Evolutionary features of ligands and their receptors via protein-protein interactions and essentiality in primates.

Protein evolution rate is negatively correlated with several effectors, such as expression level, expression distribution, protein-protein interactions (PPIs), and essentiality for survival. These effectors can characterize the signaling pathways mediated by ligand-receptor binding. However, it is unclear whether these effectors are constraining factors on the pathway-specific evolution of ligands and receptors. To clarify the relation between the effectors and protein evolution (dN /dS ratio) in ligands and their receptors considering each signaling pathway, we investigated 377 proteins in 20 peptide/protein ligand groups and their receptor groups using 15 primate sequences. The dN /dS ratios between peptide/protein ligand groups and their receptor groups were positively correlated, suggesting the protein evolution under the influence of signaling pathway to which they belong. Comparing each signaling pathway, ligands and receptors mainly related to development and growth (FGF/Hedgehog/Notch/WNT groups) showed lower dN /dS ratios, higher PPI numbers, and higher essentiality, whereas those mainly related to immune process (CSF/IFN/IL/TNF groups) showed higher dN /dS ratios, lower PPI numbers, and lower essentiality. Most ligands and receptors were poorly expressed, and expression level was not a constraining factor on the protein evolution. These findings indicate that PPI and essentiality are constraining factors that characterize the pathway-specific evolution of ligands and receptors.

rBC2LCN-reactive SERPINA3 is a glycobiomarker candidate for pancreatic ductal adenocarcinoma.

Early detection is urgently needed to improve the patient's pancreatic ductal adenocarcinoma (PDAC) survival. Previously, we identified a novel tumor-associated glycan, H-type3, which is expressed on PDAC cells and is detected by rBC2LCN (recombinant N-terminal domain of BC2L-C identified from Burkholderia cenocepacia) lectin. Here, we identified that SERPINA3 is an rBC2LCN-reactive glycoprotein (BC2-S3) secreted from PDAC cells into the blood in patients with PDAC by liquid chromatography-tandem mass spectrometry analysis and lectin blotting. In immune staining, BC2-S3 was detected specifically in the tumor but not in normal tissues of PDAC. Lectin-ELISA was then developed to measure the serum level of BC2-S3 in healthy control (HC, n = 99) and patients with PDAC (n = 88). BC2-S3 exhibited higher in patients with PDAC than in those with HC. BC2-S3 showed similar diagnostic performance in all stages of PDAC (stages IA-IV, true positive rate = 76.1%, true negative rate = 81.8%) to CA19-9 (72.7%, 75.8%). Remarkably, BC2-S3 showed a significantly higher detection rate (89.7%) for early stage PDAC (IA-IIA) than CA19-9 (62.1%, P = 0.029). The combination of BC2-S3 and CA19-9 further improved the diagnostic ability for all stages of PDAC (81.8%, 87.9%). In conclusion, BC2-S3 is a glycobiomarker candidate for PDAC.

Identification of ancestral sex chromosomes in the frog Glandirana rugosa bearing XX-XY and ZZ-ZW sex-determining systems.

Sex chromosomes constantly exist in a dynamic state of evolution: rapid turnover and change of heterogametic sex during homomorphic state, and often stepping out to a heteromorphic state followed by chromosomal decaying. However, the forces driving these different trajectories of sex chromosome evolution are still unclear. The Japanese frog Glandirana rugosa is one taxon well suited to the study on these driving forces. The species has two different heteromorphic sex chromosome systems, XX-XY and ZZ-ZW, which are separated in different geographic populations. Both XX-XY and ZZ-ZW sex chromosomes are represented by chromosome 7 (2n = 26). Phylogenetically, these two systems arose via hybridization between two ancestral lineages of West Japan and East Japan populations, of which sex chromosomes are homomorphic in both sexes and to date have not yet been identified. Identification of the sex chromosomes will give us important insight into the mechanisms of sex chromosome evolution in this species. Here, we used a high-throughput genomic approach to identify the homomorphic XX-XY sex chromosomes in both ancestral populations. Sex-linked DNA markers of West Japan were aligned to chromosome 1, whereas those of East Japan were aligned to chromosome 3. These results reveal that at least two turnovers across three different sex chromosomes 1, 3 and 7 occurred during evolution of this species. This finding raises the possibility that cohabitation of the two different sex chromosomes from ancestral lineages induced turnover to another new one in their hybrids, involving transition of heterogametic sex and evolution from homomorphy to heteromorphy.

Highly rapid and diverse sex chromosome evolution in the Odorrana frog species complex.

Sex chromosomes in poikilothermal vertebrates are characterized by rapid and diverse evolution at the species or population level. Our previous study revealed that the Taiwanese frog Odorrana swinhoana (2n = 26) has a unique system of multiple sex chromosomes created by three sequential translocations among chromosomes 1, 3, and 7. To reveal the evolutionary history of sex chromosomes in the Odorrana species complex, we first identified the original, homomorphic sex chromosomes, prior to the occurrence of translocations, in the ancestral-type population of O. swinhoana. Then, we extended the investigation to a closely related Japanese species, Odorrana utsunomiyaorum, which is distributed on two small islands. We used a high-throughput nuclear genomic approach to analyze single-nucleotide polymorphisms and identify the sex-linked markers. Those isolated from the O. swinhoana ancestral-type population were found to be aligned to chromosome 1 and showed male heterogamety. In contrast, almost all the sex-linked markers isolated from O. utsunomiyaorum were heterozygous in females and homozygous in males and were aligned to chromosome 9. Morphologically, we confirmed chromosome 9 to be heteromorphic in females, showing a ZZ-ZW sex determination system, in which the W chromosomes were heterochromatinized in a stripe pattern along the chromosome axis. These results indicated that after divergence of the two species, the ancestral homomorphic sex chromosome 1 underwent highly rapid and diverse evolution, i.e., sequential translocations with two autosomes in O. swinhoana, and turnover to chromosome 9 in O. utsunomiyaorum, with a transition from XY to ZW heterogamety and change to heteromorphy.

Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

SSEA-1-positive fibronectin is secreted by cells deviated from the undifferentiated state of human induced pluripotent stem cells.

Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1+ glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1+ deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1+ FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1+ deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine.

The rBC2LCN-positive subpopulation of PC-3 cells exhibits cancer stem-like properties.

The recombinant lectin rBC2LCN is a useful marker for discriminating the undifferentiated status of human induced or embryonic stem cells. Recently, rBC2LCN has also been used for detecting some cancers and niche cells. However, the generality of which types of cells are detected by rBC2LCN is unclear. In this study, we demonstrated the potential of rBC2LCN as a probe for detecting and isolating cancer stem-like cells. Interestingly, flow cytometric analysis of various human cell lines indicated that the human prostate cancer cell line PC-3 consisted of rBC2LCN-positive and -negative subpopulations. Compared with the rBC2LCN-negative subpopulation, the rBC2LCN-positive subpopulation possessed representative features of cancer stem cells and malignancy, such as slow proliferation, increased cell motility, anchorage-independent growth, and drug resistance. The comprehensive expression profiles revealed that the rBC2LCN-positive subpopulation expressed higher levels of cancer stem cell markers. These findings indicate that rBC2LCN is useful for detecting not only pluripotent stem cells but also the cancer stem-like subpopulation of PC-3 cells. Pluripotent and cancer cells with rBC2LCN positivity would be important for future stem cell research.

Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.

Conservatism and variability of gene expression profiles among homeologous transcription factors in Xenopus laevis.

Xenopus laevis has an allotetraploid genome of 3.1Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otx1 suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.

Genome organization of the vg1 and nodal3 gene clusters in the allotetraploid frog Xenopus laevis.

Extracellular factors belonging to the TGF-ß family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-ß family genes in response to allotetraploidization.

Clustered Xenopus keratin genes: A genomic, transcriptomic, and proteomic analysis.

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.

Molecular Evolution of Two Distinct dmrt1 Promoters for Germ and Somatic Cells in Vertebrate Gonads.

The transcription factor DMRT1 has important functions in two distinct processes, somatic-cell masculinization and germ-cell development in mammals. However, it is unknown whether the functions are conserved during evolution, and what mechanism underlies its expression in the two cell lineages. Our analysis of the Xenopus laevis and Silurana tropicalis dmrt1 genes indicated the presence of two distinct promoters: one upstream of the noncoding first exon (ncEx1), and one within the first intron. In contrast, only the ncEx1-upstream promoter was detected in the dmrt1 gene of the agnathan sand lamprey, which expressed dmrt1 exclusively in the germ cells. In X. laevis, the ncEx1- and exon 2-upstream promoters were predominantly used for germ-cell and somatic-cell transcription, respectively. Importantly, knockdown of the ncEx1-containing transcript led to reduced germ-cell numbers in X. laevis gonads. Intriguingly, two genetically female individuals carrying the knockdown construct developed testicles. Analysis of the reptilian leopard gecko dmrt1 revealed the absence of ncEx1. We propose that dmrt1 regulated germ-cell development in the vertebrate ancestor, then acquired another promoter in its first intron to regulate somatic-cell masculinization during gnathostome evolution. In the common ancestor of reptiles and mammals, only one promoter got function for both the two cell lineages, accompanied with the loss of ncEx1. In addition, we found a conserved noncoding sequence (CNS) in the dmrt1 5'-flanking regions only among amniote species, and two CNSs in the introns among most vertebrates except for agnathans. Finally, we discuss relationships between these CNSs and the promoters of dmrt1 during vertebrate evolution.

Masculinization-Related Genes and Cell-Mass Structures During Early Gonadal Differentiation in the African Clawed Frog Xenopus laevis.

The African clawed frog Xenopus laevis has a female heterogametic ZZ/ZW-type sex-determining system. We previously discovered a W-linked female sex-determining gene dm-W that is involved in ovary formation, probably through the up-regulation of the estrogen synthesis genes cyp19a1 and foxl2. We also reported that a unique "mass-in-line structure", which disappears from ZZ gonads during early testicular development, might serve as the basis for ovary differentiation in ZW gonads. However, the molecular mechanisms underlying early masculinization are poorly understood. To elucidate the development of bipotential gonads into testes after sex determination in this species, we focused on the orthologs of five mammalian sex-related genes: three nuclear factor genes, dax1, sf1 (also known as ad4bp), and sox9, and two genes encoding members of the tumor growth factor-ß (TGF-ß) family, anti-Müllerian hormone (amh) and inhibin ßb (inhbb). Quantitative RT-PCR analysis revealed that the expression of dax1, sox9, amh, and inhbb or sf1 was greatly or slightly higher in ZZ than in ZW gonads during early sex development. In situ hybridization analysis revealed that amh and inhbb mRNAs were expressed in somatic cells on the inner and outer sides of cell masses in the mass-in-line structure, respectively, in the developing ZZ gonads. Interestingly, estrogen exposure prevented the disappearance of the mass-in-line structure in early developing ZZ tadpoles. These findings suggest that TGF-ß signaling is involved in the destruction of the mass-in-line structure, which may be maintained by estrogen.

Meiotic recombination counteracts male-biased mutation (male-driven evolution).

Meiotic recombination is believed to produce greater genetic variation despite the fact that deoxyribonucleic acid (DNA)-replication errors are a major source of mutations. In some vertebrates, mutation rates are higher in males than in females, which developed the theory of male-driven evolution (male-biased mutation). However, there is little molecular evidence regarding the relationships between meiotic recombination and male-biased mutation. Here we tested the theory using the frog Rana rugosa, which has both XX/XY- and ZZ/ZW-type sex-determining systems within the species. The male-to-female mutation-rate ratio (α) was calculated from homologous sequences on the X/Y or Z/W sex chromosomes, which supported male-driven evolution. Surprisingly, each α value was notably higher in the XX/XY-type group than in the ZZ/ZW-type group, although α should have similar values within a species. Interestingly, meiotic recombination between homologous chromosomes did not occur except at terminal regions in males of this species. Then, by subdividing α into two new factors, a replication-based male-to-female mutation-rate ratio (ß) and a meiotic recombination-based XX-to-XY/ZZ-to-ZW mutation-rate ratio (γ), we constructed a formula describing the relationship among a nucleotide-substitution rate and the two factors, ß and γ. Intriguingly, the ß- and γ-values were larger and smaller than 1, respectively, indicating that meiotic recombination might reduce male-biased mutations.

Multiple Transitions between Y Chromosome and Autosome in Tago's Brown Frog Species Complex.

Sex chromosome turnover is the transition between sex chromosomes and autosomes. Although many cases have been reported in poikilothermic vertebrates, their evolutionary causes and genetic mechanisms remain unclear. In this study, we report multiple transitions between the Y chromosome and autosome in the Japanese Tago's brown frog complex. Using chromosome banding and molecular analyses (sex-linked and autosomal single nucleotide polymorphisms, SNPs, from the nuclear genome), we investigated the frogs of geographic populations ranging from northern to southern Japan of two species, Rana tagoi and Rana sakuraii (2n = 26). Particularly, the Chiba populations of East Japan and Akita populations of North Japan in R. tagoi have been, for the first time, investigated here. As a result, we identified three different sex chromosomes, namely chromosomes 3, 7, and 13, in the populations of the two species. Furthermore, we found that the transition between the Y chromosome (chromosome 7) and autosome was repeated through hybridization between two or three different populations belonging to the two species, followed by restricted chromosome introgression. These dynamic sex chromosome turnovers represent the first such findings in vertebrates and imply that speciation associated with inter- or intraspecific hybridization plays an important role in sex chromosome turnover in frogs.

Molecular evolution of vertebrate sex-determining genes.

Y-linked Dmy (also called dmrt1bY) in the teleost fish medaka, W-linked Dm-W in the African clawed frog (Xenopus laevis), and Z-linked Dmrt1 in the chicken are all sex chromosome-linked Dmrt1 homologues required for sex determination. Dmy and Dm-W both are Dmrt1 palalogues evolved through Dmrt1 duplication, while chicken Dmrt1 is a Z-linked orthologue. The eutherian sex-determining gene, Sry, evolved from an allelic gene, Sox3. Here we analyzed the exon-intron structures of the Dmrt1 homologues of several vertebrate species through information from databases and by determining the transcription initiation sites in medaka, chicken, Xenopus, and mouse. Interestingly, medaka Dmrt1 and Dmy and Xenopus Dm-W and Dmrt1 have a noncoding-type first exon, while mouse and chicken Dmrt1 do not. We next compared the 5'-flanking sequences of the Dmrt1 noncoding and coding exons 1 of several vertebrate species and found conservation of the presumptive binding sites for some transcription factors. Importantly, based on the phylogenetic trees for Dmrt1 and Sox3 homologues, it was implied that the sex-determining gene Dmy, Dm-W, and Sry have a higher substitution rate than thier prototype genes. Finally, we discuss the evolutionary relationships between vertebrate sex chromosomes and the sex-determining genes Dmy/Dm-W and Sry, which evolved by neofunctionalization of Dmrt1 and Sox3, respectively, for sex determining function. We propose a coevolution model of sex determining gene and sex chromosome, in which undifferentiated sex chromosomes easily allow replacement of a sex-determining gene with another new one, while specialized sex chromosomes are restricted a particular sex-determining gene.

Parallel Evolution of Sex-Linked Genes across XX/XY and ZZ/ZW Sex Chromosome Systems in the Frog Glandirana rugosa.

Genetic sex-determination features male (XX/XY) or female heterogamety (ZZ/ZW). To identify similarities and differences in the molecular evolution of sex-linked genes between these systems, we directly compared the sex chromosome systems existing in the frog Glandirana rugosa. The heteromorphic X/Y and Z/W sex chromosomes were derived from chromosomes 7 (2n = 26). RNA-Seq, de novo assembly, and BLASTP analyses identified 766 sex-linked genes. These genes were classified into three different clusters (XW/YZ, XY/ZW, and XZ/YW) based on sequence identities between the chromosomes, probably reflecting each step of the sex chromosome evolutionary history. The nucleotide substitution per site was significantly higher in the Y- and Z-genes than in the X- and W- genes, indicating male-driven mutation. The ratio of nonsynonymous to synonymous nucleotide substitution rates was higher in the X- and W-genes than in the Y- and Z-genes, with a female bias. Allelic expression in gonad, brain, and muscle was significantly higher in the Y- and W-genes than in the X- and Z-genes, favoring heterogametic sex. The same set of sex-linked genes showed parallel evolution across the two distinct systems. In contrast, the unique genomic region of the sex chromosomes demonstrated a difference between the two systems, with even and extremely high expression ratios of W/Z and Y/X, respectively.

Tumor necrosis factor-related apoptosis-inducing ligand 1 (TRAIL1) enhances the transition of red blood cells from the larval to adult type during metamorphosis in Xenopus.

The transition of red blood cells (RBCs) from primitive to definitive erythropoiesis is conserved across vertebrates. In anuran amphibians, the larval RBCs from primitive erythropoiesis are replaced by adult RBCs from definitive erythropoiesis during metamorphosis. The molecular mechanisms by which the primitive (larval) blood cells are specifically removed from circulation are not yet understood. In this study, we identified Xenopus tumor necrosis factor-related apoptosis-inducing ligand 1 (xTRAIL1) and xTRAIL2 as ligands of Xenopus death receptor-Ms (xDR-Ms) and investigated whether TRAIL signaling could be involved in this transition. The Trail and xDR-M genes were highly expressed in the liver and RBCs, respectively, during metamorphosis. Interestingly, xTRAIL1 enhanced the transition of the RBCs, and a dominant-negative form of the xTRAIL1 receptor attenuated it, when injected into tadpoles. Moreover, xTRAIL1 induced apoptosis in larval RBCs, but had little effect on adult RBCs in vitro. We also found that adult RBCs treated with staurosporine, a protein kinase C (PKC) inhibitor, were sensitized to xTRAIL1. The mRNAs for PKC isoforms were up-regulated in RBCs during metamorphosis. These results suggest that xTRAIL1 can cause apoptosis, probably mediated through xDR-Ms, in larval RBCs, but may not kill adult RBCs, presumably owing to PKC activation, as part of the mechanism for RBC switching.

Apoptotic and survival signaling mediated through death receptor members during metamorphosis in the African clawed frog Xenopus laevis.

The tumor necrosis factor (TNF) superfamily includes death receptor (DR) ligands, such as TNF-α, FasL, and TRAIL. Death receptors (DRs) induce intracellular signaling upon engagement of their cognate DR ligands, either leading to apoptosis, survival, or proinflammatory responses. The DR signaling is mediated by the recruitment of several death domain (DD)-containing molecules such as Fas-associated death domain (FADD) and receptor-interacting protein (RIP) 1. In this review, we describe DR signaling in mammals, and describe recent findings of DR signaling during metamorphosis in the African clawed frog Xenopus laevis. Specifically, we focus on the cell fate (apoptosis or survival) mediated through a DR ligand, TNF-α or TRAIL in endothelial cells or red blood cells (RBCs). In addition, we discuss relationships between thyroid hormone-induced metamorphosis and DR signaling.

Independent pseudogenizations and losses of sox15 during amniote diversification following asymmetric ohnolog evolution.

BACKGROUND: Four ohnologous genes (sox1, sox2, sox3, and sox15) were generated by two rounds of whole-genome duplication in a vertebrate ancestor. In eutherian mammals, Sox1, Sox2, and Sox3 participate in central nervous system (CNS) development. Sox15 has a function in skeletal muscle regeneration and has little functional overlap with the other three ohnologs. In contrast, the frog Xenopus laevis and zebrafish orthologs of sox15 as well as sox1-3 function in CNS development. We previously reported that Sox15 is involved in mouse placental development as neofunctionalization, but is pseudogenized in the marsupial opossum. These findings suggest that sox15 might have evolved with divergent gene fates during vertebrate evolution. However, knowledge concerning sox15 in other vertebrate lineages than therian mammals, anuran amphibians, and teleost fish is scarce. Our purpose in this study was to clarify the fate and molecular evolution of sox15 during vertebrate evolution. RESULTS: We searched for sox15 orthologs in all vertebrate classes from agnathans to mammals by significant sequence similarity and synteny analyses using vertebrate genome databases. Interestingly, sox15 was independently pseudogenized at least twice during diversification of the marsupial mammals. Moreover, we observed independent gene loss of sox15 at least twice during reptile evolution in squamates and crocodile-bird diversification. Codon-based phylogenetic tree and selective analyses revealed an increased dN/dS ratio for sox15 compared to the other three ohnologs during jawed vertebrate evolution. CONCLUSIONS: The findings revealed an asymmetric evolution of sox15 among the four ohnologs during vertebrate evolution, which was supported by the increased dN/dS values in cartilaginous fishes, anuran amphibians, and amniotes. The increased dN/dS value of sox15 may have been caused mainly by relaxed selection. Notably, independent pseudogenizations and losses of sox15 were observed during marsupial and reptile evolution, respectively. Both might have been caused by strong relaxed selection. The drastic gene fates of sox15, including neofunctionalization and pseudogenizations/losses during amniote diversification, might be caused by a release from evolutionary constraints.