An ATR-PrimPol pathway confers tolerance to oncogenic KRAS-induced and heterochromatin-associated replication stress.

Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRASG12V expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRASG12V-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRASG12V-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.

Promoter generation for the chimeric sex-determining gene dm-W in Xenopus frogs.

Many sex-determining genes (SDGs) were generated as neofunctionalized genes through duplication and/or mutation of gonadal formation-related genes. We previously identified dm-W as an SDG in the African clawed frog Xenopus laevis and found that a partial duplication of the masculinization gene dmrt1 created the neofunctionalized dm-W after allotetraploidization by interspecific hybridization. The allotetraploid Xenopus species have two dmrt1 genes, dmrt1.L and dmrt1.S. Xenopus laevis dm-W has four exons: two dmrt1.S-derived exons (exons 2 and 3) and two other exons (noncoding exon 1 and exon 4). Our recent work revealed that exon 4 originated from a DNA transposon, hAT-10. Here, to clarify when and how the noncoding exon 1 and its coexisting promoter evolved during the establishment of dm-W after allotetraploidization, we newly determined nucleotide sequences of the dm-W promoter region from two other allotetraploid species, X. largeni and X. petersii, and performed an evolutionary analysis. We found that dm-W acquired a new exon 1 and TATA-type promoter in the common ancestor of the three allotetraploid Xenopus species, resulting in the deletion of the dmrt1.S-derived TATA-less promoter. In addition, we demonstrated that the TATA box contributes to dm-W promoter activity in cultured cells. Collectively, these findings suggest that this novel TATA-type promoter was important for the establishment of dm-W as a sex-determining gene, followed by the degeneration of the preexisting promoter.

Heat shock factor 1 induces a short burst of transcription of the clock gene Per2 during interbout arousal in mammalian hibernation.

During winter hibernation, a diverse range of small mammals can enter prolonged torpor. They spend the nonhibernation season as a homeotherm but the hibernation season as a heterotherm. In the hibernation season, chipmunks (Tamias asiaticus) cycle regularly between 5 and 6 days-long deep torpor with a body temperature (Tb) of 5 to 7 °C and interbout arousal of ∼20 h, during which, their Tb returns to the normothermic level. Here, we investigated Per2 expression in the liver to elucidate the regulation of the peripheral circadian clock in a mammalian hibernator. In the nonhibernation season, as in mice, heat shock factor 1, activated by elevated Tb during the wake period, activated Per2 transcription in the liver, which contributed to synchronizing the peripheral circadian clock to the Tb rhythm. In the hibernation season, we determined that the Per2 mRNA was at low levels during deep torpor, but Per2 transcription was transiently activated by heat shock factor 1, which was activated by elevated Tb during interbout arousal. Nevertheless, we found that the mRNA from the core clock gene Bmal1 exhibited arrhythmic expression during interbout arousal. Since circadian rhythmicity is dependent on negative feedback loops involving the clock genes, these results suggest that the peripheral circadian clock in the liver is nonfunctional in the hibernation season.

Neofunctionalization of a Noncoding Portion of a DNA Transposon in the Coding Region of the Chimerical Sex-Determining Gene dm-W in Xenopus Frogs.

Most vertebrate sex-determining genes (SDGs) emerge as neofunctionalized genes through duplication and/or mutation of ancestral genes that are involved with sexual differentiation. We previously demonstrated dm-W to be the SDG in the African clawed frog Xenopus laevis and found that a portion of this gene emerged from the masculinization gene dmrt1 after allotetraploidization by interspecific hybridization between two ancestral species around 17-18 Ma. dm-W has four exons consisting of a noncoding exon 1, dmrt1-derived exons 2 and 3, and an orphan exon 4 (Ex4) of unknown origin that includes coding sequence (CDS). In this study, we searched for the origin of Ex4 and investigated the function of the CDS of this exon. We found that the Ex4-CDS is derived from a noncoding portion of the hAT-10 family of DNA transposon. Evolutionary analysis of transposons and determination of the Ex4 sequences from three other species indicated that Ex4 was generated before the diversification of most or all extant allotetraploid species in subgenus Xenopus, during which time we hypothesize that transposase activity of this hAT superfamily was active. Using DNA-protein binding and transfection assays, we further demonstrate that the Ex4-encoded amino acid sequence increases the DNA-binding ability and transrepression activity of DM-W. These findings suggest that the conversion of the noncoding transposon sequence to the CDS of dm-W contributed to neofunctionalization of a new chimeric SDG in the ancestor of the allotetraploid Xenopus species, offering new insights into de novo origin and functional evolution of chimerical genes.

Activation of DNA Transposons and Evolution of piRNA Genes Through Interspecific Hybridization in Xenopus Frogs.

Interspecific hybridization between two closely related species sometimes resulted in a new species with allotetraploid genomes. Many clawed frog species belonging to the Xenopus genus have diverged from the allotetraploid ancestor created by the hybridization of two closely related species with the predicted L and S genomes. There are species-specific repeated sequences including transposable elements in each genome of organisms that reproduce sexually. To understand what happened on and after the hybridization of the two distinct systems consisting of repeated sequences and their corresponding piRNAs, we isolated small RNAs from ovaries and testes of three Xenopus species consisting of allotetraploid X. laevis and X. borealis and diploid X. tropicalis as controls. After a comprehensive sequencing and selection of piRNAs, comparison of their sequences showed that most piRNA sequences were different between the ovaries and testes in all three species. We compared piRNA and genome sequences and specified gene clusters for piRNA expression in each genome. The synteny and homology analyses showed many distinct piRNA clusters among the three species and even between the two L and/or S subgenomes, indicating that most clusters of the two allotetraploid species changed after hybridization. Moreover, evolutionary analysis showed that DNA transposons including Kolobok superfamily might get activated just after hybridization and then gradually inactivated. These findings suggest that some DNA transposons and their piRNAs might greatly influence allotetraploid genome evolution after hybridization.

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.

PACT/PRKRA and p53 regulate transcriptional activity of DMRT1.

The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.

Parallel Evolution of Two dmrt1-Derived Genes, dmy and dm-W, for Vertebrate Sex Determination.

Animal sex-determining genes, which bifurcate for female and male development, are diversified even among closely related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of two sex-determining genes, the medaka dmy and the clawed frog dm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization gene dmrt1. Interestingly, we detected parallel amino acid substitutions, from serine (S) to threonine (T), on the DNA-binding domains of both ancestral DMY and DM-W, resulting from positive selection. Two types of DNA-protein binding experiments and a luciferase reporter assay demonstrated that these S-T substitutions could strengthen the DNA-binding abilities and enhance the transcriptional regulation function. These findings suggest that the parallel S-T substitutions may have contributed to the establishment of dmy and dm-W as sex-determining genes.

Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans.

Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr Ali18 are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease.

The clock components Period2, Cryptochrome1a, and Cryptochrome2a function in establishing light-dependent behavioral rhythms and/or total activity levels in zebrafish.

The circadian clock generates behavioral rhythms to maximize an organism's physiological efficiency. Light induces the formation of these rhythms by synchronizing cellular clocks. In zebrafish, the circadian clock components Period2 (zPER2) and Cryptochrome1a (zCRY1a) are light-inducible, however their physiological functions are unclear. Here, we investigated the roles of zPER2 and zCRY1a in regulating locomotor activity and behavioral rhythms. zPer2/zCry1a double knockout (DKO) zebrafish displayed defects in total locomotor activity and in forming behavioral rhythms when briefly exposed to light for 3-h. Exposing DKO zebrafish to 12-h light improved behavioral rhythm formation, but not total activity. Our data suggest that the light-inducible circadian clock regulator zCRY2a supports rhythmicity in DKO animals exposed to 12-h light. Single cell imaging analysis revealed that zPER2, zCRY1a, and zCRY2a function in synchronizing cellular clocks. Furthermore, microarray analysis of DKO zebrafish showed aberrant expression of genes involved regulating cellular metabolism, including ATP production. Overall, our results suggest that zPER2, zCRY1a and zCRY2a help to synchronize cellular clocks in a light-dependent manner, thus contributing to behavioral rhythm formation in zebrafish. Further, zPER2 and zCRY1a regulate total physical activity, likely via regulating cellular energy metabolism. Therefore, these circadian clock components regulate the rhythmicity and amount of locomotor behavior.

Circadian transcription factor HSF1 regulates differential HSP70 gene transcription during the arousal-torpor cycle in mammalian hibernation.

Mammalian hibernation is a seasonal phenomenon. The hibernation season consists of torpor periods with a reduced body temperature (Tb), interrupted by euthermic arousal periods (interbout arousal, IBA). The physiological changes associated with hibernation are assumed to be under genetic control. However, the molecular mechanisms that govern hibernation-associated gene regulation are still unclear. We found that HSP70 transcription is upregulated in the liver of nonhibernating (summer-active) chipmunks compared with hibernating (winter-torpid) ones. In parallel, HSF1, the major transcription factor for HSP70 expression, is abundant in the liver-cell nuclei of nonhibernating chipmunks, and disappears from the nuclei of hibernating ones. Moreover, during IBA, HSF1 reappears in the nuclei and drives HSP70 transcription. In mouse liver, HSF1 is regulated by the daily Tb rhythm, and acts as a circadian transcription factor. Taken together, chipmunks similarly use the Tb rhythm to regulate gene expression via HSF1 during the torpor-arousal cycle in the hibernation season.

Characteristic expressions of the natriuretic peptide family in the telencephalon of juvenile chick.

Elucidation of the genes regulating the critical (sensitive) period of imprinting behavior may shed light on the mechanism underlying neural plasticity in early childhood learning. We focused on the family of natriuretic peptides (NPs) as candidates involved in the regulation of the critical period. In avians, several structurally related molecules comprised the NP family, including renal NP (RNP), B-type NP (BNP) and C-type NP (CNP1, CNP3 and CNPP). To understand the functional roles of NPs in neural plastic changes, we aimed to characterize NPs and their receptors in chick brain. We found that CNP3 mRNA was expressed in several regions in the telencephalon, including the visual Wulst (VW, considered as mammalian visual cortex) and amygdala. CNP1 mRNA was expressed throughout the telencephalon. Using real-time PCR, the gene expression levels of NPs and their receptors (NPR1 and NPR2) were studied during and after the critical period of imprinting (post-hatching day [P]1 and P7). CNP3 mRNA was found to show higher expression in the VW of P1 chicks than in VW of P7 chicks. Moreover, the ability of these peptides to stimulate chicken NPR1 or NPR2 was tested in HEK293 cells expressing either of the receptors. The activation of NPR1 was stronger with CNP3 than with other subtypes of CNP. In the VW, CNP3-expressing cells were negative for NPR1, but they resided in the vicinity of NPR1-expressing cells. These results suggest that CNP3 and its receptor NPR1 in the VW may have functional roles in the early learning.

JLP-JNK signaling protects cancer cells from reactive oxygen species-induced cell death.

Oxidative stress, which can be caused by an overproduction of reactive oxygen species (ROS), often leads to cell death. In recent years, c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP, also known as SPAG9 or JIP4), a scaffold protein for JNK mitogen-activated protein kinase (MAPK) signaling pathways, was found to serve as a novel biomarker for cancer. However, although JNK MAPK pathways are reported to be activated in response to various stimuli, including oxidative stress, whether JLP is involved in ROS signaling remains unknown. In this study, we examined the role of JLP in hydrogen peroxide (H2O2)-induced cancer cell death, and found that JLP knockdown (KD) cells exhibit a substantially enhanced cell death response, along with increased intracellular ROS levels. This is the first demonstration of a protective role for JLP in response to cell-death stimulation. We also found that the H2O2-induced JNK activation was attenuated in JLP KD cancer cells. The decreases in cell viability and JNK activation in the JLP KD cells were almost completely reversed by expressing wild-type JLP, but not a mutant JLP lacking the JNK-binding domain. These data collectively suggest that the JLP-JNK signaling pathway counteracts ROS-induced cancer cell death.

Epigenetic regulation of hibernation-associated HP-20 and HP-27 gene transcription in chipmunk liver.

The chipmunk hibernation-related proteins (HPs) HP-20 and HP-27 are components of a 140-kDa complex that dramatically decreases in the blood during hibernation. The HP-20 and HP-27 genes are expressed specifically in the liver and are downregulated in hibernating chipmunks. Hibernation-associated physiological changes are assumed to be under genetic control. Therefore, to elucidate the molecular mechanisms of hibernation, here we examined the mechanisms behind the altered HP-20 and HP-27 gene expression in nonhibernating versus hibernating chipmunks. Chromatin immunoprecipitation (ChIP) analyses revealed that histone H3 on the HP-20 and HP-27 gene promoters was highly acetylated at lysine (K) 9 and K14 and highly trimethylated at K4 in the liver of nonhibernating chipmunks, while these active histone modifications were nearly absent in hibernating chipmunks. Furthermore, histone acetyltransferases and a histone methyltransferase were associated with the HP-20 and HP-27 gene promoters primarily in nonhibernating chipmunks. Consistent with a previous finding that HNF-1 and USF can activate HP-20 and HP-27 gene transcription by binding to the proximal promoter region, ChIP-quantitative PCR (qPCR) analyses revealed that significantly less HNF-1 and USF were bound to these gene promoters in hibernating than in nonhibernating chipmunks. These findings collectively indicated that the hibernation-associated HP-20 and HP-27 gene expression is epigenetically regulated at the transcriptional level by the binding of HNF-1 and USF to their proximal promoters, and that histone modification has a key role in hibernation-associated transcriptional regulation.

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.

HNF-4 participates in the hibernation-associated transcriptional regulation of the chipmunk hibernation-related protein gene.

The chipmunk hibernation-related protein 25 (HP-25) is involved in the circannual control of hibernation in the brain. The liver-specific expression of the HP-25 gene is repressed in hibernating chipmunks under the control of endogenous circannual rhythms. However, the molecular mechanisms that differentially regulate the HP-25 gene during the nonhibernation and hibernation seasons are unknown. Here, we show that the hibernation-associated HP-25 expression is regulated epigenetically. Chromatin immunoprecipitation analyses revealed that significantly less hepatocyte nuclear receptor HNF-4 bound to the HP-25 gene promoter in the liver of hibernating chipmunks compared to nonhibernating chipmunks. Concurrently in the hibernating chipmunks, coactivators were dissociated from the promoter, and active transcription histone marks on the HP-25 gene promoter were lost. On the other hand, small heterodimer partner (SHP) expression was upregulated in the liver of hibernating chipmunks. Overexpressing SHP in primary hepatocytes prepared from nonhibernating chipmunks caused HNF-4 to dissociate from the HP-25 gene promoter, and reduced the HP-25 mRNA level. These results suggest that hibernation-related HP-25 expression is epigenetically regulated by the binding of HNF-4 to the HP-25 promoter, and that this binding might be modulated by SHP in hibernating chipmunks.

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.

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.

Sexually dimorphic expression of Dmrt1 and γH2AX in germ stem cells during gonadal development in Xenopus laevis.

In many animals, primordial germ cells (PGCs) migrate into developing gonads. There, they proliferate and differentiate into female and male germ stem cells (GSCs), oogonia and spermatogonia, respectively. Few studies have focused on the molecular mechanisms underlying the development of GSC sex determination. Here, we investigated the expression of the transcription factor Dmrt1 and a phosphorylated form of the histone variant H2AX (γH2AX) during gonadal development in Xenopus laevis. During early sexual differentiation, Dmrt1 was expressed in the GSCs of the ZW (female) and ZZ (male) gonads as well as somatic cells of the ZZ gonads. Notably, the PGCs and primary GSCs contained large, unstructured nuclei, whereas condensed, rounder nuclei appeared only in primary oogonia during tadpole development. After metamorphosis, Dmrt1 showed its expression in secondary spermatogonia, but not in secondary oogonia. Like Dmrt1, γH2AX was expressed in the nuclei of primary GSCs in early developing gonads. However, after metamorphosis, γH2AX expression continued in primary and secondary spermatogonia, but was barely detected in the condensed nuclei of primary oogonia. Taken together, these observations indicate that spermatogonia tend to retain PGC characteristics, compared to oogonia, which undergo substantial changes during gonadal differentiation in X. laevis. Our findings suggest that Dmrt1 and γH2AX may contribute to the maintenance of stem cell identity by controlling gene expression and epigenetic changes, respectively.

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.