The Molecular Mechanism of Body Axis Induction in Lampreys May Differ from That in Amphibians.

Lamprey homologues of the classic embryonic inducer Noggin are similar in expression pattern and functional properties to Noggin homologues of jawed vertebrates. All noggin genes of vertebrates apparently originated from a single ancestral gene as a result of genome duplications. nogginA, nogginB and nogginC of lampreys, like noggin1 and noggin2 of gnathostomes, demonstrate the ability to induce complete secondary axes with forebrain and eye structures when overexpressed in Xenopus laevis embryos. According to current views, this finding indicates the ability of lamprey Noggin proteins to suppress the activity of the BMP, Nodal/Activin and Wnt/beta-catenin signaling pathways, as shown for Noggin proteins of gnathostomes. In this work, by analogy with experiments in Xenopus embryos, we attempted to induce secondary axes in the European river lamprey Lampetra fluviatilis by injecting noggin mRNAs into lamprey eggs in vivo. Surprisingly, unlike what occurs in amphibians, secondary axis induction in the lampreys either by noggin mRNAs or by chordin and cerberus mRNAs, the inductive properties of which have been described, was not observed. Only wnt8a mRNA demonstrated the ability to induce secondary axes in the lampreys. Such results may indicate that the mechanism of axial specification in lampreys, which represent jawless vertebrates, may differ in detail from that in the jawed clade.

Identification of antibacterial activity of liver-expressed antimicrobial peptide 2 (LEAP2) from primitive vertebrate lamprey.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a member of the antimicrobial peptides family and plays a key role in the innate immune system of organisms. LEAP2 orthologs have been identified from a variety of fish species, however, its function in primitive vertebrates has not been clarified. In this study, we cloned and identified Lc-LEAP2 from the primitive jawless vertebrate lamprey (Lethenteron camtschaticum) which includes a 25 amino acids signal peptide and a mature peptide of 47 amino acids. Although sequence similarity was low compared to other species, the mature Lc-LEAP2 possesses four conserved cysteine residues, forming a core structure with two disulfide bonds between the cysteine residues in the relative 1-3 (Cys 58 and Cys 69) and 2-4 (Cys 64 and Cys 74) positions. Lc-LEAP2 was most abundantly expressed in the muscle, supraneural body and buccal gland of lamprey, and was significantly upregulated during LPS and Poly I:C stimulations. The mature peptide was synthesized and characterized for its antibacterial activity against different bacteria. Lc-LEAP2 possessed inhibition of a wide range of bacteria with a dose-dependence, disrupting the integrity of bacterial cell membranes and binding to bacterial genomic DNA, although its inhibitory function is weak compared to that of higher vertebrates. These data suggest that Lc-LEAP2 plays an important role in the innate immunity of lamprey and is of great value in improving resistance to pathogens. In addition, the antimicrobial mechanism of LEAP2 has been highly conserved since its emergence in primitive vertebrates.

The hagfish genome and the evolution of vertebrates.

As the only surviving lineages of jawless fishes, hagfishes and lampreys provide a crucial window into early vertebrate evolution1-3. Here we investigate the complex history, timing and functional role of genome-wide duplications4-7 and programmed DNA elimination8,9 in vertebrates in the light of a chromosome-scale genome sequence for the brown hagfish Eptatretus atami. Combining evidence from syntenic and phylogenetic analyses, we establish a comprehensive picture of vertebrate genome evolution, including an auto-tetraploidization (1RV) that predates the early Cambrian cyclostome-gnathostome split, followed by a mid-late Cambrian allo-tetraploidization (2RJV) in gnathostomes and a prolonged Cambrian-Ordovician hexaploidization (2RCY) in cyclostomes. Subsequently, hagfishes underwent extensive genomic changes, with chromosomal fusions accompanied by the loss of genes that are essential for organ systems (for example, genes involved in the development of eyes and in the proliferation of osteoclasts); these changes account, in part, for the simplification of the hagfish body plan1,2. Finally, we characterize programmed DNA elimination in hagfish, identifying protein-coding genes and repetitive elements that are deleted from somatic cell lineages during early development. The elimination of these germline-specific genes provides a mechanism for resolving genetic conflict between soma and germline by repressing germline and pluripotency functions, paralleling findings in lampreys10,11. Reconstruction of the early genomic history of vertebrates provides a framework for further investigations of the evolution of cyclostomes and jawed vertebrates.

Hagfish genome elucidates vertebrate whole-genome duplication events and their evolutionary consequences.

Polyploidy or whole-genome duplication (WGD) is a major event that drastically reshapes genome architecture and is often assumed to be causally associated with organismal innovations and radiations. The 2R hypothesis suggests that two WGD events (1R and 2R) occurred during early vertebrate evolution. However, the timing of the 2R event relative to the divergence of gnathostomes (jawed vertebrates) and cyclostomes (jawless hagfishes and lampreys) is unresolved and whether these WGD events underlie vertebrate phenotypic diversification remains elusive. Here we present the genome of the inshore hagfish, Eptatretus burgeri. Through comparative analysis with lamprey and gnathostome genomes, we reconstruct the early events in cyclostome genome evolution, leveraging insights into the ancestral vertebrate genome. Genome-wide synteny and phylogenetic analyses support a scenario in which 1R occurred in the vertebrate stem-lineage during the early Cambrian, and 2R occurred in the gnathostome stem-lineage, maximally in the late Cambrian-earliest Ordovician, after its divergence from cyclostomes. We find that the genome of stem-cyclostomes experienced an additional independent genome triplication. Functional genomic and morphospace analyses demonstrate that WGD events generally contribute to developmental evolution with similar changes in the regulatory genome of both vertebrate groups. However, appreciable morphological diversification occurred only in the gnathostome but not in the cyclostome lineage, calling into question the general expectation that WGDs lead to leaps of bodyplan complexity.

Molecular evolution of the heat shock protein family and the role of HSP30 in immune response and wound healing in lampreys (Lethenteron reissneri).

Heat shock proteins (HSPs) are molecular chaperones that ubiquitously exist in various organisms and play essential roles in protein folding, transport, and expression. While most HSPs are highly conserved across species, a few HSPs are evolutionarily distinct in some species and may have unique functions. To explore the evolutionary history of the vertebrate HSP family, we identify members of the HSP family at the genome-wide level in lampreys (Lethenteron reissneri), a living representative of jawless vertebrates diverged from jawed vertebrates over 500 million years ago. The phylogenetic analysis reveals that the lamprey HSP family contains HSP90a1, HSP90a2, HSC70, HSP60, HSP30, HSP27, HSP17, and HSP10, which have a primitive status in the molecular evolution of vertebrate HSPs. Transcriptome analysis reveals the expression distribution of members of the HSP family in various tissues of lampreys. It is shown that HSP30, normally found in birds, amphibians, and fish, is also present in lampreys, with remarkable expansion of HSP30 gene copies in the lamprey genome. The transcription of HSP30 is significantly induced in leukocytes and heart of lampreys during various pathogens or poly(I:C) stimulation, indicating that HSP30 may be involved in the immune defense of lampreys in response to bacterial or viral infection. Immunohistochemistry demonstrates significantly increased HSP30 expression in subcutaneous muscle tissue after skin injury in lamprey models of wound repair. Furthermore, transcriptome analysis shows that ectopic expression of HSP30 in 3T3-L1 fibroblasts affect the expression of genes related to the PI3K-AKT signaling pathway, suggesting that HSP30 could serves as a negative regulator of fibrosis. These results indicate that HSP30 may play a critical role in facilitating the process of lamprey skin repair following injury. This study provides new insights into the origin and evolution of the HSP gene family in vertebrates and offers valuable clues to reveal the important role of HSP30 in immune defense and wound healing of lampreys.

Phylogenomic investigation of lampreys (Petromyzontiformes).

The history of lamprey evolution has been contentious due to limited morphological differentiation and limited genetic data. Available data has produced inconsistent results, including in the relationship among northern and southern species and the monophyly of putative clades. Here we use whole genome sequence data sourced from a public database to identify orthologs for 11 lamprey species from across the globe and build phylogenies. The phylogeny showed a clear separation between northern and southern lamprey species, which contrasts with some prior work. We also find that the phylogenetic relationships of our samples of two genera, Lethenteron and Eudontomyzon, deviate from the taxonomic classification of these species, suggesting that they require reclassification.

Evolution of the expression and regulation of the nuclear hormone receptor ERR gene family in the chordate lineage.

The Estrogen Related Receptor (ERR) nuclear hormone receptor genes have a wide diversity of roles in vertebrate development. In embryos, ERR genes are expressed in several tissues, including the central and peripheral nervous systems. Here we seek to establish the evolutionary history of chordate ERR genes, their expression and their regulation. We examine ERR expression in mollusc, amphioxus and sea squirt embryos, finding the single ERR orthologue is expressed in the nervous system in all three, with muscle expression also found in the two chordates. We show that most jawed vertebrates and lampreys have four ERR paralogues, and that vertebrate ERR genes were ancestrally linked to Estrogen Receptor genes. One of the lamprey paralogues shares conserved expression domains with jawed vertebrate ERRγ in the embryonic vestibuloacoustic ganglion, eye, brain and spinal cord. Hypothesising that conserved expression derives from conserved regulation, we identify a suite of pan-vertebrate conserved non-coding sequences in ERR introns. We use transgenesis in lamprey and chicken embryos to show that these sequences are regulatory and drive reporter gene expression in the nervous system. Our data suggest an ancient association between ERR and the nervous system, including expression in cells associated with photosensation and mechanosensation. This includes the origin in the vertebrate common ancestor of a suite of regulatory elements in the 3' introns that drove nervous system expression and have been conserved from this point onwards.

A complete prostaglandin pathway from synthesis to inactivation in the oral gland of the jawless vertebrate lamprey, Lethenteron camtschaticum.

Information on the prostaglandin pathway in lampreys is limited. Here, five genes related to the prostaglandin pathway from synthesis to inactivation, namely, phospholipase A2, cyclooxygenase-2, prostaglandin E synthase 3, prostaglandin D synthase, and 15-hydroxyprostaglandin dehydrogenase [NAD(+)], were screened and cloned from the lamprey, Lethenteron camtschaticum. Bioinformatic analysis showed that these lamprey genes are relatively conserved with teleost genes in domains, motifs, gene structure and 3D structure. Analysis of expression distribution of the genes in lamprey tissues revealed that a complete prostaglandin pathway from synthesis to inactivation exists in the oral gland of lamprey, especially the key gene of prostaglandin synthesis cyclooxygenase-2, which was highly expressed in the oral gland. Furthermore, cyclooxygenase-2 expression increased after LPS and Poly I:C stimulations. Using our established spatial metabolite database LampreyDB, six prostaglandin-related metabolites were screened from the oral gland of lamprey, four of which were highly expressed in the oral gland. This study provides new insights into prostaglandin synthesis and inactivation pathways in lamprey, thereby improving our understanding of the origin and evolution of the prostaglandin pathway and contributing to the recognition of lamprey regulatory mechanisms in development and immunity.

ß Pore-forming Protein-based Evolutionary Divergence of Gnathostomata from Agnatha.

INTRODUCTION: The first vertebrates were jawless fish, or Agnatha, whose evolution diverged into jawed fish, or Gnathostomes, around 550 million years ago. METHODS: In this study, we investigated ß PFT proteins' evolutionary divergence of lamprey immune protein from Agnatha, reportedly possessing anti-cancer activity, into Dln1 protein from Gnathostomes. Both proteins showed structural and functional divergence, and shared evolutionary origin. Primary, secondary and tertiary sequences were compared to discover functional domains and conserved motifs in order to study the evolution of these two proteins. The structural and functional information relevant to evolutionary divergence was revealed using hydrophobic cluster analysis. RESULTS: The findings demonstrate that two membrane proteins with only a small degree of sequence identity can have remarkably similar hydropathy profiles, pointing towards conserved and similar global structures. When facing the lipid bilayer or lining the pore lumen, the two proteins' aerolysin domains' corresponding residues displayed a similar and largely conserved pattern. Aerolysin-like proteins from different species can be identified using a fingerprint created by PIPSA analysis of the pore-forming protein. CONCLUSION: We were able to fully understand the mechanism of action during pore formation through structural studies of these proteins.

Identification and characterization of tryptophan-kynurenine pathway-related genes involving lamprey (Lampetra japonica) innate immunity.

The tryptophan-kynurenine (TRP-KYN) pathway is involved in several biological functions, including immunosuppression, inflammatory response, and tumor suppression. Six TRP-KYN pathway-related genes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 2 (IDO2), aminoadipate aminotransferase (AADAT), glutamate oxaloacetate transaminase 2 (GOT2), kynurenine monooxygenase (KMO), and kynureninase (KYNU) have been identified and cloned from the jawless vertebrate lamprey (Lampetra japonica) to gain insights into their evolution and characterization. Expression distribution showed that the key gene Lj-TDO was highly expressed in the oral gland. Real-time quantitative PCR showed that TRP-KYN pathway-related genes were significantly overexpressed after multi-stimulation. RNA interference showed that Lj-IDO2 knockdown regulated the expression of inflammatory factors. In conclusion, our study successfully clarified the ancestral features and functions of the TRP-KYN pathway, while providing valuable insights into the involvement of this pathway in the immune responses of a jawless vertebrate.

Identification and characterization of Toll-like receptor 14d in Northeast Chinese lamprey (Lethenteron morii).

Toll-like receptors (TLRs) play an important role in innate immunity of defense against bacterial or viral pathogens. To study the biological characteristics and functions of the TLR genes, TLR14d was identified from Northeast Chinese lamprey (Lethenteron morii) and named LmTLR14d. LmTLR14d coding sequence (cds) is 3285 bp in length and encodes 1094 amino acids (aa). The results showed that LmTLR14d has the typical structure of TLR molecule, which contains the extracellular domain of leucine-rich repeats (LRR), transmembrane domain, and intracellular domain of Toll/interleukin-1 receptor (TIR). The phylogenetic tree showed that LmTLR14d is a homologous gene of TLR14/18 in bony fish. Quantitative real-time PCR (qPCR) revealed that LmTLR14d was expressed in various healthy tissues, including immune and non-immune tissues. Pseudomonas aeruginosa infection up-regulated LmTLR14d in the supraneural body (SB), gill, and kidney tissues of infected Northeast Chinese lamprey. Immunofluorescence results showed that LmTLR14d was located in the cytoplasm of HEK 293T cells in clusters, and its subcellular localization was determined by the TIR domain. The immunoprecipitation results showed that LmTLR14d could recruit L.morii MyD88 (LmMyD88) but not L.morii TRIF (LmTRIF). Dual luciferase reporter results showed that LmTLR14d significantly enhanced the activity of L.morii NF-κß (LmNF-κß) promoter. Furthermore, co-transfection of LmTLR14d with MyD88 significantly enhanced the L.morii NF-κß (LmNF-κß) promoter activity. LmTLR14d can induce the expression of inflammatory cytokine genes il-6 and tnf-α downstream of NF-κB signal. This study suggested that LmTLR14d might play an important role in the innate immune signal transduction process of lamprey and revealed the origin and function of teleost-specific TLR14.

High-quality total RNA extraction from early-stage lamprey embryos.

High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.

Molecular evolution and gene expression of ferritin family involved in immune defense of lampreys.

Ferritin, one of the key regulators of iron homeostasis, is widely present throughout almost all species. The vertebrate ferritin family, which originates from a single gene in the ancestral invertebrates, contains the widest variety of ferritin subtypes among all animal species. However, the evolutionary history of the vertebrate ferritin family remains to be further clarified. In this study, genome-wide identification of the ferritin homologs is performed in lampreys, which are the extant representatives of jawless vertebrates that diverged from the future jawed vertebrates more than 500 million years ago. Molecular evolutionary analyses show that four members of the lamprey ferritin family, L-FT1-4, are derived from a common ancestor with jawed vertebrate ferritins prior to the divergence of the jawed vertebrate ferritin subtypes. The lamprey ferritin family shares evolutionarily conserved characteristics of the ferritin H subunit with higher vertebrates, but certain members such as L-FT1 additionally accumulate some features of the M or L subunits. Expression profiling reveals that lamprey ferritins are highly expressed in the liver. The transcription of L-FT1 is significantly induced in the liver and heart during lipopolysaccharide stimulation, indicating that L-FTs may play a role in the innate immune response to bacterial infection in lampreys. Furthermore, the transcriptional expression of L-FT1 in quiescent and LPS-activated leukocytes is up- and down-regulated by the lamprey TGF-ß2, an essential regulator of the inflammatory response, respectively. Our results provide new insights into the origin and evolution of the vertebrate ferritin family and reveal that lamprey ferritins may be involved in immune regulation as target genes of the TGF-ß signaling pathway.

Cloning and Characterization of Cyp7a1 and Cyp27a1 Genes from the Non-Parasitic Japanese Lamprey Lethenteron reissneri.

Two cytochrome P450 genes homologous to human CYP7A1 and CYP27A1 were cloned from the non-parasitic Japanese lamprey Lethenteron reissneri. Lamprey cyp7a1 mRNA had varied expression levels among individuals: about four orders of magnitude differences in larval liver and nearly three orders of magnitude differences in male adult liver. Overexpressed Cyp7a1 protein tagged with green fluorescent protein (GFP) was localized to the endoplasmic reticulum. Lamprey cyp27a1 mRNA had relatively constant expression levels: within two orders of magnitude differences in larvae and adult liver and intestine. GFP-tagged Cyp27a1 protein was localized to mitochondria. The expression profiles of lamprey cyp7a1 and cyp27a1 genes and the cellular localizations of their products were in good agreement with their counterparts in mammals, where these two P450s catalyze initial hydroxylation reactions of cholesterol in classical and alternative pathways of bile acid synthesis, respectively. The cyp7a1 mRNA levels in adult male liver showed significant negative correlations to both body weight and total length of the animal, implying the involvement of the gene in the production of female-attractive pheromones in sexually matured male livers. The lamprey Cyp7a1 contains a long extension of 116 amino acids between helices D and E of the protein. Possible roles of this extension in regulating the enzymatic activity of lamprey Cyp7a1 are discussed.

Phylogeny and expression of ADAM10 and ADAM17 homologs in lamprey.

The ADAMs (a disintegrin and metalloproteinase) play regulatory roles in cell adhesion, migration and proteolysis. To explore the origin and evolution of ADAMs, this study identified the homologs of adam10 and adam17 in Lampetra morii and Lampetra japonica. Sequence analysis revealed that they share the same genomic structures with their counterparts in jawed vertebrates. The putative proteins possess conserved motifs, including a furin cut site (RXXR) for precursor processing, an enzyme catalytic motif (HEXGEHXXGXXH) for hydrolysis, and a Ca2+-binding motif (CGNXXXEXGEXCD) for stabilizing protein structure. In addition, a substrate recognition domain is present at the membrane-proximal region of lamprey ADAM17. The cytoplasmic region of lamprey ADAM10 contains a potential threonine phosphorylation site which has been shown to be activated by protein kinase C (PKC) in mammals. Both the adam10 and adam17 genes were constitutively expressed in the brain, kidney, and gills and were differentially regulated in the primary blood leukocytes by lipopolysaccharide (LPS) and pokeweed mitogen (PWM). Adam10 was induced by LPS but not PWM; conversely, adam17 was induced by PWM but not LPS. Taken together, our results suggest that the activation pathways and functions of ADAM10 and ADAM17 are conserved in agnathans.

Phylogenetic and functional properties of hagfish neurohypophysial hormone receptors distinct from their jawed vertebrate counterparts.

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.

Molecular characterization and expression analysis of a novel cold-inducible RNA-binding protein (CIRBP) gene in lamprey (Lethenteron reissneri).

Cold-inducible RNA-binding protein (CIRBP) responds to a wide array of cellular stresses such as cold shock, hypoxia, and inflammatory responses. However, functional studies of CIRBP in jawless vertebrates are limited. In this study, a CIRBP homolog from the jawless vertebrate lamprey (Lethenteron reissneri) was cloned and characterized (named Lr-CIRBP). The cDNA fragment of Lr-CIRBP has a 516 bp open reading frame (ORF) that encodes 171 amino acids, comprising a glycine-rich region at the C-terminal, similar to higher vertebrates but slightly shorter, and an RNA recognition motif (RRM) domain at the N-terminus. The predicted Lr-CIRBP sequence had 51.4 ~ 70.6% similarity with CIRBPs from other vertebrates. Further phylogenetic analysis revealed that Lr-CIRBP is located in the outgroup of vertebrates and is the ancestor of vertebrates. Based on real-time quantitative PCR experimental analysis, Lr-CIRBP expression was highest in leukocytes and increased significantly after multi-stimulation, peaking at 12 h. RNA interference showed that Lr-CIRBP knockdown can down-regulate the expression of inflammatory factors in Lethenteron reissneri. In conclusion, our study successfully clarifies the ancestral features and functions of CIRBP, while revealing valuable insight into how the protein is involved in the immune responses of a jawless vertebrate.

Phylogenetics and the Cenozoic radiation of lampreys.

The development of a movable jaw is one of the most important transitions in the evolutionary history of animals.1 Jawed vertebrates rapidly diversified after appearing approximately 470 million years ago. Today, only lampreys and hagfishes represent the once dominant jawless grade2,3,4 and comprise less than 1% of living vertebrate species. Their relationship to other vertebrates ranks among the more contentious problems in animal phylogenetics.5,6,7,8,9,10,11,12 Further, the phylogenetic relationships within lampreys and hagfishes remain unclear,13,14,15 and the ages of their living lineages are largely unexplored.16,17 Because of their importance for the genomic and developmental changes that prefigured jawed vertebrate diversity,18,19,20,21 the evolutionary history of lampreys and hagfishes is a major frontier of organismal biology. Of these two clades, lampreys22 are more ecologically diverse, exhibiting freshwater, anadromous, and fully marine forms, as well as parasitic and nonparasitic species.23,24 Here, we present a new phylogeny and historical biogeographic reconstruction of all living lampreys. Whereas the early diversification of this clade tracks Pangaean fragmentation, lampreys also rapidly radiated in the northern hemisphere during the mid-Cretaceous and directly after the Cretaceous-Paleogene extinction. These radiations mirrored concurrent ones in other animals and plants and coincided with changes to lamprey ecology and feeding behavior. Our results suggest that 80% of living lamprey clades appeared in the last 20 million years of Earth history. Rather than gradually accumulating since the oldest stem-group forms appeared in the early Paleozoic, living lamprey biodiversity results from diversifications extending from the Cretaceous to present.

Molecular Characterization of a B Cell Adaptor for Phosphoinositide 3-Kinase Homolog in Lamprey (Lampetra japonica) and Its Function in the Immune Response.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.