Unleashing diversity through flexibility: The evolutionary journey of sex chromosomes in amphibians and reptiles.

Sex determination systems have greatly diversified between amphibians and reptiles, with such as the different sex chromosome compositions within a single species and transition between temperature-dependent sex determination (TSD) and genetic sex determination (GSD). In most sex chromosome studies on amphibians and reptiles, the whole-genome sequence of Xenopous tropicalis and chicken have been used as references to compare the chromosome homology of sex chromosomes among each of these taxonomic groups, respectively. In the present study, we reviewed existing reports on sex chromosomes, including karyotypes, in amphibians and reptiles. Furthermore, we compared the identified genetic linkages of sex chromosomes in amphibians and reptiles with the chicken genome as a reference, which is believed to resemble the ancestral tetrapod karyotype. Our findings revealed that sex chromosomes in amphibians are derived from genetic linkages homologous to various chicken chromosomes, even among several frogs within single families, such as Ranidae and Pipidae. In contrast, sex chromosomes in reptiles exhibit conserved genetic linkages with chicken chromosomes, not only across most species within a single family, but also within closely related families. The diversity of sex chromosomes in amphibians and reptiles may be attributed to the flexibility of their sex determination systems, including the ease of sex reversal in these animals.

Diversity and Evolution of Highly Repetitive DNA Sequences Constituting Chromosome Site-Specific Heterochromatin in Two Gerbillinae Species.

Constitutive heterochromatin, consisting of repetitive sequences, diverges very rapidly; therefore, its nucleotide sequences and chromosomal distributions are often largely different, even between closely related species. The chromosome C-banding patterns of two Gerbillinae species, Meriones unguiculatus and Gerbillus perpallidus, vary greatly, even though they belong to the same subfamily. To understand the evolution of C-positive heterochromatin in these species, we isolated highly repetitive sequences, determined their nucleotide sequences, and characterized them using chromosomal and filter hybridization. We obtained a centromeric repeat (MUN-HaeIII) and a chromosome 13-specific repeat (MUN-EcoRI) from M. unguiculatus. We also isolated a centromeric/pericentromeric repeat (GPE-MBD) and an interspersed-type repeat that was predominantly amplified in the X and Y chromosomes (GPE-EcoRI) from G. perpallidus. GPE-MBD was found to contain a 17-bp motif that is essential for binding to the centromere-associated protein CENP-B. This indicates that it may play a role in the formation of a specified structure and/or function of centromeres. The nucleotide sequences of the three sequence families, except GPE-EcoRI, were conserved only in Gerbillinae. GPE-EcoRI was derived from the long interspersed nuclear elements 1 retrotransposon and showed sequence homology throughout Muridae and Cricetidae species, indicating that the repeat sequence occurred at least in the common ancestor of Muridae and Cricetidae. Due to a lack of assembly data of highly repetitive sequences constituting heterochromatin in whole-genome sequences of vertebrate species published to date, the knowledge obtained in this study provides useful information for a deep understanding of the evolution of repetitive sequences in not only rodents but also in mammals.

Elasmobranch genome sequencing reveals evolutionary trends of vertebrate karyotype organization.

Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum, an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality.

Molecular Cytogenetic Characterization of C-Band-Positive Heterochromatin of the Greater Long-Tailed Hamster (Tscherskia triton, Cricetinae).

The greater long-tailed hamster (Tscherskia triton, Cricetinae) has a unique karyotype (2n = 28), containing 11 pairs of acrocentric chromosomes with large C-band-positive centromeric heterochromatin blocks. To understand the origin and evolutionary process of heterochromatin in this species, we isolated novel families of chromosome site-specific highly repetitive DNA sequences from TaqI-digested genomic DNA and then characterized them by chromosome in situ and filter hybridization. The TaqI-families of repetitive sequences were classified into 2 types by their genome organization and chromosomal distribution: the 110-bp repeated sequence organized in large tandem arrays (as satellite DNA), localized to centromeric C-positive heterochromatin of acrocentric autosomes (chromosomes 1-11) and submetacentric X chromosome, and the 405-bp repeated sequence that was composed of 30-32-bp internal repeats, distributed in the pericentromeric region on the short arms of X and Y chromosomes. The repetitive sequences did not cross-hybridize with genomic DNA of any genera of Cricetinae (Mesocricetus, Cricetulus, and Phodopus). These results suggest that the 110-bp and 405-bp repeats rapidly diverged in the lineage of T. triton, evolving in a concerted manner among autosomes and X chromosome and within X and Y chromosomes, respectively. The 110-bp centromeric repeat contained a 17-bp motif in which 9 bases are essential for binding with the centromere-associated protein CENP-B, suggesting the possibility that the 110-bp major satellite DNA carrying the 17-bp motif may have a role in the formation of specified structure and/or function of centromeres in T. triton.

Squalomix: shark and ray genome analysis consortium and its data sharing platform.

The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.

Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis.

Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.

Molecular cytogenetic characterization of repetitive sequences comprising centromeric heterochromatin in three Anseriformes species.

The highly repetitive DNA sequence of centromeric heterochromatin is an effective molecular cytogenetic marker for investigating genomic compartmentalization between macrochromosomes and microchromosomes in birds. We isolated four repetitive sequence families of centromeric heterochromatin from three Anseriformes species, viz., domestic duck (Anas platyrhynchos, APL), bean goose (Anser fabalis, AFA), and whooper swan (Cygnus cygnus, CCY), and characterized the sequences by molecular cytogenetic approach. The 190-bp APL-HaeIII and 101-bp AFA-HinfI-S sequences were localized in almost all chromosomes of A. platyrhynchos and A. fabalis, respectively. However, the 192-bp AFA-HinfI-L and 290-bp CCY-ApaI sequences were distributed in almost all microchromosomes of A. fabalis and in approximately 10 microchromosomes of C. cygnus, respectively. APL-HaeIII, AFA-HinfI-L, and CCY-ApaI showed partial sequence homology with the chicken nuclear-membrane-associated (CNM) repeat families, which were localized primarily to the centromeric regions of microchromosomes in Galliformes, suggesting that ancestral sequences of the CNM repeat families are observed in the common ancestors of Anseriformes and Galliformes. These results collectively provide the possibility that homogenization of centromeric heterochromatin occurred between microchromosomes in Anseriformes and Galliformes; however, homogenization between macrochromosomes and microchromosomes also occurred in some centromeric repetitive sequences.

Overexpression of microRNAs from the Gtl2-Rian locus contributes to postnatal death in mice.

The Dlk1-Dio3 imprinted domain functions in embryonic development but the roles of noncoding RNAs expressed from this domain remain unclear. We addressed this question by generating transgenic (TG) mice harbouring a BAC carrying IG-DMR (intergenic-differentially methylated region), Gtl2-DMR, Gtl2, Rtl1/Rtl1as, and part of Rian. High postnatal lethality (>85%) of the BAC-TG pups was observed in the maternally transmitted individuals (MAT-TG), but not following paternal transmission (PAT-TG). The DNA methylation status of IG-DMR and Gtl2-DMR in the BAC-allele was paternally imprinted similar to the genomic allele. The mRNA-Seq and miRNA-Seq analysis revealed marked expression changes in the MAT-TG, with 1,500 upregulated and 2,131 downregulated genes. The long noncoding RNAs and 12 miRNAs containing the BAC locus were markedly enhanced in the MAT-TG. We identified the 24 target genes of the overexpressed miRNAs and confirmed the downregulation in the MAT-TG. Notably, overexpression of mir770, mir493, and mir665 from Gtl2 in the MAT-TG embryos led to decreased expression of the 3 target genes, Col5a1, Pcgf2, and Clip2. Our results suggest that decreased expression of the 3 target genes concomitant with overexpression of the miRNAs within Gtl2 may be involved in the postnatal death in the MAT-TG. Because this imprinted domain is well conserved between mice and humans, the results of genetic and molecular analysis in mice hold important implications for related human disorders such as Temple syndrome.

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.

Molecular cytogenetic characterization of chromosome site-specific repetitive sequences in the Arctic lamprey (Lethenteron camtschaticum, Petromyzontidae).

All extant lamprey karyotypes are characterized by almost all dot-shaped microchromosomes. To understand the molecular basis of chromosome structure in lampreys, we performed chromosome C-banding and silver staining and chromosome mapping of the 18S-28S and 5S ribosomal RNA (rRNA) genes and telomeric TTAGGG repeats in the Arctic lamprey (Lethenteron camtschaticum). In addition, we cloned chromosome site-specific repetitive DNA sequences and characterized them by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. Three types of repetitive sequences were detected; a 200-bp AT-rich repetitive sequence, LCA-EcoRIa that co-localized with the 18S-28S rRNA gene clusters of 3 chromosomal pairs; a 364-bp AT-rich LCA-EcoRIb sequence that showed homology to the EcoRI sequence family from the sea lamprey (Petromyzon marinus), which contains short repeats as centromeric motifs; and a GC-rich 702-bp LCA-ApaI sequence that was distributed on nearly all chromosomes and showed significant homology with the integrase-coding region of a Ty3/Gypsy family long terminal repeat (LTR) retrotransposon. All three repetitive sequences are highly conserved within the Petromyzontidae or within Petromyzontidae and Mordaciidae. Molecular cytogenetic characterization of these site-specific repeats showed that they may be correlated with programed genome rearrangement (LCA-EcoRIa), centromere structure and function (LCA-EcoRIb), and site-specific amplification of LTR retroelements through homogenization between non-homologous chromosomes (LCA-ApaI).

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.

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.

A Genetic Map for the Only Self-Fertilizing Vertebrate.

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.

No Interstitial Telomeres on Autosomes but Remarkable Amplification of Telomeric Repeats on the W Sex Chromosome in the Sand Lizard (Lacerta agilis).

Telomeres are repeat (TTAGGG) n sequences that form terminal ends of chromosomes and have several functions, such as protecting the coding DNA from erosion at mitosis. Due to chromosomal rearrangements through evolutionary history (e.g., inversions and fusions), telomeric sequences are also found between the centromere and the terminal ends (i.e., at interstitial telomeric sites, ITSs). ITS telomere sequences have been implicated in heritable disease caused by genomic instability of ITS polymorphic variants, both with respect to copy number and sequence. In the sand lizard (Lacerta agilis), we have shown that telomere length is predictive of lifetime fitness in females but not males. To assess whether this sex specific fitness effect could be traced to ITSs differences, we mapped (TTAGGG) n sequences using fluorescence in situ hybridization in fibroblast cells cultured from 4 specimens of known sex. No ITSs could be found on autosomes in either sex. However, females have heterogametic sex chromosomes in sand lizards (ZW, 2n = 38) and the female W chromosome showed degeneration and remarkable (TTAGGG) n amplification, which was absent in the Z chromosomes. This work warrants further research on sex chromosome content, in particular of the degenerate W chromosome, and links to female fitness in sand lizards.

Karyotype Reorganization in the Hokou Gecko (Gekko hokouensis, Gekkonidae): The Process of Microchromosome Disappearance in Gekkota.

The Hokou gecko (Gekko hokouensis: Gekkonidae, Gekkota, Squamata) has the chromosome number 2n = 38, with no microchromosomes. For molecular cytogenetic characterization of the gekkotan karyotype, we constructed a cytogenetic map for G. hokouensis, which retains the ancestral karyotype of Gekkota, with 86 functional genes, and compared it with cytogenetic maps for four Toxicofera species that have many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) and that for a lacertid species (Lacerta agilis) with only one pair of autosomal microchromosomes. Ten pairs of G. hokouensis chromosomes [GHO1, 2, 3, Z(4), 6, 7, 8, 13, 14, and 15] showed highly conserved linkage homology with macrochromosomes and/or macrochromosome arms of the four Toxicofera species and corresponded to eight L. agilis macrochromosomes (LAG). However, GHO5, GHO9, GHO10, GHO11, and LAG6 were composed of chromosome segments that have a homology with Toxicofera microchromosomes, and no homology was found in the chromosomes between G. hokouensis and L. agilis. These results suggest that repeated fusions of microchromosomes may have occurred independently in each lineage of Gekkota and Lacertidae, leading to the disappearance of microchromosomes and appearance of small-sized macrochromosomes.

Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae).

Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

Molecular cloning and characterization of oocyte-specific Pat1a in Rana rugosa frogs.

The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a germ cell-specific protein-and Pat1a, Vasa-immunopositive signals were observed in all of the germ cells, whereas Pat1a signals were confined to the growing oocytes (50-200 µm in diameter), and absent from small germ cells (<50 µm in diameter). Forty days after testosterone injection into tadpoles to induce female-to-male sex-reversal, Pat1a-immunoreactive oocytes had disappeared completely from the sex-reversed gonad, but Vasa-positive small germ cells persisted. Thus, Pat1a would be a good marker for identifying the sexual status of the sex-reversing gonad in amphibians. In addition, fluorescence in situ hybridization analysis showed Pat1a to have an autosomal locus, suggesting that Pat1a transcription is probably regulated by a tissue-specific transcription factor in R. rugosa.

Extraordinary Diversity in the Origins of Sex Chromosomes in Anurans Inferred from Comparative Gene Mapping.

Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.

Distribution of the T2-MITE Family Transposons in the Xenopus (Silurana) tropicalis Genome.

The T2 family of miniature inverted-repeat transposable elements (T2-MITE) is a prevalent MITE family found in both Xenopus(Silurana) tropicalis and X. laevis. Some subfamilies, particularly T2-A1 and T2-C, may have originated prior to the diversification of the 2 Xenopus lineages and currently include active members in X. tropicalis, whereas another subfamily, T2-E, may have lost its transposition activity even earlier. The distribution of each T2-MITE subfamily in X. tropicalis was investigated and compared to evaluate the evolutionary dynamics of the T2-MITE subfamilies. The subfamilies showed differences in chromosomal distribution, uniformity of insertion density on scaffolds, ratios of upstream to downstream insertions with respect to genes, and their distance from genes. Among these, the T2-C subfamily was interesting because it was frequently inserted upstream and close to genes and because genes with close insertions of this subfamily showed high correlations in spatial expression patterns. This unique distribution and long-lived transposition activity may reflect a mutual relationship evolved between this subfamily and the host.