Shuuji Mawaribuchi

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.

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.

Tumor Necrosis Factor-α Attenuates Thyroid Hormone-Induced Apoptosis in Vascular Endothelial Cell Line XLgoo Established from Xenopus Tadpole Tails

Amphibian metamorphosis induced by T(3) involves programmed cell death and the differentiation of various types of cells in degenerated and reconstructed tissues. However, the signaling pathway that directs the T(3)-dependent cell-fate determinations remains unclear. TNF-alpha is a pleiotropic cytokine that affects diverse cellular responses. Engagement of TNF-alpha with its receptor (TNFR1) causes intracellular apoptotic and/or survival signaling. To investigate TNF signaling functions during anuran metamorphosis, we first identified Xenopus laevis orthologs of TNF (xTNF)-alpha and its receptor. We found that xTNF-alpha activated nuclear factor-kappaB in X. laevis A6 cells through the Fas-associated death domain and receptor-interacting protein 1. Interestingly, xTNF-alpha mRNA in blood cells showed prominent expression at prometamorphosis during metamorphosis. Next, to elucidate the apoptotic and/or survival signaling induced by xTNF-alpha in an in vitro model of metamorphosis, we established a vascular endothelial cell line, XLgoo, from X. laevis tadpole tail. XLgoo cells formed actin stress fibers and elongated in response to xTNF-alpha. T(3) induced apoptosis in these cells, but the addition of xTNF-alpha blocked the T(3)-induced apoptosis. In addition, treatment of the cells with T(3) for 2 d induced the expression of thyroid hormone receptor-beta and caspase-3, and this thyroid hormone receptor-beta induction was drastically repressed by xTNF-alpha. Furthermore, in organ culture of the tail, xTNF-alpha significantly attenuated the tail degeneration induced by T(3). These findings suggested that xTNF-alpha could protect vascular endothelial cells from apoptotic cell death induced by T(3) during metamorphosis and thereby participate in the regulation of cell fate.

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.

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.

Cell-Mass Structures Expressing the Aromatase Gene Cyp19a1 Lead to Ovarian Cavities in Xenopus laevis

The African clawed frog, Xenopus laevis, has a ZZ/ZW-type sex-determination system. We previously reported that a W-linked gene, Dm-W, can determine development as a female. However, the mechanisms of early sex differentiation remain unclear. We used microarrays to screen for genes with sexually dimorphic expression in ZZ and ZW gonads during early sex differentiation in X laevis and found several steroidogenic genes. Importantly, the steroid 17α-hydroxylase gene Cyp17a1 and the aromatase gene Cyp19a1 were highly expressed in ZZ and ZW gonads, respectively, just after sex determination. At this stage, we found that Cyp17a1, Cyp19a1, or both were expressed in the ZZ and ZW gonads in a unique mass-in-line structure, in which several masses of cells, each surrounded by a basement membrane, were aligned along the anteroposterior axis. In fact, during sex differentiation, ovarian cavities formed inside each mass of Cyp17a1- and Cyp19a1-positive cells in the ZW gonads. However, the mass-in-line structure disappeared during testicular development in the ZZ testes. These results suggested that the mass-in-line structure found in both ZZ and ZW gonads just after sex determination might be formed in advance to produce ovarian cavities and then oocytes. Consequently, we propose a view that the default sex may be female in the morphological aspect of gonads in X laevis.

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

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.

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.

Apoptosis and differentiation of Xenopus tail-derived myoblasts by thyroid hormone

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.