAK1 gene family. The further qRT-PCR analysis has shown that AmphiTAK1 is widely expressed in six investigated tissues (gonad, gill, hepatic cecum, intestine, muscle and notochord) of Branchiostoma belcheri, with high expression in notochord and gonad, moderate in gill and hepatic cecum. Notably, the expression level of AmphiTAK1 is significantly up-regulated after LPS stimulation. Specially, we also find that AmphiTAK1 protein can interact with AmphiTAB1 by immunoprecipitation assay. These findings reveal that AmphiTAK1 might interact with AmphiTAB1 to involve in innate immune response of Branchiostoma belcheri. Taken together, our present works provide a new insight into evolution and innate immune response mechanism of AmphiTAK1 gene in Branchiostoma belcheri.

The similarities between amphioxus and vertebrate brains, in their regional subdivision, cell types and circuitry, make the former a useful benchmark for understanding the evolutionary innovations that shaped the latter. Locomotory control systems were already well developed in basal chordates, with the ventral neuropile of the dien-mesencephalon serving to set levels of activity and initiate locomotory actions. A chief deficit in amphioxus is the absence of complex vertebrate-type sense organs. Hence, much of vertebrate story is one of progressive improvement both to these and to sensory experience more broadly. This has two aspects: (i) anatomical and neurocircuitry innovations in the organs of special sense and the brain centres that process and store their output, and (ii) the emergence of primary consciousness, i.e. sentience. With respect to the latter, a bottom up, evolutionary perspective has a different focus from a top down human-centric one. At issue: the obstacles to the emergence of sentience in the first instance, the sequence of addition of new contents to evolving consciousness, and the homology relationship between them. A further question, and a subject for future investigation, is how subjective experience is optimized for each sensory modality.This article is part of the theme issue ‘Systems neuroscience through the lens of evolutionary theory’.

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 glycinergic or glutamatergic neurons, respectively. Glycinergic neurotransmission in invertebrates has so far only been investigated in a very limited number of species, and, although it was suggested that its functions are to some extent conserved with vertebrates, the evolution of glycinergic neurotransmission remains very 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 possesses three glycine transporter genes, two of which (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, while the other (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. While expression of GlyT2.2 is predominantly non-neural, GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are characterized by differential expression in neurons and glia, respectively. However, in vertebrates, glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by complex genetic remodeling leading to the paralog-specific inversion of gene expression. Albeit this genetic divergence between amphioxus and vertebrates, we found strong evidence for a general conservation of the role of glycinergic neurotransmission during larval swimming, allowing us to hypothesize that the neural networks controlling the rhythmic movement of chordate bodies are homologous.

The fibroblast growth factor (FGF) signalling pathway plays various roles during vertebrate embryogenesis, from mesoderm formation to brain patterning. This diversity of functions relies on the fact that vertebrates possess the largest FGF gene complement among metazoans. In the cephalochordate amphioxus, which belongs to the chordate clade together with vertebrates and tunicates, we have previously shown that the main role of FGF during early development is the control of rostral somite formation. Inhibition of this signalling pathway induces the loss of these structures, resulting in an embryo without anterior segmented mesoderm, as in the vertebrate head. Here, by combining several approaches, we show that the anterior presumptive paraxial mesoderm cells acquire an anterior axial fate when FGF signal is inhibited and that they are later incorporated in the anterior notochord. Our analysis of notochord formation in wild type and in embryos in which FGF signalling is inhibited also reveals that amphioxus anterior notochord presents transient prechordal plate features. Altogether, our results give insight into how changes in FGF functions during chordate evolution might have participated to the emergence of the complex vertebrate head.

The organ pancreas is characteristic of all vertebrates, but its evolutionary origin remains largely unknown. The serine proteinases trypsin, chymotrypsin and elastase are produced primarily in the pancreas in vertebrates. Here we clearly demonstrate by enzymatic activity assay, histochemical staining and in situ hybridization that trypsin, chymotrypsin and elastase are widely distributed in the digestive tract of the amphioxus Branchiostoma japonicum, especially in the hepatic caecum and mid-gut. This suggests that an extensive region of the amphioxus digestive tract including the hepatic caecum is homologous to the vertebrate exocrine pancreatic cells, providing a new angle for the study of the origin and evolution of vertebrate pancreas.

Vertebrate evolution was accompanied by two rounds of whole-genome duplication followed by functional divergence in terms of regulatory circuits and gene expression patterns. As a basal and slow-evolving chordate species, amphioxus is an ideal paradigm for exploring the origin and evolution of vertebrates. Single-cell sequencing has been widely used to construct the developmental cell atlas of several representative species of vertebrates (human, mouse, zebrafish, and frog) and tunicates (sea squirts). Here, we perform single-nucleus RNA sequencing (snRNA-seq) and single-cell assay for transposase accessible chromatin sequencing (scATAC-seq) for different stages of amphioxus (covering embryogenesis and adult tissues). With the datasets generated, we constructed a developmental tree for amphioxus cell fate commitment and lineage specification and characterize the underlying key regulators and genetic regulatory networks. The data are publicly available on the online platform AmphioxusAtlas.

During animal ontogenesis, body axis patterning is finely regulated by complex interactions between several signaling pathways. Nitric Oxide (NO) and Retinoic Acid (RA) are potent morphogens that play a pivotal role in vertebrate development. Their involvement in axial patterning of head and pharynx shows conserved features in the chordate phylum. Indeed, in the cephalochordate amphioxus NO and RA are crucial for the correct development of pharyngeal structures. Here we demonstrate the functional cooperation between NO and RA occurring in amphioxus embryogenesis. During neurulation, NO modulates RA production through the transcriptional regulation of Aldh1a.2 that irreversibly converts retinaldehyde into RA. On the other hand, RA regulates the transcription of Nos genes, probably through RA Response Elements found in their regulatory regions. The reciprocal regulation of NO and RA pathways results to be essential for the normal pharyngeal development in amphioxus and suggests that this regulatory crosstalk could be conserved in vertebrates.

Background Left-right (LR) asymmetry is an essential feature of bilateral animals. Studies in vertebrates show that LR asymmetry formation comprises three major steps: symmetry breaking, asymmetric gene expression, and LR morphogenesis. Although much progress has been made in the first two events, mechanisms underlying asymmetric morphogenesis remain largely unknown due to the complex developmental processes deployed by vertebrate organs. Results We here addressed this question by studying Pitx gene function in the basal chordate amphioxus whose asymmetric organogenesis, unlike that in vertebrates, occurs essentially in situ and does not rely on cell migration. Pitx null mutation in amphioxus causes loss of all left-sided organs and incomplete ectopic formation of all right-sided organs on the left side, whereas Pitx partial loss-of-function leads to milder phenotypes with only some LR organs lost or ectopically formed. At the N1 to N3 stages, Pitx expression is gradually expanded from the dorsal anterior domain to surrounding regions. This leads to activation of genes like Lhx3 and/or Prop1 and Pit, which are essential for left-side organs, and downregulation of genes like Hex and/or Nkx2.1 and FoxE4, which are required for right-side organs to form ectopically on the left side. In Pitx mutants, the left-side expressed genes are not activated, while the right-side genes fail to decrease expression on the left side. In contrast, in embryos overexpressing Pitx genes, the left-side genes are induced ectopically on the right side, and the right-side genes are inhibited. Several Pitx binding sites are identified in the upstream sequences of the left-side and right-side genes which are essential for activation of the former and repression of the latter by Pitx. Conclusions Our results demonstrate that (1) Pitx is a major (although not the only) determinant of asymmetric morphogenesis in amphioxus, (2) the development of different LR organs have distinct requirements for Pitx activity, and (3) Pitx controls amphioxus LR morphogenesis probably through inducing left-side organs and inhibiting right-side organs directly. These findings show much more dependence of LR organogenesis on Pitx in amphioxus than in vertebrates. They also provide insight into the molecular developmental mechanism of some vertebrate LR organs like the lungs and atria, since they show a right-isomerism phenotype in Pitx2 knockout mice like right-sided organs in Pitx mutant amphioxus. Our results also explain why some organs like the adenohypophysis are asymmetrically located in amphioxus but symmetrically positioned in vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates