Pan-Vertebrate Toll-Like Receptors During Evolution

Human toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) to raise innate immune responses. The human TLR family was discovered because of its sequence similarity to fruit fly (Drosophila) Toll, which is involved in an anti-fungal response. In this review, we focus on the origin of the vertebrate TLR family highlighted through functional and phylogenetic analyses of TLRs in non-mammalian vertebrates. Recent extensive genome projects revealed that teleosts contain almost all subsets of TLRs that correspond to human TLRs (TLR1, 2, 3, 4, 5, 7, 8, and 9), whereas the urochordate Ciona intestinalis contains only a few TLR genes. Therefore, mammals likely obtained almost all TLR family members at the beginning of vertebrate evolution. This premise is further supported by several functional analyses of non-mammalian TLRs. We have summarized several teleost TLRs with unique properties distinct from mammalian TLRs to outline their specific roles. According to Takifugu rubripes genome project, the puffer fish possesses fish-specific TLR21 and 22. Surprisingly, phylogenetic analyses indicate that TLR21 and 22 emerged during an early period of vertebrate evolution in parallel with other TLRs and that the mammalian ancestor lost TLR21 and 22 during evolution. Our laboratory recently revealed that TLR22 recognizes double-strand RNA and induces interferon production through the TICAM-1 adaptor, as in TLR3, but unlike TLR3, TLR22 localizes to the cell surface. Therefore, differential expression of TLR3 and TLR22, rather than simple redundancy of RNA sensors, may explain the effective protection of fish from RNA virus infection in the water. In this review, we summarize the similarities and differences of the TLR family in various vertebrates and introduce these unique TLRs for a possible application to the field of clinical practices for cancer or virus infection.     

Immunogenetics of the NKG2D ligand gene family

NKG2D ligands (NKG2DLs) are a group of major histocompatibility complex (MHC) class I-like molecules, the expression of which is induced by cellular stresses such as infection, tumorigenesis, heat shock, tissue damage, and DNA damage. They act as a molecular danger signal alerting the immune system for infected or neoplastic cells. Mammals have two families of NKG2DL genes: the MHC-encoded MIC gene family and the ULBP gene family encoded outside the MHC region in most mammals. Rodents such as mice and rats lack the MIC family of ligands. Interestingly, some mammals have NKG2DL-like molecules named MILL that are phylogenetically related to MIC, but do not function as NKG2DLs. In this paper, we review our current knowledge of the MIC, ULBP, and MILL gene families in representative mammalian species and discuss the origin and evolution of the NKG2DL gene family.     

High-mobility-group family genes from Lampetra japonica reveal their early origin and molecular evolution in the vertebrate lineage

High-mobility group family (HMG) genes are ubiquitous in vertebrates, including mammals, birds, amphibians and fishes. To elucidate the molecular phylogeny of the HMG genes in the primitive vertebrate, we have cloned three homologues of HMG-box genes, called Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX, from a cDNA library generated from lymphocyte-like cells of the Japanese lamprey (Lampetra japonica), an Agnathan that occupies a critical phylogenetic position between invertebrates and vertebrates. The open reading frames of Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX contained 627 bp, 585 bp and 678 bp, respectively. The analysis of the deduced amino acid sequences indicated that these three putative Lj-HMGB proteins contain four domains: HMG-box A, HMG-box B, an acidic carboxyl-terminal tail and a linker. A phylogenetic analysis revealed that the Lj-HMGB proteins fall outside the vertebrate clade; Lj-HMGBX is descended from a gene ancestral to the mammalian HMGB1/2/3. This discovery implies that there was a gene duplication event in the HMGB1/2/3 gene family that occurred after the divergence of the vertebrates (Cyclostomata) from the Cephalochordata and Urochordata at least 450 million years ago (MYA). The Lj-HMGB1, Lj-HMGB2 and Lj-HMGBX genes were detected in most tissues of the lamprey by RT-PCR. Our findings provide insight into the phylogeny of the HMGB genes in vertebrates.     

Phospholipase C and Diacylglycerol Mediate Olfactory Responses to Amino Acids in the Main Olfactory Epithelium of an Amphibian

The semi-aquatic lifestyle of amphibians represents a unique opportunity to study the molecular driving forces involved in the transition of aquatic to terrestrial olfaction in vertebrates. Most amphibians have anatomically segregated main and vomeronasal olfactory systems, but at the cellular and molecular level the segregation differs from that found in mammals. We have recently shown that amino acid responses in the main olfactory epithelium (MOE) of larval Xenopus laevis segregate into a lateral and a medial processing stream, and that the former is part of a vomeronasal type 2 receptor expression zone in the MOE. We hypothesized that the lateral amino acid responses might be mediated via a vomeronasal-like transduction machinery. Here we report that amino acid-responsive receptor neurons in the lateral MOE employ a phospholipase C (PLC) and diacylglycerol-mediated transduction cascade that is independent of Ca²⁺ store depletion. Furthermore, we found that putative transient receptor potential (TRP) channel blockers inhibit most amino acid-evoked responses in the lateral MOE, suggesting that ion channels belonging to the TRP family may be involved in the signaling pathway. Our data show, for the first time, a widespread PLC- and diacylglycerol-dependent transduction cascade in the MOE of a vertebrate already possessing a vomeronasal organ.     

Prediction of Functionally Important Phospho-Regulatory Events in Xenopus laevis Oocytes

The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 2636 phosphosites. We used structural information and phosphoproteomic data for 13 other species in order to predict functionally important phospho-regulatory events. We found that the degree of conservation of phosphosites across species is predictive of sites with known molecular function. In addition, we predicted kinase-protein interactions for a set of cell-cycle kinases across all species. The degree of conservation of kinase-protein interactions was found to be predictive of functionally relevant regulatory interactions. Finally, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. Our analysis suggests that a small class of phosphosites occurs in positions that have the potential to regulate protein conformation.     

Development and Degeneration of Retinal Ganglion Cell Axons in Xenopus tropicalis

Neurons make long-distance connections via their axons, and the accuracy and stability of these connections are crucial for brain function. Research using various animal models showed that the molecular and cellular mechanisms underlying the assembly and maintenance of neuronal circuitry are highly conserved in vertebrates. Therefore, to gain a deeper understanding of brain development and maintenance, an efficient vertebrate model is required, where the axons of a defined neuronal cell type can be genetically manipulated and selectively visualized in vivo. Placental mammals pose an experimental challenge, as time-consuming breeding of genetically modified animals is required due to their in utero development. Xenopus laevis, the most commonly used amphibian model, offers comparative advantages, since their embryos ex utero during which embryological manipulations can be performed. However, the tetraploidy of the X. laevis genome makes them not ideal for genetic studies. Here, we use Xenopus tropicalis, a diploid amphibian species, to visualize axonal pathfinding and degeneration of a single central nervous system neuronal cell type, the retinal ganglion cell (RGC). First, we show that RGC axons follow the developmental trajectory previously described in X. laevis with a slightly different timeline. Second, we demonstrate that co-electroporation of DNA and/or oligonucleotides enables the visualization of gene function-altered RGC axons in an intact brain. Finally, using this method, we show that the axon-autonomous, Sarm1-dependent axon destruction program operates in X. tropicalis. Taken together, the present study demonstrates that the visual system of X. tropicalis is a highly efficient model to identify new molecular mechanisms underlying axon guidance and survival.     

Virus-Host Coevolution: Common Patterns of Nucleotide Motif Usage in Flaviviridae and Their Hosts

Virus-host biological interaction is a continuous coevolutionary process involving both host immune system and viral escape mechanisms. Flaviviridae family is composed of fast evolving RNA viruses that infects vertebrate (mammals and birds) and/or invertebrate (ticks and mosquitoes) organisms. These host groups are very distinct life forms separated by a long evolutionary time, so lineage-specific anti-viral mechanisms are likely to have evolved. Flaviviridae viruses which infect a single host lineage would be subjected to specific host-induced pressures and, therefore, selected by them. In this work we compare the genomic evolutionary patterns of Flaviviridae viruses and their hosts in an attempt to uncover coevolutionary processes inducing common features in such disparate groups. Especially, we have analyzed dinucleotide and codon usage patterns in the coding regions of vertebrate and invertebrate organisms as well as in Flaviviridae viruses which specifically infect one or both host types. The two host groups possess very distinctive dinucleotide and codon usage patterns. A pronounced CpG under-representation was found in the vertebrate group, possibly induced by the methylation-deamination process, as well as a prominent TpA decrease. The invertebrate group displayed only a TpA frequency reduction bias. Flaviviridae viruses mimicked host nucleotide motif usage in a host-specific manner. Vertebrate-infecting viruses possessed under-representation of CpG and TpA, and insect-only viruses displayed only a TpA under-representation bias. Single-host Flaviviridae members which persistently infect mammals or insect hosts (Hepacivirus and insect-only Flavivirus, respectively) were found to posses a codon usage profile more similar to that of their hosts than to related Flaviviridae. We demonstrated that vertebrates and mosquitoes genomes are under very distinct lineage-specific constraints, and Flaviviridae viruses which specifically infect these lineages appear to be subject to the same evolutionary pressures that shaped their host coding regions, evidencing the lineage-specific coevolutionary processes between the viral and host groups.     

Phylogenetic Analysis of the MS4A and TMEM176 Gene Families

Background

The MS4A gene family in humans includes CD20 (MS4A1), FcRβ (MS4A2), Htm4 (MS4A3), and at least 13 other syntenic genes encoding membrane proteins, most having characteristic tetraspanning topology. Expression of MS4A genes is variable in tissues throughout the body; however, several are limited to cells in the hematopoietic system where they have known roles in immune cell functions. Genes in the small TMEM176 group share significant sequence similarity with MS4A genes and there is evidence of immune function of at least one of the encoded proteins. In this study, we examined the evolutionary history of the MS4A/TMEM176 families as well as tissue expression of the phylogenetically earliest members, in order to investigate their possible origins in immune cells.

Principal Findings

Orthologs of human MS4A genes were found only in mammals; however, MS4A gene homologs were found in most jawed vertebrates. TMEM176 genes were found only in mammals and bony fish. Several unusual MS4A genes having 2 or more tandem MS4A sequences were identified in the chicken (Gallus gallus) and early mammals (opossum, Monodelphis domestica and platypus, Ornithorhyncus anatinus). A large number of highly conserved MS4A and TMEM176 genes was found in zebrafish (Danio rerio). The most primitive organism identified to have MS4A genes was spiny dogfish (Squalus acanthus). Tissue expression of MS4A genes in S. acanthias and D. rerio showed no evidence of expression restricted to the hematopoietic system.

Conclusions/Significance

Our findings suggest that MS4A genes first appeared in cartilaginous fish with expression outside of the immune system, and have since diversified in many species into their modern forms with expression and function in both immune and nonimmune cells.     

Genetics,Morphology, Advertisement Calls,and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus,Pipidae) from West and Central Africa

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).     

5 S DNAs of Xenopus laevis and Xenopus mulleri: evolution of a gene family

The 5 S DNA which contains the genes for 5 S RNA has been purified from the frog Xenopus mulleri and compared with the 5 S DNA of Xenopus laevis. Both DNAs contain highly repetitive sequences in which the gene sequence that codes for 5 S RNA alternates with a spacer sequence. The 5 S DNAs of X. laevis and X. mulleri comprise about 0.7% of the total DNA or about 24,000 and 9000 repeating sequences, respectively. The average repeat length within native X. laevis and X. mulleri 5 S DNA is about 0.5 to 0.6 and 1.2 to 1.5 × 10⁶ daltons, respectively, each repeat of which contains a single gene sequence for 5 S RNA (0.08 × 10⁶ daltons). The two DNAs differ in the average length of their spacers and no cross homology can be detected by heterologous hybridization of the two DNAs, except within the 5 S RNA gene regions. Despite their differences, the spacer sequences of X. laevis and X. mulleri 5 S DNA resemble each other enough to conclude that they have diverged from a common ancestral sequence.The multiple repeating sequences of 5 S DNA in each species have evolved as a family of similar, but not identical sequences. It is known that 5 S DNA is located at the ends (telomeres) of the long arms of most, if not all, X. laevis chromosomes. It is proposed that multiple gene sequences located on the ends of many chromosomes can evolve together as a family if there is extensive and unequal exchange of DNA sequences between homologous and non-homologous chromosomes at their ends.     

Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.     

Prickle1 is necessary for the caudal migration of murine facial branchiomotor neurons

Facial branchiomotor neurons (FBMs) of vertebrates typically develop in rhombomere 4 (r4), and in mammals and several other vertebrate taxa, migrate caudally into r6 and subsequently laterally and ventrally to the pial surface. How similar or dissimilar these migratory processes between species are at a molecular level remains unclear. In zebrafish and mouse, mutations in certain PCP genes disrupt normal caudal migration of FBMs. Zebrafish prickle1a (prickle-like 1a) and prickle1b, two orthologs of Prickle1, act non-cell-autonomously and cell-autonomously, respectively, to regulate FBM migration. Here, we show that, in Prickle1 ^C251X/C251X mice which have reduced Prickle1 expression, the caudal migration of FBMs is affected. Most FBM neurons do not migrate caudally along the floor plate. However, some neurons perform limited caudal migration such that the neurons eventually lie near the pial surface from r4 to anterior r6. FBMs in Prickle1 ^C251X/C251X mice survive until P0 and form an ectopic nucleus dorsal to the olivo-cochlear efferents of r4. Ror2, which modifies the PCP pathway in other systems, is expressed by the migrating mouse FBMs, but is not required for FBM caudal migration. Our results suggest that, in mice, Prickle1 is part of a molecular mechanism that regulates FBM caudal migration and separates the FBM and the olivo-cochlear efferents. This defective caudal migration of FBMs in Prickle1C251X mutants resembles Vangl2 mutant defects. In contrast to other developing systems that show similar defects in Prickle1, Wnt5a and Ror2, the latter two only have limited or no effect on FBM caudal migration.     

Members of the Ikaros gene family are present in early representative vertebrates

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.     

Characterization of a divergent non-classical MHC class I gene in sharks

Sharks are the most ancient group of vertebrates known to possess members of the major histocompatibility complex (MHC) gene family. For this reason, sharks provide a unique opportunity to gain insight into the evolution of the vertebrate immune system through comparative analysis. Two genes encoding proteins related to the MHC class I gene family were isolated from splenic cDNA derived from spiny dogfish shark ( Squalus acanthias). The genes have been designated MhcSqac-UAA*01 and MhcSqac-UAA*NC1. Comparative analysis demonstrates that the Sqac-UAA*01 protein sequence clusters with classical MHC class I of several shark species and has structural elements common to most classical MHC class I molecules. In contrast, Sqac-UAA*NC1 is highly divergent from all vertebrate classical MHC class I proteins, including the Sqac-UAA *01 sequence and those of other shark species. Although Sqac-UAA*NC1 is clearly related to the MHC class I gene family, no orthologous genes from other species were identified due to the high degree of sequence divergence. In fact, the Sqac NC1 protein sequence is the most divergent MHC class-I-like protein identified thus far in any shark species. This high degree of divergence is similar in magnitude to some of the MHC class-I-related genes found in mammals, such as MICA or CD1. These data support the existence of a class of highly divergent non-classical MHC class I genes in the most primitive vertebrates known to possess homologues of the MHC and other components of the adaptive immune system.     

Evolution of Vertebrate Adam Genes; Duplication of Testicular Adams from Ancient Adam9/9-like Loci

Members of the disintegrin metalloproteinase (ADAM) family have important functions in regulating cell-cell and cell-matrix interactions as well as cell signaling. There are two major types of ADAMs: the somatic ADAMs (sADAMs) that have a significant presence in somatic tissues, and the testicular ADAMs (tADAMs) that are expressed predominantly in the testis. Genes encoding tADAMs can be further divided into two groups: group I (intronless) and group II (intron-containing). To date, tAdams have only been reported in placental mammals, and their evolutionary origin and relationship to sAdams remain largely unknown. Using phylogenetic and syntenic tools, we analyzed the Adam genes in various vertebrates ranging from fishes to placental mammals. Our analyses reveal duplication and loss of some sAdams in certain vertebrate species. In particular, there exists an Adam9-like gene in non-mammalian vertebrates but not mammals. We also identified putative group I and group II tAdams in all amniote species that have been examined. These tAdam homologues are more closely related to Adams 9 and 9-like than to other sAdams. In all amniote species examined, group II tAdams lie in close vicinity to Adam9 and hence likely arose from tandem duplication, whereas group I tAdams likely originated through retroposition because of their lack of introns. Clusters of multiple group I tAdams are also common, suggesting tandem duplication after retroposition. Therefore, Adam9/9-like and some of the derived tAdam loci are likely preferred targets for tandem duplication and/or retroposition. Consistent with this hypothesis, we identified a young retroposed gene that duplicated recently from Adam9 in the opossum. As a result of gene duplication, some tAdams were pseudogenized in certain species, whereas others acquired new expression patterns and functions. The rapid duplication of Adam genes has a major contribution to the diversity of ADAMs in various vertebrate species.