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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.

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.

Introduction Tumourigenesis attributed to residual undifferentiated cells in a graft is considered to be a significant issue in cell therapy using human pluripotent stem cells. To ensure the safety of regenerative medicine derived from pluripotent stem cells, residual undifferentiated cells must be eliminated in the manufacturing process. We previously described the lectin probe rBC2LCN, which binds harmlessly and specifically to the cell surface of human pluripotent stem cells. We report here a technique using rBC2LCN to remove pluripotent cells from a heterogenous population to reduce the chance of teratoma formation. Methods We demonstrate a method for separating residual tumourigenic cells using rBC2LCN-bound magnetic beads. This technology is a novel use of their previous discovery that rBC2LCN is a lectin that selectively binds to pluripotent cells. We optimize and validate a method to remove hPSCs from a mixture with human fibroblasts using rBC2LCN-conjugated magnetic beads. Results Cells with the potential to form teratoma could be effectively eliminated from a heterogeneous cell population with biotin-labelled rBC2LCN and streptavidin-bound magnetic beads. The efficiency was measured by FACS, ddPCR, and animal transplantation, suggesting that magnetic cell separation using rBC2LCN is quite efficient for eliminating hPSCs from mixed cell populations. Conclusions The removal of residual tumourigenic cells based on rBC2LCN could be a practical option for laboratory use and industrialisation of regenerative medicine using human pluripotent stem cells., Highlights • A method using rBC2LCN-conjugated magnetic beads was developed to select hPSCs. • This method is applicable to eliminate hPSCs from a heterogeneous cell population. • The potential for teratoma formation was reduced by using this method.

Extracellular factors belonging to the TGF-β 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-β family genes in response to allotetraploidization.

The potential applications of human pluripotent stem cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells in cell therapy and regenerative medicine have been widely studied. The precise definition of pluripotent stem cell status during culture using biomarkers is essential for basic research and regenerative medicine. Culture conditions, including extracellular matrices, influence the balance between self-renewal and differentiation. Accordingly, to explore biomarkers for defining and monitoring the pluripotent substates during culture, we established different substates in H9 human ES cells by changing the extracellular matrix from vitronectin to Matrigel. The substate was characterised by low and high expression of the pluripotency marker R-10G epitope and the mesenchymal marker vimentin, respectively. Immunohistochemistry, induction of the three germ layers, and exhaustive expression analysis showed that the substate was ectoderm-biased, tended to differentiate into nerves, but retained the potential to differentiate into the three germ layers. Further integrated analyses of mRNA and miRNA microarrays and qPCR analysis showed that nine genes (COL9A2, DGKI, GBX2, KIF26B, MARCH1, PLXNA4, SLC24A4, TLR4, and ZHX3) were upregulated in the ectoderm-biased cells as ectoderm-biased biomarker candidates in pluripotent stem cells. Our findings provide important insights into ectoderm-biased substates of human pluripotent stem cells in the fields of basic research and regenerative medicine.

The metamorphosis of anuran amphibians is induced by thyroid hormone (TH). To study the molecular mechanisms underlying tail regression during metamorphosis, we established a cell line, XL-B4, from a Xenopus laevis tadpole tail at a premetamorphic stage. The cells expressed myoblast markers and differentiated into myotubes in differentiation medium. XL-B4 cells expressing fluorescent proteins were transplanted into tadpole tails. At 5 days post-transplantation, fluorescence was observed in myotube-like structures, indicating that the myoblastic cells could contribute to skeletal muscle. Exposure of XL-B4 cells to the TH triiodothyronine (T3) for several days significantly induced apoptotic cell death. We then examined an early response of expression of genes involved in apoptosis or myogenesis to T3. Treatment of the cells with T3 increased transcription of genes for matrix metalloproteinase-9 (MMP-9) and thyroid hormone receptor beta. Interestingly, the T3-treatment also increased myoD transcripts, but decreased the amounts of myogenin mRNA and myosin heavy chain. Importantly, we also observed upregulation of myoD expression and downregulation of myogenin expression in tails, but not in hind limbs, when tadpoles at a premetamorphic stage were treated with T3 for 1 day. These results indicated that T3 could not only induce apoptosis, but also attenuate myogenesis in tadpole tails during metamorphosis.

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.

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.

Xenopus laevis has an allotetraploid genome of 3.1 Gb, 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.

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.

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.