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Recent advances in DNA sequencing technology have enabled the precise decoding of genomes in non-model organisms, providing a basis for unraveling the patterns and mechanisms of sex chromosome evolution. Studies of different species have yielded conflicting results regarding the traditional theory that sex chromosomes evolve from autosomes via the accumulation of deleterious mutations and degeneration of the Y (or W) chromosome. The concept of the 'sex chromosome cycle,' emerging from this context, posits that at any stage of the cycle (i.e., differentiation, degeneration, or loss), sex chromosome turnover can occur while maintaining stable sex determination. Thus, understanding the mechanisms that drive both the persistence and turnover of sex chromosomes at each stage of the cycle is crucial. In this review, we integrate recent findings on the mechanisms underlying maintenance and turnover, with a special focus on several organisms having unique sex chromosomes. Our review suggests that the diversity of sex chromosomes in the maintenance of stable sex determination is underappreciated and emphasizes the need for more research on the sex chromosome cycle.
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Cromosomas Sexuales , Procesos de Determinación del Sexo , Cromosomas Sexuales/genética , Procesos de Determinación del Sexo/genética , Animales , Humanos , Femenino , MasculinoRESUMEN
Xenopus is one of the essential model systems for studying vertebrate development. However, one drawback of this system is that, because of the opacity of Xenopus embryos, 3D imaging analysis is limited to surface structures, explant cultures, and post-embryonic tadpoles. To develop a technique for 3D tissue/organ imaging in whole Xenopus embryos, we identified optimal conditions for using placental alkaline phosphatase (PLAP) as a transgenic reporter and applied it to the correlative light microscopy and block-face imaging (CoMBI) method for visualization of PLAP-expressing tissues/organs. In embryos whose endogenous alkaline phosphatase activities were heat-inactivated, PLAP staining visualized various tissue-specific enhancer/promoter activities in a manner consistent with green fluorescent protein (GFP) fluorescence. Furthermore, PLAP staining appeared to be more sensitive than GFP fluorescence as a reporter, and the resulting expression patterns were not mosaic, in striking contrast to the mosaic staining pattern of ß-galactosidase expressed from the lacZ gene that was introduced by the same transgenesis method. Owing to efficient penetration of alkaline phosphatase substrates, PLAP activity was detected in deep tissues, such as the developing brain, spinal cord, heart, and somites, by whole-mount staining. The stained embryos were analyzed by the CoMBI method, resulting in the digital reconstruction of 3D images of the PLAP-expressing tissues. These results demonstrate the efficacy of the PLAP reporter system for detecting enhancer/promoter activities driving deep tissue expression and its combination with the CoMBI method as a powerful approach for 3D digital imaging analysis of specific tissue/organ structures in Xenopus embryos.
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Fosfatasa Alcalina , Calor , Animales , Femenino , Embarazo , Xenopus laevis , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/análisis , Placenta , Animales Modificados GenéticamenteRESUMEN
The mitochondrial genome (mt-genome) is one of the promising molecular markers for phylogenetics and population genetics. Recently, various mt-genomes have been determined rapidly by using massively parallel sequencers. However, the control region (CR, also called D-loop) in mt-genomes remain difficult to precisely determine due to the presence of repeat regions. Here, using Nanopore sequencing, we succeeded in rapid and collective determination of complete mt-genome of the hot-spring frog, Buergeria japonica, and found that its mt-genome size was 22,274 bp including CR (6,929 bp) with two types of tandem repeat motifs forming repeat regions. Comparison of assembly strategies revealed that the long- and short-read data combined together enabled efficient determination of the CR, but the short-read data alone did not. The B. japonica CR was longer than that of a congenic species inhabiting cooler climate areas, Buergeria buergeri, because of the long repeat regions in the former. During the thermal adaptation of B. japonica, the longer repeat regions in its CR may have accumulated within a period after divergence from B. buergeri.
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Genoma Mitocondrial , Secuenciación de Nanoporos , Animales , Análisis de Secuencia de ADN , Anuros/genética , FilogeniaRESUMEN
Cancer treatment is still challenging because the disease is often caused by multiple mutations. Although genomic studies have identified many oncogenes and tumor suppressor genes, gene sets involved in tumorigenesis remain poorly understood. Xenopus, a genus of aquatic frogs, is a useful model to identify gene sets because it can be genetically and experimentally analyzed. Here, we analyzed gene expression in tumor tissues of three individuals in Xenopus tropicalis and identified 55 differentially expressed genes (DEGs). Gene ontology (GO) analysis showed that the upregulated genes in the tumor tissues were enriched in GO terms related to the extracellular matrix and collagen fibril organization. Hierarchical clustering showed that the gene expression patterns of tumor tissues in X. tropicalis were comparable to those of human connective, soft, and subcutaneous tissue-derived cancers. Additionally, pathway analysis revealed that these DEGs were associated with multiple pathways, including the extracellular matrix, collagen fibril organization, MET signaling, and keratan sulfate. We also found that the expression tendency of some DEGs that have not been well analyzed in the cancer field clearly determines the prognosis of human cancer patients. This study provides a remarkable reference for future experimental work on X. tropicalis to identify gene sets involved in human cancer.
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Perfilación de la Expresión Génica , Genes Relacionados con las Neoplasias , Humanos , Animales , Xenopus/genética , Xenopus/metabolismo , Biología Computacional , Análisis de Secuencia de ARN , Colágeno/genética , Colágeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Biomarcadores de Tumor/genética , Ontología de GenesRESUMEN
Since CRISPR-based genome editing technology works effectively in the diploid frog Xenopus tropicalis, a growing number of studies have successfully modeled human genetic diseases in this species. However, most of their targets were limited to non-syndromic diseases that exhibit abnormalities in a small fraction of tissues or organs in the body. This is likely because of the complexity of interpreting the phenotypic variations resulting from somatic mosaic mutations generated in the founder animals (crispants). In this study, we attempted to model the syndromic disease campomelic dysplasia (CD) by generating sox9 crispants in X. tropicalis. The resulting crispants failed to form neural crest cells at neurula stages and exhibited various combinations of jaw, gill, ear, heart, and gut defects at tadpole stages, recapitulating part of the syndromic phenotype of CD patients. Genotyping of the crispants with a variety of allelic series of mutations suggested that the heart and gut defects depend primarily on frame-shift mutations expected to be null, whereas the jaw, gill, and ear defects could be induced not only by such mutations but also by in-frame deletion mutations expected to delete part of the jawed vertebrate-specific domain from the encoded Sox9 protein. These results demonstrate that Xenopus crispants are useful for investigating the phenotype-genotype relationships behind syndromic diseases and examining the tissue-specific role of each functional domain within a single protein, providing novel insights into vertebrate jaw evolution.
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Displasia Campomélica , Animales , Humanos , Xenopus laevis/metabolismo , Displasia Campomélica/genética , Xenopus/genética , Xenopus/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Fenotipo , GenotipoRESUMEN
The chytrid fungus Batrachochytrium dendrobatidis (Bd) is a major threat to amphibians, yet there are no reports of major disease impacts in East Asian frogs. Genetic variation of the major histocompatibility complex (MHC) has been associated with resistance to Bd in frogs from East Asia and worldwide. Using transcriptomic data collated from 11 Japanese frog species (one individual per species), we isolated MHC class I and IIb sequences and validated using molecular cloning. We then compared MHC from Japanese frogs and other species worldwide, with varying Bd susceptibility. Supertyping analysis, which groups MHC alleles based on physicochemical properties of peptide binding sites, identified that all examined East Asian frogs contained at least one MHC-IIb allele belonging to supertype ST-1. This indicates that, despite the large divergence times between some Japanese frogs (up to 145 million years), particular functional properties in the peptide binding sites of MHC-II are conserved among East Asian frogs. Furthermore, preliminary analysis using NetMHCIIpan-4.0, which predicts potential Bd-peptide binding ability, suggests that MHC-IIb ST-1 and ST-2 have higher overall peptide binding ability than other supertypes, irrespective of whether the peptides are derived from Bd, other fungi, or bacteria. Our findings suggest that MHC-IIb among East Asian frogs may have co-evolved under the same selective pressure. Given that Bd originated in this region, it may be a major driver of MHC evolution in East Asian frogs.
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BACKGROUND: Determining the association between radiographic spinal instability assessment and lower back lumbar diseases with lower limb symptoms can contribute to evidence-based assessment and treatment in clinical practice and rehabilitation. Therefore, radiological evidence of lumbar spine instability assessment, such as sagittal translation (ST) and segmental angulation (SA), is clinically important. OBJECTIVE: To identify factors associated with the assessment of spinal instability in lumbar disc herniation with leg pain and discogenic low back pain using ST and SA. METHODS: We examined 112 patients with lumbar disc herniation with leg pain and 116 with discogenic low back pain at our clinic from 2016 to 2021. Data on age, gender, sports activities, and occupation were collected from medical records. Additionally, ST and SA of L4 and L5 during maximum trunk flexion and extension were measured using radiography. Simple and multiple logistic regression analyses were used for statistical analysis. RESULTS: Simple logistic regression analysis showed that ST and SA (odds ratio [OR]: 1.11; 95% confidence interval [CI]: 1.03-1.19) were associated with lumbar disc herniation. Multiple logistic regression analysis showed that only ST was associated with lumbar disc herniation (OR: 2.29; 95% CI: 1.78-3.00). CONCLUSION: Multiple logistic regression analysis showed that ST was associated with lumbar disc herniation with leg pain and had a stronger association than SA.
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Desplazamiento del Disco Intervertebral , Dolor de la Región Lumbar , Enfermedades de la Columna Vertebral , Humanos , Dolor de la Región Lumbar/diagnóstico por imagen , Dolor de la Región Lumbar/tratamiento farmacológico , Desplazamiento del Disco Intervertebral/complicaciones , Desplazamiento del Disco Intervertebral/diagnóstico por imagen , Pierna , Región Lumbosacra , Vértebras Lumbares/diagnóstico por imagenRESUMEN
To evaluate the degree of postmating isolation and the evolutionary relationships among frog species in the genus Fejervarya from Indonesia (Lesser Sunda), Bangladesh, China, and Japan, crossing experiments and molecular phylogenetic analyses were carried out. Crossing experiments revealed that reciprocal hybrids among F. iskandari, F. verruculosa, and F. sp. large type and between F. multistriata and F. kawamurai are viable through metamorphosis, while those between the F. iskandari group and F. limnocharis group were completely or partially inviable at the tadpole stage and those between Southeast Asian and South Asian Fejervarya groups were completely inviable at the embryonic stage. The mature reciprocal hybrids between F. iskandari and F. verruculosa from Lesser Sunda, Indonesia, showed some degree of abnormality in spermatogenesis. In phylogenetic analyses based on mtDNA Cytb sequences, F. iskandari formed a sister clade with F. verruculosa from Lesser Sunda, Indonesia, with 8.1% sequence divergence. F. multistriata from China formed a clade with populations of F. limnocharis in Thailand, Malaysia, and Indonesia (topotype), and these taxa showed sister relationships to F. kawamurai from Japan with 8.9% sequence divergence. Fejervarya sp. small type from Bangladesh formed a clade with the other South Asian members of the Fejervarya group and formed a sister clade with the Southeast Asian Fejervarya group, with 23.1% sequence divergence in the Cytb gene. These results showed that the degree of postmating isolation reflects molecular phylogenetic relationships and that F. iskandari and F. verruculosa from Indonesia (Lesser Sunda) are reproductively isolated by abnormalities in spermatogenesis and show genetic differentiation.
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BACKGROUND: Cervical kyphosis mechanically influences the intervertebral discs, which may lead to neck, shoulder, and scapular pain. OBJECTIVES: To investigate the incidence of cervical kyphosis and posterior displacement of cervical vertebrae (cervical posterior translation index) in patients with radiologically evident degenerative disc disease. DESIGN: Retrospective-cohort study. METHODS: Ninety-five patients with radiologically evident degenerative disc disease were recruited at our clinic from 2015 to 2019. Radiography images were used to analyze the straight-line index in each patient in the upright posture. The straight-line index was calculated by drawing a reference line connecting the posterior inferior edges of C2 to C7 and normalizing the addition of the distances from the posterior inferior edges of C3, C4, C5, and C6 to the reference line, respectively. Straight-line index <0 was defined as cervical kyphosis and ≥0 as cervical lordosis, the cervical kyphosis. In cervical kyphosis group, the distance from the posterior inferior edges of C3, C4, C5, and C6 to the reference line was normalized by the reference line distance to determine posterior displacement. Multiple comparison tests aside from simple and multiple linear regression analysis were performed. RESULTS: The incidence of cervical kyphosis in patients with radiologically evident degenerative disc disease was 60% (57/95 patients). Multiple comparison tests revealed that the cervical posterior translation indexes of C4 and C5 were significantly higher than those of C3, C4, and C6, in cervical kyphosis group. CONCLUSIONS: Cervical kyphosis was present in 60% of patients with radiologically evident degenerative disc disease, with C5 showing the most posterior displacement.
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Degeneración del Disco Intervertebral , Cifosis , Humanos , Dolor de Cuello/diagnóstico por imagen , Dolor de Cuello/epidemiología , Dolor de Cuello/etiología , Degeneración del Disco Intervertebral/diagnóstico por imagen , Degeneración del Disco Intervertebral/epidemiología , Degeneración del Disco Intervertebral/complicaciones , Estudios Retrospectivos , Estudios de Cohortes , Cifosis/diagnóstico por imagen , Cifosis/epidemiología , Vértebras Cervicales/diagnóstico por imagenRESUMEN
Candidatus Vesicomyosocius okutanii is a currently uncultured endosymbiotic bacterium of Phreagena okutanii, a clam that inhabits deep-sea vent environments. The genome of Ca. V. okutanii encodes a sulfur-oxidizing (Sox) enzyme complex, presumably generating biological energy for the host from inorganic sulfur compounds. Here, Ca. V. okutanii SoxX (VoSoxX), a mono-heme cytochrome c component of the Sox complex, was shown to be phylogenetically related to its homologous counterpart (HcSoxX) from a free-living deep-sea bacterium, Hydrogenovibrio crunogenus. Both proteins were heterologously expressed in Escherichia coli co-expressing cytochrome c maturation genes for comparative biochemical analysis. The VoSoxX recombinant had significantly lower thermal stability than HcSoxX, reflecting the difference in growth conditions of the source bacteria. The endosymbiont inhabits a mild intracellular environment, whereas the free-living bacterium dwells in a harsh environment. This study represents the first successful case of heterologous expression of genes from Ca. V. okutanii, allowing further biochemical studies of the molecular mechanism of sulfur oxidation in deep-sea environments.
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Bivalvos , Gammaproteobacteria , Animales , Bacterias/genética , Bivalvos/genética , Bivalvos/metabolismo , Citocromos c , Filogenia , Piscirickettsiaceae , Azufre/metabolismo , Compuestos de AzufreRESUMEN
Environmental temperature is a critical factor for all forms of life, and thermal tolerance defines the habitats utilized by a species. Moreover, the evolutionary tuning of thermal perception can also play a key role in habitat selection. Yet, the relative importance of thermal tolerance and perception in environmental adaptation remains poorly understood. Thermal conditions experienced by anuran tadpoles differ among species due to the variation in breeding seasons and water environments selected by parental frogs. In the present study, heat tolerance and avoidance temperatures were compared in tadpoles from five anuran species that spatially and temporally inhabit different thermal niches. These two parameters were positively correlated with each other and were consistent with the thermal conditions of habitats. The species difference in avoidance temperature was 2.6 times larger than that in heat tolerance, suggesting the importance of heat avoidance responses in habitat selection. In addition, the avoidance temperature increased after warm acclimation, especially in the species frequently exposed to heat in their habitats. Characterization of the heat-sensing transient receptor potential ankyrin 1 (TRPA1) ion channel revealed an amphibian-specific alternatively spliced variant containing a single valine insertion relative to the canonical alternative spliced variant of TRPA1, and this novel variant altered the response to thermal stimuli. The two alternatively spliced variants of TRPA1 exhibited different thermal responses in a species-specific manner, which are likely to be associated with a difference in avoidance temperatures among species. Together, our findings suggest that the functional change in TRPA1 plays a crucial role in thermal adaptation processes.
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Calor , Taxia , Aclimatación/genética , Animales , Ancirinas , Anuros/genética , Reacción de PrevenciónRESUMEN
Cytochrome c'-ß is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel ß-sheet fold. Here, the crystal structure of cytochrome c'-ß from the thermophilic Thermus thermophilus (TTCP-ß) is reported at 1.74â Å resolution. TTCP-ß has a typical antiparallel ß-sheet fold similar to that of cytochrome c'-ß from the moderately thermophilic Methylococcus capsulatus (MCCP-ß). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-ß and MCCP-ß, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-ß exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-ß. Mutational analysis reveals that the increased homodimeric interface area of TTCP-ß contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-ß loop regions, possibly contribute to the rigid loop structure compared with MCCP-ß, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-ß, including TTCP-ß, are optimized for function under the high-temperature conditions in which the source organisms live.
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Citocromos c' , Thermus thermophilus , Secuencia de Aminoácidos , Cristalografía por Rayos X , Citocromos c , Filogenia , Prolina , Thermus thermophilus/químicaRESUMEN
The CRISPR/Cas9 method has become popular for gene disruption experiments in Xenopus laevis. However, the experimental conditions that influence the efficiency of CRISPR/Cas9 remain unclear. To that end, we developed an image analysis technique for the semi-quantitative evaluation of the pigment phenotype resulting from the disruption of tyrosinase genes in X. laevis using a CRISPR/Cas9 approach, and then examined the effects of varying five experimental parameters (timing of the CRISPR reagent injection into developing embryos; amount of Cas9 mRNA in the injection reagent; total injection volume per embryo; number of injection sites per embryo; and the culture temperature of the injected embryos) on the gene disruption efficiency. The results of this systematic analysis suggest that the highest possible efficiency of target gene disruption can be achieved by injecting a total of 20 nL of the CRISPR reagent containing 1500 pg of Cas9 mRNA or 4 ng of Cas9 protein into two separate locations (10 nL each) of one-cell stage embryos cultured at 22°C. This study also highlights the importance of balancing the experimental parameters for increasing gene disruption efficiency and provides valuable insights into the optimal conditions for applying the CRISPR/Cas9 system to new experimental organisms.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Fenotipo , ARN Mensajero/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
BACKGROUND: Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo-injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo-injury procedures are common in mouse and zebrafish. RESULTS: In the present study, we induced cryo-damage on the ventricle in Iberian ribbed newts using a liquid nitrogen-chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo-injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures. CONCLUSIONS: Notably, we established a cryo-injury procedure for the newt and confirmed that regeneration of the cryo-damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo-injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models.
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Pleurodeles , Pez Cebra , Animales , Ratones , Pleurodeles/genética , Regeneración/genética , Salamandridae/genética , Transcriptoma , Pez Cebra/genéticaRESUMEN
Hynobius dunni is a salamander species of the genus Hynobius endemically distributed in eastern Kyushu in southwestern Japan. In this study, we determined the complete mitochondrial genome sequence and clarified the phylogenetic position of this species. The mitochondrial genome was 16,47 bp in length and encoded 13 protein, 2 ribosomal RNA, and 22 transfer RNA genes. Phylogenetic tree based on 13 protein-coding genes revealed that H. nebulosus were the most closely related species within the Hynobius species. The data identified in this study will be useful for population and conservation genetic studies of Hynobius species.
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Japanese indigenous chickens have a long breeding history, possibly beginning 2000 years ago. Genetic characterization of Japanese indigenous chickens has been performed using mitochondrial D-loop region and microsatellite DNA markers. Their phylogenetic relationships with chickens worldwide and genetic variation within breeds have not yet been examined. In this study, the genetic characteristics of 38 Japanese indigenous chicken breeds were assessed by phylogenetic analyses of mitochondrial D-loop sequences compared with those of indigenous chicken breeds overseas. To evaluate the genetic relationships among Japanese indigenous chicken breeds, a STRUCTURE analysis was conducted using 27 microsatellite DNA markers. D-loop sequences of Japanese indigenous chickens were classified into five major haplogroups, A-E, among 15 haplogroups found in chickens worldwide. The haplogroup composition suggested that Japanese indigenous chickens originated mainly from China, with some originating from Southeast Asia. The STRUCTURE analyses revealed that Japanese indigenous chickens are genetically differentiated from chickens overseas; Japanese indigenous chicken breeds possess distinctive genetic characteristics, and Jidori breeds, which have been reared in various regions of Japan for a long time, are genetically close to each other. These results provide new insights into the history of chickens around Asia in addition to novel genetic data for the conservation of Japanese indigenous chickens.