Characterization of Amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm

Structure and developmental expression are described for amphioxus AmphiVent, a homolog of vertebrate Vent genes. In amphioxus, AmphiVent-expressing ventral mesoderm arises at midneurula by outgrowth from the paraxial mesoderm, but in vertebrates, Vent-expressing ventral mesoderm originates earlier, at the gastrula stage. In other embryonic tissues (nascent paraxial mesoderm, neural plate, endoderm, and tailbud), AmphiVent and its vertebrate homologs are expressed in similar spatiotemporal domains, indicating conservation of many Vent gene functions during chordate evolution. The ventral mesoderm evidently develops precociously in vertebrates because their relatively large embryos probably require an early and extensive deployment of the mesoderm-derived circulatory system. The vertebrate ventral mesoderm, in spite of its strikingly early advent, still resembles the nascent ventral mesoderm of amphioxus in expressing Vent homologs. This coincidence may indicate that Vent homologs in vertebrates and amphioxus play comparable roles in ventral mesoderm specification.     

Cloning and characterization of NM23-Bbt2 gene from amphioxus Branchiostoma belcheri tsingtauense

A full-length amphioxus (Branchiostoma belcheri tsingtauense) NM23-Bbt2, NM23-H2 homologue, cDNA was isolated from the cDNA library and sequenced. The obtained amphioxus NM23-Bbt2 cDNA contains an open reading frame coding for 171 amino acids. Sequence analysis showed that the amphioxus NM23-Bbt2 was highly conserved with that of other species, and all of them contained highly conserved motifs that play important roles in the function of NM23. RT-PCR revealed that NM23-Bbt2 is expressed in the neuronal tissues and is expressed in all stages during the embryogenesis. Nucleoside kinases are thought to have a critical role in regulatory processes such as signal transduction, proliferation, and differentiation. Taken together, these results suggest that nucleoside diphosphate kinases have an important role to play in embryogenic development in amphioxus. Phylogenetic analysis showed that the amphioxus group 1 NDPKs (Bf1-4) may be precursors of the human group 1 NDPKs, NM23-Bf5, NM23-Bf6, NM23-Bf7 and NM23-Bf8 may be precursors of NM23-H5, NM23-H6, NM23-H7 and M23-H8, respectively. Our finding of nine NM23 genes in Branchiostoma floride, the precursor of vertebrates, strongly suggests that the ancestral gene corresponding to each of vertebrates NM23 genes generated before the appearance of vertebrates. Comparison of the gene structures of NM23-H2 homologue from invertebrates to vertebrates suggests that the locations of three of the four introns are conserved in amphioxus and vertebrates.     

EST analysis of the immune-relevant genes in Chinese amphioxus challenged with lipopolysaccharide

It is generally accepted that the adaptive immune system is only present in vertebrates but not in invertebrates. Amphioxus is the most basal chordate and hence is an important reference to the evolution of the adaptive immune system. Here, a cDNA library of lipopolysaccharide-challenged amphioxus was constructed in order to identify immune genes. A total of 3024 expressed sequence tags (ESTs) were examined and 63 out of 398 annotated genes (16.3%) appeared related to immunity. Most of them encode cell adhesion molecules or signal proteins that are involved in immune responses. Although the key molecules such as TCR, MHC, Ig or VLR involved in the adaptive immune system were not identified in our database, we demonstrated the presence of histocompatibility-relevant genes and lymphocyte immune signaling-relevant genes. These findings support the statement that amphioxus presents some components that may be recruited by adaptive immune processes.     

文昌鱼抗金葡菌感染差减文库的构建及免疫相关基因分析

适应性免疫的起源一直是免疫学研究的关键问题.文昌鱼被认为是最接近于脊椎动物的祖先 自从被发现以来一直是研究脊椎动物起源与进化机制的经典模式动物.为了在文昌鱼中寻找适应性免疫系统的分子证据,采用金黄色葡萄球菌感染文昌鱼以调查免疫的起源.应用抑制性差减杂交(SSH)技术,通过对差减文库克隆序列的测定,共获得588个表达序列标签(EST).对这些EST进行生物信息学分析和进一步功能分类,发现了一些免疫上调基因,如免疫调控基因、凋亡相关基因、细胞黏附相关基因、转录相关基因、信号传导相关基因等,以及一些非免疫相关基因;这些基因在文昌鱼中绝大多数为首次报道.金黄色葡萄球菌差减文库的成功构建,为调查文昌鱼抗细菌感染的分子事件提供了重要线索,对于这些新发现基因的进一步研究将有助于深入了解免疫系统起源与进化的机制.     

The amphioxus genome provides unique insight into the evolution of immunity

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.     

Actors of the tyrosine kinase receptor downstream signaling pathways in amphioxus

SUMMARY One of the major goals of evo-developmentalists is to understand how the genetic mechanisms controlling embryonic development have evolved to create the current diversity of bodyplans that we encounter in the animal kingdom. Tyrosine kinase receptors (RTKs) are transmembrane receptors present in all metazoans known to control several developmental processes. They act via the activation of various cytoplasmic signaling cascades, including the mitogen-activated protein kinase (MAPK), the PI3K/Akt, and the phospholipase C-γ (PLCγ)/protein kinase C (PKC) pathways. In order to address the evolution of these three pathways and their involvement during embryogenesis in chordates, we took advantage of the complete genome sequencing of a key evolutionarily positioned species, the cephalochordate amphioxus, and searched for the complete gene set of the three signaling pathways. We found that the amphioxus genome contains all of the most important modules of the RTK-activated cascades, and looked at the embryonic expression of two genes selected from each cascade. Our data suggest that although the PI3K/Akt pathway may have ubiquitous functions, the MAPK and the PLCγ/PKC cascades may play specific roles in amphioxus development. Together with data known in vertebrates, the expression pattern of PKC in amphioxus suggests that the PLCγ/PKC cascade was implicated in neural development in the ancestor of all chordates.     

Following the genes: a framework for animal modeling of psychiatric disorders

Background

The caspase family, which plays a central role in apoptosis in metazoans, has undergone an expansion in amphioxus, increasing to 45 members through domain recombination and shuffling.

Results

In order to shed light on the conservation and uniqueness of this family in amphioxus, we cloned three representative caspase genes, designated as bbtCaspase-8, bbtCaspase-1/2 and bbtCaspase3-like, from the amphioxus Branchiostoma belcheri tsingtauense. We found that bbtCaspase-8 with conserved protein architecture is involved in the Fas-associated death domain-Caspase-8 mediated pro-apoptotic extrinsic pathway, while bbtCaspase3-like may mediate a nuclear apoptotic pathway in amphioxus. Also, bbtCaspase-1/2 can co-localize with bbtFADD2 in the nucleus, and be recruited to the cytoplasm by amphioxus apoptosis associated speck-like proteins containing a caspase recruitment domain, indicating that bbtCaspase-1/2 may serve as a switch between apoptosis and caspase-dependent innate immune response in invertebrates. Finally, amphioxus extrinsic apoptotic pathway related caspases played important roles in early embryogenesis.

Conclusions

Our study not only demonstrates the conservation of bbtCaspase-8 in apoptosis, but also reveals the unique features of several amphioxus caspases with novel domain architectures arose some 500 million years ago.     

Cloning,characterization and expression of tyrosinase-like gene in amphioxus Branchiostoma japonicum

Prophenoloxidase (tyrosinase) widely distributed in invertebrates and vertebrates, and plays a crucial role in the innate immune. In the present study, the full-length cDNA of a tyrosinase-like (designated AmphiTYR) was cloned from amphioxus Branchiostoma japonicum by PCR techniques. The full-length cDNA of AmphiTYR is 2314 bp, and its predicted open-reading frame codes for a protein of 544 amino acids with a predicted molecular mass of approximately 60.9 kDa and an isoelectric point of 5.65. It has a conserved putative copper-binding domain with six histidines in tyrosinase proteins. Six potential N-linked glycosylation sites and 14 conserved cysteine residues were also predicted to be present in B. japonicum tyrosinase. Homology analysis revealed that AmphiTYR was higher similar to vertebrates tyrosinases (32.5–40.5%) than to invertebrates phenoloxidase (6.4–25.4%). In the adult, AmphiTYR mRNA was expressed in the muscle, epidermis, notochord, ovary, hepatic caecum, pharynx and gill, but not in the neural tube, intestines and testis. During the different development stages from unfertilized egg to larvae of amphioxus, AmphiTYR expressed during all the amphioxus development. These results indicated that AmphiTYR gene not only play a pivotal role in innate immune but also play an important role during embryonic development of cephalochordate amphioxus.     

The evolution and regulation of the mucosal immune complexity in the basal chordate amphioxus

Both amphioxus and the sea urchin encode a complex innate immune gene repertoire in their genomes, but the composition and mechanisms of their innate immune systems, as well as the fundamental differences between two systems, remain largely unexplored. In this study, we dissect the mucosal immune complexity of amphioxus into different evolutionary-functional modes and regulatory patterns by integrating information from phylogenetic inferences, genome-wide digital expression profiles, time course expression dynamics, and functional analyses. With these rich data, we reconstruct several major immune subsystems in amphioxus and analyze their regulation during mucosal infection. These include the TNF/IL-1R network, TLR and NLR networks, complement system, apoptosis network, oxidative pathways, and other effector genes (e.g., peptidoglycan recognition proteins, Gram-negative binding proteins, and chitin-binding proteins). We show that beneath the superficial similarity to that of the sea urchin, the amphioxus innate system, despite preserving critical invertebrate components, is more similar to that of the vertebrates in terms of composition, expression regulation, and functional strategies. For example, major effectors in amphioxus gut mucous tissue are the well-developed complement and oxidative-burst systems, and the signaling network in amphioxus seems to emphasize signal transduction/modulation more than initiation. In conclusion, we suggest that the innate immune systems of amphioxus and the sea urchin are strategically different, possibly representing two successful cases among many expanded immune systems that arose at the age of the Cambrian explosion. We further suggest that the vertebrate innate immune system should be derived from one of these expanded systems, most likely from the same one that was shared by amphioxus.     

Ancestral genetic complexity of arachidonic acid metabolism in Metazoa

Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus.     

Role of toll-like receptors gene polymorphism in hepatocellular carcinoma

Abstract

Toll-like receptors (TLRs), evolutionarily conserved innate, play important roles in the development of autoimmunity. TLRs proteins are localized on the cell surface or in endosomes and play critical roles in innate immune responses against different pathogens. Aberrant stimulation of the innate immune system through intracellular TLRs may lead to hyperactive immune responses and contribute to the pathogenesis of hepatocellular carcinoma (HCC). HCC is the seventh most common cancer and the third leading cause of cancer deaths worldwide, and innate immune takes a most important role in HCC. There was no review to sum up the role of TLRs gene polymorphism in HCC. This review was performed to sum up the role of TLRs gene polymorphism in HCC.     

Ectopic expression of micro-RNA-1, 21 and 125a in peripheral blood immune cells is associated with chronic HBV infection

Micro-RNAs (miRNAs) play key roles in regulating genes of the immune system. The aim of this study was to examine the expression of miR-1, 21 and 125a in the immune cells taken from the peripheral blood of patients suffering from chronic HBV infection (CHB). This cross-sectional study was performed on 60 CHB patients and 60 healthy controls and expression of miR-1, 21 and 125a was evaluated using quantitative Real-Time PCR. The results showed that expression of miR-1, 21 and 125a was significantly increased in CHB patients in comparison to healthy controls. Based on our results it may be concluded that increased expression of miR-1, 21 and 125a is significantly associated with CHB and may play key roles in the induction of impaired immune responses in CHB patients.     

Functional characterization of a ficolin-mediated complement pathway in amphioxus

The ficolin-mediated complement pathway plays an important role in vertebrate immunity, but it is not clear whether this pathway exists in invertebrates. Here we identified homologs of ficolin pathway components from the cephalochordate amphioxus and investigated whether they had been co-opted into a functional ficolin pathway. Four of these homologs, ficolin FCN1, serine protease MASP1 and MASP3, and complement component C3, were highly expressed in mucosal tissues and gonads, and were significantly up-regulated following bacterial infection. Recombinant FCN1 could induce hemagglutination, discriminate among sugar components, and specifically recognize and aggregate several bacteria (especially gram-positive strains) without showing bactericidal activity. This suggested that FCN1 is a dedicated pattern-recognition receptor. Recombinant serine protease MASP1/3 formed complexes with recombinant FCN1 and facilitated the activation of native C3 protein in amphioxus humoral fluid, in which C3 acted as an immune effector. We conclude that amphioxus have developed a functional ficolin-complement pathway. Because ficolin pathway components have not been reported in non-chordate species, our findings supported the idea that this pathway may represent a chordate-specific innovation in the evolution of the complement system.     

Identification and expression of amphioxus <Emphasis Type="Italic">AmphiSmad1/5/8</Emphasis> and <Emphasis Type="Italic">AmphiSmad4</Emphasis>

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily. In this study, we identified two novel members of the Smad family, termed as AmphiSmad1/5/8 and AmphiSmad4, from Chinese amphioxus. Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains. Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1, 5 and 8 subgroup of the R-Smad subfamily, and AmphiSmad4 in the Co-Smad subfamily. The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord, myotome and alimentary canal, and may help to establish the specification of dorsal-ventral axis of amphioxus. Moreover, AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined, suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.     

Inhibitory leukocyte immunoglobulin-like receptors in cancer development

Inhibitory leukocyte immunoglobulin-like receptors(LILRB1-5) signal through immunoreceptor tyrosine-based inhibitory motifs(ITIMs) in their intracellular domains and recruit phosphatases protein tyrosine phosphatase, non-receptor type 6(PTPN6, SHP-1), protein tyrosine phosphatase, non-receptor type 6(PTPN6, SHP-2), or Src homology 2 domain containing inositol phosphatase(SHIP) to negatively regulate immune cell activation. These receptors are known to play important regulatory roles in immune and neuronal functions. Recent studies demonstrated that several of these receptors are expressed by cancer cells. Importantly, they may directly regulate development, drug resistance, and relapse of cancer, and the activity of cancer stem cells. Although counterintuitive, these findings are consistent with the generally immune-suppressive and thus tumor-promoting roles of the inhibitory receptors in the immune system. This review focuses on the ligands, expression pattern, signaling, and function of LILRB family in the context of cancer development. Because inhibition of the signaling of certain LILRBs directly blocks cancer growth and stimulates immunity that may suppress tumorigenesis, but does not disturb normal development, LILRB signaling pathways may represent ideal targets for treating hematological malignancies and perhaps other tumors.