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The vertebral column is a characteristic structure of vertebrates. Genetic studies in mice have shown that Hox-mediated patterning plays a key role in specifying discrete anatomical regions of the vertebral column. Expression pattern analyses in several vertebrate embryos have provided correlative evidence that the anterior boundaries of Hox expression coincide with distinct anatomical vertebrae. However, because functional analyses have been limited to mice, it remains unclear which Hox genes actually function in vertebral patterning in other vertebrates. In this study, various zebrafish Hox mutants were generated for loss-of-function phenotypic analysis to functionally decipher the Hox code responsible for the zebrafish anterior vertebrae between the occipital and thoracic vertebrae. We found that Hox genes in HoxB- and HoxC-related clusters participate in regulating the morphology of the zebrafish anterior vertebrae. In addition, medaka hoxc6a was found to be responsible for anterior vertebral identity, as in zebrafish. Based on phenotypic similarities with Hoxc6 knockout mice, our results suggest that the Hox patterning system, including at least Hoxc6, may have been functionally established in the vertebral patterning of the common ancestor of ray-finned and lobe-finned fishes.
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Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Columna Vertebral , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/embriología , Columna Vertebral/embriología , Tipificación del Cuerpo/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Genes Homeobox/genética , Oryzias/genética , Oryzias/embriología , RatonesRESUMEN
The dorsal and anal fins can vary widely in position and length along the anterior-posterior axis in teleost fishes. However, the molecular mechanisms underlying the diversification of these fins remain unknown. Here, we used genetic approaches in zebrafish and medaka, in which the relative positions of the dorsal and anal fins are opposite, to demonstrate the crucial role of hox genes in the patterning of the teleost posterior body, including the dorsal and anal fins. By the CRISPR-Cas9-induced frameshift mutations and positional cloning of spontaneous dorsalfinless medaka, we show that various hox mutants exhibit the absence of dorsal or anal fins, or a stepwise posterior extension of these fins, with vertebral abnormalities. Our results indicate that multiple hox genes, primarily from hoxc-related clusters, encompass the regions responsible for the dorsal and anal fin formation along the anterior-posterior axis. These results further suggest that shifts in the anterior boundaries of hox expression which vary among fish species, lead to diversification in the position and size of the dorsal and anal fins, similar to how modulations in Hox expression can alter the number of anatomically distinct vertebrae in tetrapods. Furthermore, we show that hox genes responsible for dorsal fin formation are different between zebrafish and medaka. Our results suggest that a novel mechanism has occurred during teleost evolution, in which the gene network responsible for fin formation might have switched to the regulation downstream of other hox genes, leading to the remarkable diversity in the dorsal fin position.
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Aletas de Animales , Genes Homeobox , Proteínas de Homeodominio , Oryzias , Pez Cebra , Animales , Oryzias/genética , Pez Cebra/genética , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Tipificación del Cuerpo/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismoRESUMEN
The neuromast is a sensory structure of the lateral line system in aquatic vertebrates, which consists of hair cells and supporting cells. Hair cells are mechanosensory cells, generally arranged with bidirectional polarity. Here, we describe a neuromast with hair cells arranged radially instead of bidirectionally in the first cranial neuromast of four teleost species: red seabream (Pagrus major), spotted halibut (Verasper variegatus), brown sole (Pseudopleuronectes herzensteini), and marbled sole (Pseudopleuronectes yokohamae). In these four species, this polarity was identified only in the first cranial neuromast, where it appeared at the rostral edge of the otic vesicle before hatching. We investigated the initial appearance and fate of this unique neuromast using scanning electron microscopy. We also assessed characteristics of radial neuromast pertaining to morphogenesis, development, and innervation using a vital fluorescent marker and immunohistochemistry in V. variegatus. The kinocilium initially appears at the center of each hair cell, then moves to its outer perimeter to form radial polarity by around 7 days postfertilization. However, hair cells arranged radially disappear about 15 days after hatching. This is followed by the appearance of bidirectionally arranged hair cells, indicating that polarity replacement from radial to bidirectional has occurred. In P. herzensteini, both afferent and efferent synapses between the nerve fibers and hair cells were observed by transmission electron microscopy, suggesting that radial neuromast is functional. Our discovery suggests that neuromasts with radial polarity could enable larval fish to assimilate multiaxial stimuli during this life stage, potentially assisting them in detecting small water vibrations or water pressure changes.
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Sistema de la Línea Lateral , Mecanorreceptores , Animales , Peces , Cráneo , Microscopía Electrónica de Rastreo , Agua , Pez CebraRESUMEN
The interactions between the host immune system and intestinal microorganisms have been studied in many animals, including fish. However, a detailed analysis has not been performed in medaka, an established fish model for biological studies. Here, we investigated the effect of immunodeficiency on the microbiota composition and the effect of gut bacteria on intestinal epithelial development and immune responses in medaka. Chronological analysis of the intestinal microbiota of interleukin 2 receptor subunit gamma (il2rg) mutant medaka showed a gradual decrease in the evenness of operational taxonomic units, mainly caused by the increased abundance of the Aeromonadaceae family. Exposure of wild-type medaka to high doses of an intestine-derived opportunistic bacterium of the Aeromonadaceae family induced an inflammatory response, suggesting a harmful effect on adult il2rg mutants. In addition, we established germ-free conditions in larval medaka and observed large absorptive vacuoles in intestinal epithelial cells, indicating a block in epithelial maturation. Transcriptome analysis revealed a decrease in the expression of genes involved in the defense response, including the antimicrobial peptide gene hepcidin, whose expression is induced by lipopolysaccharide stimulation in normal larvae. These results show that reciprocal interactions between the microbiome and the intestinal tract are required for the maturation of the medaka immune system.
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Microbioma Gastrointestinal , Microbiota , Oryzias , Animales , Crecimiento y Desarrollo , Sistema InmunológicoRESUMEN
In ELISA, blocking reagents and stabilizers are important to improve the sensitivity and/or quantitative nature of the measurement system. Usually, biological substances such as bovine serum albumin and casein are used, but they still have problems such as lot-to-lot differences and biohazard. Here, we describe the methods using a chemically synthesized polymer, BIOLIPIDURE®, as a new blocking agent and stabilizer that can solve these problems.
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Caseínas , Polímeros , Ensayo de Inmunoadsorción Enzimática/métodos , Caseínas/análisis , Albúmina Sérica BovinaRESUMEN
The INSPIRIS RESILIA valve is designed to dilate its valve annulus in transcatheter aortic valve-in-surgical aortic valve (TAV-in-SAV), a catheter therapy for biological valve deterioration. RESILIA tissue has improved anti-calcification properties. An 83-year-old man on hemodialysis undergoing surgical aortic valve replacement (SAVR) with a 25-mm INSPIRIS for severe aortic stenosis 22 months ago presented with general malaise. Transthoracic echocardiography revealed severe bioprosthetic stenosis (peak velocity: 3.5 m/s, mean pressure gradient: 32 mmHg, and effective orifice area: 0.45 cm2) and severely reduced left ventricular function (ejection fraction: 17%). Because redo-SAVR was extremely risky (society of thoracic surgeons [STS] risk score: 31%), the patient underwent transfemoral-TAV-in-SAV using a 26-mm SAPIEN 3ï¸. Pre- and postoperative computed tomography showed that the internal diameter of the INSPIRIS had expanded from 22.2 mm to 24.2 mm. This case demonstrated the dilatable design of INSPIRIS but not the durability of RESILIA tissue.
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Hermansky-Pudlak syndrome is an autosomal recessive disease characterized by albinism, visual impairment, and blood platelet dysfunction. One of the genes responsible for Hermansky-Pudlak syndrome, hps1, regulates organelle biogenesis and thus plays important roles in melanin production, blood clotting, and the other organelle-related functions in humans and mice. However, the function of hps1 in other species remains poorly understood. In this study, we discovered albino medaka fish during the maintenance of a wild-derived population and identified hps1 as the responsible gene using positional cloning. In addition to the specific absence of melanophore pigmentation, the hps1 mutant showed reduced blood coagulation, suggesting that hps1 is involved in clotting caused by both mammalian platelets and fish thrombocytes. Together, the findings of our study demonstrate that hps1 has an evolutionarily conserved role in melanin production and blood coagulation. In addition, our study presents a useful vertebrate model for understanding the molecular mechanisms of Hermansky-Pudlak syndrome.
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Síndrome de Hermanski-Pudlak , Oryzias , Albinismo , Animales , Coagulación Sanguínea/genética , Trastornos Hemorrágicos , Síndrome de Hermanski-Pudlak/genética , Humanos , Mamíferos , Melaninas/genética , Proteínas de la Membrana/genética , Ratones , Mutación , Oryzias/genéticaRESUMEN
Many studies have reported the complete mitochondrial genome sequences of Chinese Megascolecidae earthworms, however, there have been no reports on sequences originating from Japanese Megascolecidae earthworms. In this study, we determined complete mitochondrial genome sequences of two Japanese earthworms belonging to the Pheretima complex within the Megascolecidae family. Metaphire hilgendorfi is one of the most common earthworms in Japan and Amynthas yunoshimensis was found to be morphologically similar to M. hilgendorfi. The complete mitochondrial genomes of M. hilgendorfi (15,186 bp; LC573968) and A. yunoshimensis (15,109 bp; LC573969) contained typical 13 protein coding genes, 22 tRNA genes, and 2 rRNA genes. The phylogenetic analysis revealed that these two species were sister species. Therefore, our findings will further contribute to phylogenetic and genetic diversity analyses of Megascolecidae earthworms.
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SIGNIFICANCE: Differential interference contrast (DIC) microscopes allow noninvasive in vivo observation of transparent microstructures in tissue without the use of fluorescent dyes or genetic modification. We show how to modify a DIC microscope to measure the sample phase distribution accurately and in real-time even deep inside sample tissue. AIM: Our aim is to improve the DIC microscope's phase measurement to remove the phase bias that occurs in the presence of strong scattering. APPROACH: A quarter-wave plate was added in front of the polarization camera, allowing a modified phase calculation to incorporate all four polarization orientation angles (0 deg, 45 deg, 90 deg, and 135 deg) captured simultaneously by the polarization camera, followed by deconvolution. RESULTS: We confirm that the proposed method reduces phase measurement error in the presence of scattering and demonstrate the method using in vivo imaging of a beating heart inside a medaka egg and the whole-body blood circulation in a young medaka fish. CONCLUSIONS: Modifying a polarization-camera DIC microscope with a quarter-wave plate allows users to image deep inside samples without phase bias due to scattering effects.
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Microscopía , Animales , Microscopía de InterferenciaRESUMEN
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.
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[This corrects the article on p. 1273 in vol. 10, PMID: 30891345.].
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This paper describes how to take advantage of the replacement of an intensity camera with a polarization camera in a standard differential interference contrast (DIC) microscope. Using a polarization camera enables snapshot quantitative phase analysis so that real-time imaging of living transparent tissues become possible. Using our method, we quantify the phase measurement accuracy using a phantom consisting of glass beads embedded in lacquer. In order to demonstrate these advantages, we image the pumping heart and blood flow in a living medaka egg.
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An α-l-fucosidase (Pap-Alf) was purified from the pancreas of a starfish Patiria pectinifera by ammonium sulfate precipitation followed by several column chromatographies. The molecular mass of the purified enzyme was estimated to be 52.6â¯kDa by SDS-PAGE, although gel filtration analysis of the native enzyme suggests it exists as a homodimer in solution. The purified enzyme showed maximal activity at pH 5.0 and 70⯰C. The enzyme was highly specific toward a fucosyl-monosaccharide (Fuc-α-pNP), but it also showed activity toward 2-sulfo-Fuc-α-pNP and fucosyl-α-lactosides (Fuc-α-Galß1â4Glc-ß-pNP). We determined the primary structure of the α-l-fucosidase and validated its expression level in starfish tissue. Whole genome sequence analysis of P. pectinifera was also performed in the present study. Detailed primary structural analysis using bioinformatics tools revealed Pap-Alf lacks the C-terminal region that is otherwise conserved in all previously described α-l-fucosidases. Quantitative gene expression analysis of Pap-Alf in each tissue indicated that the expression of Pap-Alf gene in pancreas was 5-fold higher than in ovary.
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Clonación Molecular , Páncreas/enzimología , alfa-L-Fucosidasa/química , alfa-L-Fucosidasa/genética , Animales , Regulación Enzimológica de la Expresión Génica/genética , Estrellas de Mar , alfa-L-Fucosidasa/metabolismoRESUMEN
In the genetic sex determination of vertebrates, the gonadal sex depends on the combination of sex chromosomes that a zygote possesses. Despite the discovery of the sex-determining gene (SRY/Sry) in mammals in 1990s, the sex-determining gene in non-mammalian vertebrates remained an enigma for over a decade. In most mammals, the male-inducing master sex-determining gene is located on the Y chromosome and is therefore absent from XX females. A second sex-determining gene, Dmy, was described in the Oryzias latipes in 2002 and has a DNA-binding motif that is different from the motif in the mammalian sex-determining gene SRY or Sry. Dmy is also located on the Y chromosome and is therefore absent in XX females. Seven other sex-determining genes, including candidate genes, are now known in birds, a frog species, and 5 fish species. These findings over the past twenty years have increased our knowledge of sex-determining genes and sex chromosomes among vertebrates. Here, we review recent advances in our understanding of sex-determining genes and genetic sex determination systems in fish, especially those of the Oryzias species, which are described in detail. The facts suggest some patterns of how new sex-determining genes emerged and evolved. We believe that these facts are common not only in Oryzias but also in other fish species. This knowledge will help to elucidate the conserved mechanisms from which various sex-determining mechanisms have evolved.
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Evolución Molecular , Genes sry , Oryzias/genética , Procesos de Determinación del Sexo/genética , Animales , Femenino , Masculino , Mutación , Oryzias/clasificación , Cromosomas Sexuales , Factores de Transcripción/genética , Cromosoma YRESUMEN
Gonochoristic species have a bipotential gonad that develops into a testis or an ovary. In species whose sex is determined by a genetic factor, the expression of a sex-determining gene is the first cue that directs the development of a bipotential gonad. Subsequent expression of downstream genes induces the gonad to develop into a testis or an ovary. The TGF-ß family member Gonadal soma-derived factor (Gsdf) is thought to be an important gene for gonadal development in teleost fish, and it is expressed at higher levels in the testis than in the ovary from early to mature stages. However, there is little functional information about the gene. In this study, we targeted the Gsdf coding region in the medaka fish Oryzias latipes using transcription activator-like effector nucleases (TALENs) and studied the phenotypes of the Gsdf mutant medaka. Although normal and heterozygous XY gonads developed into a testis, all XY gonads with a homozygous mutation in Gsdf developed into an ovary at early developmental stages. However, two-thirds of Gsdf mutant XY gonads developed into testes in the adult stages. These results demonstrate that although a gonad can develop into a complete testis in the absence of Gsdf, Gsdf function is critical for directing the bipotential gonad at early developmental stages. Therefore, Gsdf is an endogenous inducer of testicular development similar to a master sex-determining gene.
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Gónadas/crecimiento & desarrollo , Mutación , Oryzias/crecimiento & desarrollo , Oryzias/genética , Procesos de Determinación del Sexo/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , ADN/genética , Femenino , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Masculino , Datos de Secuencia Molecular , Ovario/crecimiento & desarrollo , Fenotipo , Testículo/crecimiento & desarrollo , Factor de Crecimiento Transformador beta/genética , Cromosoma X/genética , Cromosoma Y/genéticaRESUMEN
We propose ultra-high resolution optical coherence tomography to study the morphological development of internal organs in medaka fish in the post-embryonic stages at micrometer resolution. Different stages of Japanese medaka were imaged after hatching in vivo with an axial resolution of 2.8 µm in tissue. Various morphological structures and organs identified in the OCT images were then compared with the histology. Due to the medaka's close resemblance to vertebrates, including humans, these morphological features play an important role in morphogenesis and can be used to study diseases that also occur in humans.
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R-spondin (Rspo) encodes a multi-domain protein that modulates the Wnt-signaling pathway. Two distinct rspo2 zebrafish mutants were generated by TALEN-mediated mutagenesis: a null mutant, rspo2(null), lacking all functional domains, and a hypomorphic mutant, rspo2(tsp), lacking the two N-terminal domains. Mutants were analyzed mainly for abnormalities in the skeletal system. Fin ray skeletons were formed normally in the rspo2(tsp) mutants, but were absent from the rspo2(null) mutants. Hypoplasia of the neural/hemal arches and ribs was observed in both mutants. Thus, the two rspo2 mutants help to identify the functions of Rspo2 in skeletogenesis, as well as functional differences among multiple Rspo2 domains.
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Enzimas de Restricción del ADN/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mutagénesis , Rajidae/crecimiento & desarrollo , Rajidae/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Rajidae/anomalías , Proteínas de Pez Cebra/químicaRESUMEN
Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.
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Reacción de Prevención/fisiología , Habénula/fisiología , Vías Nerviosas/fisiología , Núcleos del Rafe/fisiología , Neuronas Serotoninérgicas/fisiología , 5,7-Dihidroxitriptamina/metabolismo , Potenciales de Acción/fisiología , Adaptación Psicológica/fisiología , Animales , Animales Modificados Genéticamente , Condicionamiento Clásico/fisiología , Señales (Psicología) , Miedo/fisiología , Habénula/citología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Neurotransmisores/metabolismo , Núcleos del Rafe/citología , Serotonina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/genética , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.
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Elementos Transponibles de ADN , Genoma , Células Germinativas , Oryzias/genética , Alelos , Animales , Secuencia de Bases , Cruzamiento , Femenino , Sitios Genéticos , Genotipo , Masculino , Datos de Secuencia Molecular , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.