A pre-vertebrate endodermal origin of calcitonin-producing neuroendocrine cells.

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.

Neuropeptides and receptors in the cephalochordate: A crucial model for understanding the origin and evolution of vertebrate neuropeptide systems.

Genomes and transcriptomes from diverse organisms are providing a wealth of data to explore the evolution and origin of neuropeptides and their receptors in metazoans. While most neuropeptide-receptor systems have been extensively studied in vertebrates, there is still a considerable lack of understanding regarding their functions in invertebrates, an extraordinarily diverse group that account for the majority of animal species on Earth. Cephalochordates, commonly known as amphioxus or lancelets, serve as the evolutionary proxy of the chordate ancestor. Their key evolutionary position, bridging the invertebrate to vertebrate transition, has been explored to uncover the origin, evolution, and function of vertebrate neuropeptide systems. Amphioxus genomes exhibit a high degree of sequence and structural conservation with vertebrates, and sequence and functional homologues of several vertebrate neuropeptide families are present in cephalochordates. This review aims to provide a comprehensively overview of the recent findings on neuropeptides and their receptors in cephalochordates, highlighting their significance as a model for understanding the complex evolution of neuropeptide signaling in vertebrates.

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.

A novel C-type lectin protein (BjCTL5) interacts with apoptosis stimulating proteins of p53 (ASPP) to activate NF-κB signaling pathway in primitive chordate.

C-type lectin proteins (CTLs), a group of pattern recognition receptors (PRRs), play pivotal roles in immune responses. However, the signal transduction and regulation of CTLs in cephalochordates have yet to be explored. In this study, we examined the composition of CTLs in Branchiostoma japonicum, identifying a total of 272 CTLs. These CTLs underwent further analysis concerning domain arrangement, tandem and segmental duplication events. A multidomain C-type lectin gene, designated as BjCTL5, encompassing CLECT, KR, CUB, MAM, and SR domains, was the focal point of our investigation. BjCTL5 exhibits ubiquitous expression across all detected tissues and is responsive to stimulation by LPS, mannose, and poly (I:C). The recombinant protein of BjCTL5 can bind to Escherichia coli and Staphylococcus aureus, inducing their agglutination and inhibiting the proliferation of S. aureus. Yeast two-hybrid, CoIP, and confocal immunofluorescence experiments revealed the interaction between BjCTL5 and apoptosis-stimulating proteins of p53, BjASPP. Intriguingly, BjCTL5 was observed to induce the luciferase activity of the NF-κB promoter in HEK293T cells. These results suggested a potential interaction between BjCTL5 and BjASPP, implicating that they involve in the activation of the NF-κB signaling pathway, which provides an evolutionary viewpoint on NF-κB signaling pathway in primitive chordate.

Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA.

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site 'HxE-PCxxC' and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

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.

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.

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.

Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.

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.

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.

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.

The Origination of Growth Hormone/Insulin-Like Growth Factor System: A Story From Ancient Basal Chordate Amphioxus.

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.

Identification of amphioxus protein disulfide isomerase as both an enzyme and an immunocompotent factor.

Previous studies have shown that protein disulfide isomerase (PDI), a member of the thioredoxin (TRX) superfamily, are broadly associated with immune responses in a variety of animals. However, it remains largely unknown about the direct roles of PDIs during a bacterial infection. In this study, we identified the presence of a single pdi gene in the amphioxus Branchiostoma japonicum, Bjpdi. The deduced protein BjPDI is structurally characterized by the presence of four Trx-like domains in the order of a, b, b' and a' and a short acidic C-terminal tail, that are characteristic of PDIs. We demonstrated that rBjPDI displayed both thiol reductase and disulfide bond isomerase activities, indicating comparability of BjPDI with PDIs in term of enzymatic activities. We also showed that rBjPDI induced bacterial agglutination and exhibited a lectin-like activity capable of binding both bacteria (E. coli and S. aureus) and their signature molecules LPS and LTA. Furthermore, BjPDI could kill S. aureus via inducing membrane depolarization and intracellular ROS production in vitro, and treatment of amphioxus with a blocking anti-PDI antibody in vivo markedly reduced the survival rate of amphioxus following attack by S. aureus. Collectively, our study demonstrates that amphioxus protein disulfide isomerase acts as both an enzyme and an immunocompotent factor, and reports the specific function and mode of action of PDIs in immune responses.

Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor.

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

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.

Differential expression pattern of two Brachyury genes in amphioxus embryos.

Cephalochordate amphioxus contain two Brachyury genes (AmphiBra1 and AmphiBra2). Using probes from the highly conserved coding regions, a summation of their expression profiles in amphioxus embryos have been investigated by several previous studies. However, their respective expression patterns have not been determined up to date. We here address this issue using both qRT-PCR and in situ hybridization methods (with probes from the divergent untranslated regions). qRT-PCR detected a very low maternal expression for AmphiBra2, but not for AmphiBra1. Zygotic expression of both genes are activated around early gastrula stage and change in a similar pattern at subsequent stages. However, compared to AmphiBra1, the expression level of AmphiBra2 is much higher in all examined stages of embryos; in some extreme cases an over fifty-times difference is observed. In situ hybridization and embryonic sections reveal that while AmphiBra2 is highly expressed in the blastopore, the tail bud and the notochord, AmphiBra1 is weakly transcribed only in the notochord. Our results show that the two Brachyury genes, resulted from a lineage-specific duplication in amphioxus, have evolved different embryonic expression profiles.

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.

Wnt/ß-catenin signaling is an evolutionarily conserved determinant of chordate dorsal organizer.

Deciphering the mechanisms of axis formation in amphioxus is a key step to understanding the evolution of chordate body plan. The current view is that Nodal signaling is the only factor promoting the dorsal axis specification in the amphioxus, whereas Wnt/ß-catenin signaling plays no role in this process. Here, we re-examined the role of Wnt/ßcatenin signaling in the dorsal/ventral patterning of amphioxus embryo. We demonstrated that the spatial activity of Wnt/ß-catenin signaling is located in presumptive dorsal cells from cleavage to gastrula stage, and provided functional evidence that Wnt/ß-catenin signaling is necessary for the specification of dorsal cell fate in a stage-dependent manner. Microinjection of Wnt8 and Wnt11 mRNA induced ectopic dorsal axis in neurulae and larvae. Finally, we demonstrated that Nodal and Wnt/ß-catenin signaling cooperate to promote the dorsal-specific gene expression in amphioxus gastrula. Our study reveals high evolutionary conservation of dorsal organizer formation in the chordate lineage.

Chordate PIAS proteins act as conserved repressors of the TRAF6 self-polyubiquitination.

In mammals, PIAS proteins are important SUMO E3 ligases and act as versatile regulators of over sixty different proteins, including components from the NF-κB pathways. But the PIAS functions are not well-understood due to complicated molecular mechanisms and multiple gene paralogs with overlapping roles, which is especially true in lower vertebrates where dedicated studies are scarce. As a basal chordate with a single PIAS gene, amphioxus is a convenient model to study PIAS from the evolutionary perspective. TRAF6 is a critical adaptor of the NF-κB pathways but it is not known whether TRAF6 is regulated by PIAS. Here we discover that in mammalian cells, amphioxus PIAS inhibited NF-κB activation by co-localizing and binding with TRAF6. The interaction relied on the N-terminal SAP and PINIT domains of PIAS. TRAF6 is an E3 ubiquitin ligase, which initiates downstream NF-κB signaling by promoting its self-ubiquitination. Both amphioxus SUMO1 and Ubc9 (SUMO E2 ligase) could suppress TRAF6 self-ubiquitination and NF-κB activation, suggesting that the SUMOylation activity competed away the ubiquitination activity of TRAF6. However, we show that the wild-type PIAS and the mutant PIAS without SUMO E3 ligase activity both could inhibit TRAF6-mediated NF-κB activation by reducing TRAF6 self-ubiquitination. This implies that SUMO ligase activity is not the only mechanism for PIAS to negatively regulate TRAF6. Finally, we tested the interactions between human PIAS1-4 and TRAF6. It reveals that human PIAS1, 3 and 4, but not 2, were able to repress NF-κB activation by reducing TRAF6 self-ubiquitination. Taken together, our study discovers a conserved regulatory interaction between chordate PIAS and TRAF6. It therefore sheds light on the complicated role of PIAS in immune regulation, and may help to understand the PIAS functions in other lower chordate taxa, such as jawless and jawed fishes.