Ion regulation at gills precedes gas exchange and the origin of vertebrates.

Gas exchange and ion regulation at gills have key roles in the evolution of vertebrates1-4. Gills are hypothesized to have first acquired these important homeostatic functions from the skin in stem vertebrates, facilitating the evolution of larger, more-active modes of life2,3,5. However, this hypothesis lacks functional support in relevant taxa. Here we characterize the function of gills and skin in a vertebrate (lamprey ammocoete; Entosphenus tridentatus), a cephalochordate (amphioxus; Branchiostoma floridae) and a hemichordate (acorn worm; Saccoglossus kowalevskii) with the presumed burrowing, filter-feeding traits of vertebrate ancestors6-9. We provide functional support for a vertebrate origin of gas exchange at the gills with increasing body size and activity, as direct measurements in vivo reveal that gills are the dominant site of gas exchange only in ammocoetes, and only with increasing body size or challenges to oxygen supply and demand. Conversely, gills of all three taxa are implicated in ion regulation. Ammocoete gills are responsible for all ion flux at all body sizes, whereas molecular markers for ion regulation are higher in the gills than in the skin of amphioxus and acorn worms. This suggests that ion regulation at gills has an earlier origin than gas exchange that is unrelated to vertebrate size and activity-perhaps at the very inception of pharyngeal pores in stem deuterostomes.

An ancient defense mechanism: Conservation of gasdermin-mediated pyroptosis.

The gasdermins are a family of pore-forming proteins involved in various cellular processes such as cell death and inflammation. A new study in PLOS Biology explores the evolutionary history of gasdermins across metazoans, highlighting the conservation and divergence of gasdermin E.

Characterization of GSDME in amphioxus provides insights into the functional evolution of GSDM-mediated pyroptosis.

Members of the gasdermin (GSDM) family are pore-forming effectors that cause membrane permeabilization and pyroptosis, a lytic proinflammatory type of cell death. To reveal the functional evolution of GSDM-mediated pyroptosis at the transition from invertebrates to vertebrates, we conducted functional characterization of amphioxus GSDME (BbGSDME) and found that it can be cleaved by distinct caspase homologs, yielding the N253 and N304 termini with distinct functions. The N253 fragment binds to cell membrane, triggers pyroptosis, and inhibits bacterial growth, while the N304 performs negative regulation of N253-mediated cell death. Moreover, BbGSDME is associated with bacteria-induced tissue necrosis and transcriptionally regulated by BbIRF1/8 in amphioxus. Interestingly, several amino acids that are evolutionarily conserved were found to be important for the function of both BbGSDME and HsGSDME, shedding new lights on the functional regulation of GSDM-mediated inflammation.

Functions of the FGF signalling pathway in cephalochordates provide insight into the evolution of the prechordal plate.

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.

Functional characterization of growth hormone releasing hormone and its receptor in amphioxus with implication for origin of hypothalamic-pituitary axis.

Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek's pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek's pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates.

Cilia-driven asymmetric Hedgehog signalling determines the amphioxus left-right axis by controlling Dand5 expression.

Cilia rotation-driven nodal flow is crucial for the left-right (L-R) break in symmetry in most vertebrates. However, the mechanism by which the flow signal is translated to asymmetric gene expression has been insufficiently addressed. Here, we show that Hedgehog (Hh) signalling is asymmetrically activated (L

SUMO and PIAS repress NF-κB activation in a basal chordate.

Small ubiquitin-like modifier (SUMO) regulates various biological processes, including the MyD88/TICAMs-IRAKs-TRAF6-NF-κB pathway, one of the core immune pathways. However, its functions are inconsistent between invertebrates and vertebrates and have rarely been investigated in lower chordates, including amphioxus and fishes. Here, we investigated the SUMOylation gene system in the amphioxus, a living basal chordate. We found that amphioxus has a SUMOylation system that has a complete set of genes and preserves several ancestral traits. We proceeded to study their molecular functions using the mammal cell lines. Both amphioxus SUMO1 and SUMO2 were shown to be able to attach to NF-κB Rel and to inhibit NF-κB activation by 50-75% in a dose-dependent fashion. The inhibition by SUMO2 could be further enhanced by the addition of the SUMO E2 ligase UBC9. In comparison, while human SUMO2 inhibited RelA, human SUMO1 slightly activated RelA. We also showed that, similar to human PIAS1-4, amphioxus PIAS could serve as a SUMO E3 ligase and promote its self-SUMOylation. This suggests that amphioxus PIAS is functionally compatible in human cells. Moreover, we showed that amphioxus PIAS is not only able to inhibit NF-κB activation induced by MyD88, TICAM-like, TRAF6 and IRAK4 but also able to suppress NF-κB Rel completely in the presence of SUMO1/2 in a dose-insensitive manner. This suggests that PIAS could effectively block Rel by promoting Rel SUMOylation. In comparison, in humans, only PIAS3, but not PIAS1/2/4, has been reported to promote NF-κB SUMOylation. Taken together, the findings from amphioxus, together with those from mammals and other species, not only offer insights into the functional volatility of the animal SUMO system, but also shed light on its evolutionary transitions from amphioxus to fish, and ultimately to humans.

Identification of Nodal-dependent enhancer of amphioxus Chordin sufficient to drive gene expression into the chordate dorsal organizer.

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.

Co-option of the PRDM14-CBFA2T complex from motor neurons to pluripotent cells during vertebrate evolution.

Gene regulatory networks underlying cellular pluripotency are controlled by a core circuitry of transcription factors in mammals, including POU5F1. However, the evolutionary origin and transformation of pluripotency-related transcriptional networks have not been elucidated in deuterostomes. PR domain-containing protein 14 (PRDM14) is specifically expressed in pluripotent cells and germ cells, and is required for establishing embryonic stem cells (ESCs) and primordial germ cells in mice. Here, we compared the functions and expression patterns of PRDM14 orthologues within deuterostomes. Amphioxus PRDM14 and zebrafish PRDM14, but not sea urchin PRDM14, compensated for mouse PRDM14 function in maintaining mouse ESC pluripotency. Interestingly, sea urchin PRDM14 together with sea urchin CBFA2T, an essential partner of PRDM14 in mouse ESCs, complemented the self-renewal defect in mouse Prdm14 KO ESCs. Contrary to the Prdm14 expression pattern in mouse embryos, Prdm14 was expressed in motor neurons of amphioxus embryos, as observed in zebrafish embryos. Thus, Prdm14 expression in motor neurons was conserved in non-tetrapod deuterostomes and the co-option of the PRDM14-CBFA2T complex from motor neurons into pluripotent cells may have maintained the transcriptional network for pluripotency during vertebrate evolution.This article has an associated 'The people behind the papers' interview.

Nodal and Hedgehog synergize in gill slit formation during development of the cephalochordate Branchiostoma floridae.

The larval pharynx of the cephalochordate Branchiostoma (amphioxus) is asymmetrical. The mouth is on the left, and endostyle and gill slits are on the right. At the neurula, Nodal and Hedgehog (Hh) expression becomes restricted to the left. To dissect their respective roles in gill slit formation, we inhibited each pathway separately for 20 min at intervals during the neurula stage, before gill slits penetrate, and monitored the effects on morphology and expression of pharyngeal markers. The results pinpoint the short interval spanning the gastrula/neurula transition as the critical period for specification and positioning of future gill slits. Thus, reduced Nodal signaling shifts the gill slits ventrally, skews the pharyngeal domains of Hh, Pax1/9, Pax2/5/8, Six1/2 and IrxC towards the left, and reduces Hh and Tbx1/10 expression in endoderm and mesoderm, respectively. Nodal auto-regulates. Decreased Hh signaling does not affect gill slit positions or Hh or Nodal expression, but it does reduce the domain of Gli, the Hh target, in the pharyngeal endoderm. Thus, during the neurula stage, Nodal and Hh cooperate in gill slit development - Hh mediates gill slit formation and Nodal establishes their left-right position.

Progress on the left-right asymmetry patterning in amphioxus.

The mechanisms underlying the establishment of left-right (L-R) asymmetry in bilaterians is one of the central enigmas in developmental biology. Amphioxus is an important model in studying the mechanisms of animal asymmetry specification due to its particular phylogenetic position, vertebrate-like embryogenesis and body plan. Recently, with the establishments of artificial breeding technology, high-efficiency microinjection method and gene knockout technology, researchers have successfully dissected the mechanisms of amphioxus L-R asymmetry development. In this review, we summarize the major progress in understanding L-R asymmetry specification in amphioxus and propose a model of regulation of L-R asymmetry in this species. Hh protein is transported dominantly to the right side by cilia movement, leading to R>L Hh signaling andCerexpression. Cer inhibits expression of Nodal, leading to the asymmetric expression of Nodal-dependent genes. The L-R differences in the propagation of the Nodal pathway result in the correct morphological L-R asymmetry development in amphioxus embryo. BMP signaling probably does not provide the asymmetric cue, but is necessary for correct expression ofCer andNodal.

Interplay between Lefty and Nodal signaling is essential for the organizer and axial formation in amphioxus embryos.

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.

The EJC component Magoh in non-vertebrate 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.

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

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

Canonical Wnt/ß-catenin and Notch signaling regulate animal/vegetal axial patterning in the cephalochordate amphioxus.

In bilaterians, animal/vegetal axial (A/V) patterning is a fundamental early developmental event for establishment of animal/vegetal polarity and following specification of the germ layers (ectoderm, mesoderm, endoderm), of which the evolutionary origin is enigmatic. Understanding A/V axial patterning in a basal animal from each phylum would help to reconstruct the ancestral state of germ layer specification in bilaterians and thus, the evolution of mesoderm, the third intermediate cell layer. Herein, data show that the canonical Wnt/ß-catenin (cWnt) and Notch signaling pathways control mesoderm specification from the early endomesoderm in the basal chordate amphioxus. Amphioxus belongs to the deuterostome, one of the main superphyla in Bilateria. In the present study, genes (tcf, dsh, axin, gsk3ß) encoding cWnt components were expressed in the endomesoderm during the gastrula stages. Excess cWnt signaling by BIO, a GSK3 inhibitor, expanded the expression domains of outer endomesodermal genes that include future mesodermal ones and suppressed inner endomesodermal and ectodermal genes. Interfering Notch signaling by DAPT, a γ-secretase inhibitor, resulted in decreased expression of ectodermal and endomesodermal markers. These results suggest that cWnt and Notch have important roles in mesoderm specification in amphioxus embryos. The evolution of the mesoderm is also discussed.

Identification of circular RNAs and their altered expression under poly(I:C) challenge in key antiviral immune pathways in amphioxus.

Amphioxus is a key model for studying comparative immunity of vertebrates. Circular RNA (circRNA), as RNAs with a circular structure, has received little attention until recently, where several studies have reported that circRNA expression changes are involved in the immune response in animals. However, circRNA and its immune role in amphioxus have not been previously studied. Here, circRNAs in Chinese amphioxus (Branchiostoma belcheri) were sequenced, and 1859 circRNAs were identified using two algorithms (find_circ and CIRI). The analysis of miRNA target sites on circRNAs showed that 332 circRNAs may function as miRNA sponges. Furthermore, we identified circRNAs that were conserved between B. belcheri and vertebrates, tracing the origin of these circRNAs within chordates. Additionally, in combination with several key antiviral immune (poly(I:C), pIC) pathways identified in our previous B. belcheri studies, nine circRNAs potentially involved in these pathways were identified using bioinformatic predictions. Among these nine circRNAs, eight were selected to examine their expression response in B. belcheri challenged by pIC in comparison to control using real-time quantitative PCR. The results showed that four circRNAs were induced as part of the antiviral response against pIC, while expression of two circRNAs was decreased, and the expression levels of the remaining two were not significantly altered after pIC challenge. This work is the first to identify circRNAs and reveal their antiviral role in amphioxus. Therefore, it opens a new window to explore the comparative immunology of circRNAs in chordates and the regulatory roles of circRNAs in antiviral immunity in amphioxus.

The amphioxus ERK2 gene is involved in innate immune response to LPS stimulation.

The ERK2 gene is a member of the MAPK family, which plays very important roles in responses to external environmental pressures. However, the ERK2 has yet not been identified in amphioxus to date. To further illuminate the function and evolutionary mechanism of the ERK2 gene, in this present study, we have cloned the full length of the ERK2 gene of Branchiostoma belcheri (designed as AmphiERK2), which is highly homologous to these vertebrate ERK2 genes. The AmphiERK2 protein contains the conserved S_TKc domain and the TEY motif, and its 3D structure is also highly similar to human ERK2 protein. Taken together, our results indicate that the AmphiERK2 gene belongs to a member of the ERK2 gene family. We further use qRT-PCR technology to detect an ubiquitous expression of AmphiERK2 gene in all five investigated tissues (muscle, notochord, gill, hepatic caecum and intestine), and the expression level of AmphiERK2 in both notochord and muscle is significantly higher than the other three tissues. Meanwhile our results also demonstrate that LPS stimulation can induce the up-regulation expression of AmphiERK2 gene and significantly increase the phosphorylation level of AmphiERK2 protein, which seems to imply that the AmphiERK2 may be involved in amphioxus innate immune responses. Overall, our findings provide an important insight into amphioxus innate immune function and evolution of the ERK2 gene family.

Involvement of JNK signaling pathway in lipopolysaccharide-induced complement C3 transcriptional activation from amphioxus Branchiostoma belcheri.

Complement C3 is a pivotal component of three cascades of complement activation. C3 in circulation is mainly provided by the hepatic cecum. The expression and secretion of C3 by hepatocytes is increased during acute inflammation. The detailed information on the regulationary mechanism underlying C3 transcriptional activation is limited. Here, we characterized the 5'-flanking region of the amphioxus C3 gene. To functionally analyze the upstream regulatory region of the C3 gene, a series of luciferase reporter gene constructs containing deleted or mutant regulatory elements were prepared. Using luciferase assay, we revealed that a potential C-JUN-1 binding sites within the proximal promoter region were necessary for full activation of the C3 promoter, whereas NF-κB, AP-1, C-JUN-2 and NFAT transcription factor binding sites played roles in governing the promoter activity at a homeostatic level. Our data also indicated that sp600125, a c-Jun N-terminal kinase (JNK) inhibitor, decreased lipopolysaccharide (LPS)-stimulated C3 promoter activity, mRNA expression and protein secretion using western blotting and quantitative real-time PCR analysis. These findings demonstrated that JNK signaling pathway is involved in the regulation of C3 gene transcription by targeting C-JUN transcription factor binding sites in the 5'-flanking promoter region, leading to LPS-induced C3 activation and therefore providing a potential target for regulating C3 expression.

Genome-wide organization, evolutionary diversification of the COMMD family genes of amphioxus (Branchiostoma belcheri) with the possible role in innate immunity.

The COMMD (COpper Metabolism gene MURR1 Domain) gene family with ten members participates in various biological processes, such as the regulation of copper and sodium transport, NF-κB activity and cell cycle progression. However, studies on the COMMD gene family in amphioxus (Branchiostoma belcheri) are yet largely unknown. In this study, we have identified and characterized the ten COMMD family members from amphioxus (designated as AmphiCOMMDs). Firstly, we clone the full length of AmphiCOMMDs, and all AmphiCOMMD proteins contain the conserved COMM domain with two NES (Nuclear Export Signal) motifs. Secondly, the genomic structure analysis demonstrates that genes of the COMMD family have undergone intron loss and gain during the process of divergence from amphioxus to vertebrates. Thirdly, phylogenetic analysis indicates that AmphiCOMMDs are more closely related to vertebrates, implying the AmphiCOMMDs may be the ancestor of the vertebrate COMMDs. Fourthly, AmphiCOMMDs are ubiquitously and differentially expressed in five investigated tissues (muscles, gills, intestine, heaptic cecum and notochord). Finally, our results show that expression levels of AmphiCOMMD genes are fluctuating after LPS stimulation to some different extent. Taken together, our studies have elaborated the evolutionary dynamic and the innate immune role of the COMMD family genes in amphioxus.

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.