RESUMEN
BACKGROUND: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. RESULTS: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. CONCLUSIONS: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.
Asunto(s)
Elementos Transponibles de ADN/fisiología , Células Germinativas/metabolismo , Factores de Transcripción SOXD/biosíntesis , Cromosomas Sexuales/metabolismo , Procesos de Determinación del Sexo/fisiología , Animales , Animales Modificados Genéticamente , Femenino , Masculino , Oryzias , Factores de Transcripción SOXD/genética , Cromosomas Sexuales/genéticaRESUMEN
Assessing allele-specific gene expression (ASE) on a large scale continues to be a technically challenging problem. Certain biological phenomena, such as X chromosome inactivation and parental imprinting, affect ASE most drastically by completely shutting down the expression of a whole set of alleles. Other more subtle effects on ASE are likely to be much more complex and dependent on the genetic environment and are perhaps more important to understand since they may be responsible for a significant amount of biological diversity. Tools to assess ASE in a diploid biological system are becoming more reliable. Non-diploid systems are, however, not uncommon. In humans full or partial polyploid states are regularly found in both healthy (meiotic cells, polynucleated cell types) and diseased tissues (trisomies, non-disjunction events, cancerous tissues). In this work we have studied ASE in the medaka fish model system. We have developed a method for determining ASE in polyploid organisms from RNAseq data and we have implemented this method in a software tool set. As a biological model system we have used nuclear transplantation to experimentally produce artificial triploid medaka composed of three different haplomes. We measured ASE in RNA isolated from the livers of two adult, triploid medaka fish that showed a high degree of similarity. The majority of genes examined (82%) shared expression more or less evenly among the three alleles in both triploids. The rest of the genes (18%) displayed a wide range of ASE levels. Interestingly the majority of genes (78%) displayed generally consistent ASE levels in both triploid individuals. A large contingent of these genes had the same allele entirely suppressed in both triploids. When viewed in a chromosomal context, it is revealed that these genes are from large sections of 4 chromosomes and may be indicative of some broad scale suppression of gene expression.
Asunto(s)
Alelos , Regulación de la Expresión Génica , Técnicas Genéticas , Oryzias/genética , Triploidía , Desequilibrio Alélico , Animales , Secuencia de Bases , Cromosomas/genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ADNRESUMEN
Mechanisms generating diverse cell types from multipotent progenitors are crucial for normal development. Neural crest cells (NCCs) are multipotent stem cells that give rise to numerous cell-types, including pigment cells. Medaka has four types of NCC-derived pigment cells (xanthophores, leucophores, melanophores and iridophores), making medaka pigment cell development an excellent model for studying the mechanisms controlling specification of distinct cell types from a multipotent progenitor. Medaka many leucophores-3 (ml-3) mutant embryos exhibit a unique phenotype characterized by excessive formation of leucophores and absence of xanthophores. We show that ml-3 encodes sox5, which is expressed in premigratory NCCs and differentiating xanthophores. Cell transplantation studies reveal a cell-autonomous role of sox5 in the xanthophore lineage. pax7a is expressed in NCCs and required for both xanthophore and leucophore lineages; we demonstrate that Sox5 functions downstream of Pax7a. We propose a model in which multipotent NCCs first give rise to pax7a-positive partially fate-restricted intermediate progenitors for xanthophores and leucophores; some of these progenitors then express sox5, and as a result of Sox5 action develop into xanthophores. Our results provide the first demonstration that Sox5 can function as a molecular switch driving specification of a specific cell-fate (xanthophore) from a partially-restricted, but still multipotent, progenitor (the shared xanthophore-leucophore progenitor).
Asunto(s)
Cresta Neural/crecimiento & desarrollo , Oryzias/crecimiento & desarrollo , Pigmentación/genética , Factores de Transcripción SOXD/genética , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica/genética , Melanóforos/fisiología , Cresta Neural/fisiología , Oryzias/fisiología , Factor de Transcripción PAX7/genética , Fenotipo , Pigmentación/fisiología , Células Madre/fisiologíaRESUMEN
Previously, we successfully generated fully grown, cloned medaka (the Japanese rice fish, Oryzias latipes) using donor nuclei from primary culture cells of adult caudal fin tissue and nonenucleated recipient eggs that were heat shock-treated to induce diploidization of the nuclei. However, the mechanism of clone formation using this method is unknown, and the rate of adult clone formation is not high enough for studies in basic and applied sciences. To gain insight into the mechanism and increase the success rate of this method of clone formation, we tested two distinct nuclear transfer protocols. In one protocol, the timing of transfer of donor nuclei was changed, and in the other, the size of the donor cells was changed; each protocol was based on our original methodology. Ultimately, we obtained an unexpectedly high rate of adult clone formation using the protocol that differed with respect to the timing of donor nuclei transfer. Specifically, 17% of the transplants that developed to the blastula stage ultimately developed into adult clones. The success rate with this method was 13 times higher than that obtained using the original method. Analyses focusing on the reasons for this high success rate of clone formation will help to elucidate the mechanism of clone formation that occurs with this method.
Asunto(s)
Células Clonales , Técnicas de Transferencia Nuclear , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Femenino , Fluorescencia , Marcadores Genéticos , Masculino , Oryzias , Reacción en Cadena de la PolimerasaRESUMEN
Intracellular transport is spatiotemporally controlled by microtubule-dependent motor proteins, including kinesins. In order to elucidate the mechanisms controlling kinesin expression, it is important to analyze their genomic regulatory regions. In this study, we cloned the neuronal tissue-specific kinesin in medaka fish and generated transgenic fish which mimic endogenous neuronal kinesin expression in order to elucidate the mechanisms which regulate kinesin expression. Searches for medaka neuronal orthologues by RT-PCR identified a candidate gene expressed only in neuronal tissues. Using BAC clones, we determined the cDNA sequence and the gene structure of the candidate neuronal kinesin. Evolutionary analysis indicated that the candidate gene encoded medaka KIF5Aa. The endogenous medaka orthologue was found to be expressed only in the nervous system, including the brain and spinal cord, while expression of KIF5Ab was not exclusive to neuronal tissues. Transgenic (Tg) medaka that expressed EGFP under the control of the 6.9 kbp 5' and 1.9kbp 3' flanking regions of the KIF5Aa gene showed characteristic expression throughout the nervous system, including the brain, spinal cord, olfactory pit, eye and cranial nerve. Immunohistological analysis showed that EGFP expression in Tg fish co-localized with expression of HuC/D, a neuronal marker. These results demonstrate that the 6.9 kbp 5' and 1.9 kbp 3' flanking regions of medaka KIF5Aa have neuronal-specific promoter activity mimicking endogenous expression of medaka KIF5Ab. This transgenic fish strain will be useful for further functional analysis of the effects of these regulatory regions on gene expression.
Asunto(s)
Encéfalo/metabolismo , Cinesinas/metabolismo , Neuronas/metabolismo , Oryzias/metabolismo , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Clonación Molecular , Expresión Génica , Cinesinas/genética , Oryzias/genética , Regiones Promotoras GenéticasRESUMEN
Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.
Asunto(s)
Aletas de Animales/embriología , Proteínas de Peces/genética , Morfogénesis , Oryzias/embriología , Oryzias/genética , Factores de Transcripción/genética , Aletas de Animales/metabolismo , Animales , Evolución Biológica , Elementos Transponibles de ADN , Proteínas de Peces/metabolismo , Mesodermo/embriología , Mesodermo/metabolismo , Datos de Secuencia Molecular , Mutación , Oryzias/metabolismo , Reacción en Cadena de la Polimerasa , Cola (estructura animal)/embriología , Cola (estructura animal)/metabolismo , Factores de Transcripción/metabolismo , Dedos de ZincRESUMEN
We evaluated the effects of 17(-ethinylestradiol (EE(2)) on sexual differentiation in transgenic olvas-GFP/STII-YI medaka (Oryzias latipes) in terms of the proliferative activity of germ cells. This strain contains the green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene, and germ cell-specific expression of GFP can be visualized in living (transparent) individuals. From 0 days post-hatch (0 dph) onwards, juveniles were exposed to graded concentrations of EE(2) (25.2-1710 ng/L) for 35 days. The gonads of live specimens were monitored by measuring their size and calculating their GFP-fluorescence area. GFP-fluorescent area in control females was about 10 times that in control males at 10 days posthatch (dph) whereas the gonadal size of 10 dph males that had been exposed to 158 ng/L of EE(2) significantly increased up to twice the size of control males, indicating that abnormal sexual differentiation towards female might occur in these individuals. Histological examination and identification of the sex-linked marker SL1 indicated that male to female sex reversal occurred at EE(2) exposure ≥45.1 ng/L at 35 dph. These results suggest that observation of proliferative activity of germ cells in the olvas-GFP/STII-YI strain could be applied to facilitated screening fish model to detect adverse effects on sexual differentiation as early as 10 dph juveniles.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Etinilestradiol/toxicidad , Células Germinativas/efectos de los fármacos , Oryzias/fisiología , Diferenciación Sexual/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Femenino , Células Germinativas/fisiología , Gónadas/efectos de los fármacos , Gónadas/crecimiento & desarrollo , Gónadas/patología , MasculinoRESUMEN
We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.
Asunto(s)
Técnicas de Transferencia Nuclear , Pez Cebra/embriología , Animales , Antiportadores/genética , Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Diploidia , Fertilidad , Genotipo , Óvulo/citología , Óvulo/ultraestructura , Triploidía , Pez Cebra/anatomía & histología , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Ciliary defects lead to various diseases, such as primary ciliary dyskinesia (PCD) and polycystic kidney disease (PKD). We isolated a medaka mutant mii, which exhibits defects in the left-right (LR) polarity of organs, and found that mii encodes dynein axonemal intermediate chain 2a (dnai2a). Ortholog mutations were recently reported to cause PCD in humans. mii mutant embryos exhibited loss of nodal flow in Kupffer's Vesicle (KV), which is equivalent to the mammalian node, and abnormal expression of the left-specific gene. KV cilia in the mii mutant were defective in their outer dynein arms (ODAs), indicating that Dnai2a is required for ODA formation in KV cilia. While the mii mutant retained motility of the renal cilia and failed to show PKD, the loss of dnai2a and another dnai2 ortholog dnai2b led to PKD. These findings demonstrate that Dnai2 proteins control LR polarity and kidney formation through regulation of ciliary motility.
Asunto(s)
Dineínas Axonemales/metabolismo , Tipificación del Cuerpo , Riñón/embriología , Oryzias/embriología , Animales , Secuencia de Bases , Cilios/metabolismo , Cilios/patología , Embrión no Mamífero/ultraestructura , Regulación del Desarrollo de la Expresión Génica , Riñón/metabolismo , Riñón/patología , Datos de Secuencia Molecular , Mutación/genética , Especificidad de Órganos , Oryzias/genética , Fenotipo , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In addition to altering the phenotypes of gene-modified animals, transgenesis also has the potential to facilitate access to the various mechanisms underlying the development and functioning of specific phenotypes and genes, respectively. Myostatin (MSTN) is implicated in double-muscling when mutated in mammals, indicating that MSTN is a negative regulator of skeletal muscle formation. In order to elucidate the role of an MSTN equivalent in fish muscle formation, we created a transgenic medaka strain that expresses dominant-negative MSTN exclusively in skeletal muscle, d-rR-Tg(OlMA1-C315Y-MSTN-hrGFPII-FLAG). The transgenic fish exhibited increased production of skeletal muscle fibers at the adult stage (hyperplasia), although gross muscle mass was not altered. During embryogenesis, ectopic accumulation and misalignment of muscle fibers, possibly due to muscle-fiber hypertrophy, were observed in the transgenic medaka. Our findings suggest that MSTN function is required for regulating the appropriate growth of skeletal muscle in medaka. Unlike in mammals, MSTN loss-of-function failed to induce double-muscling in medaka, despite the highly conserved nature of MSTN function among taxa.
Asunto(s)
Proteínas de Peces/genética , Fibras Musculares Esqueléticas/metabolismo , Miostatina/genética , Oryzias/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Western Blotting , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hiperplasia , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Músculo Esquelético/crecimiento & desarrollo , Músculo Esquelético/patología , Mutación , Miostatina/metabolismo , Oryzias/embriología , Oryzias/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
We performed in ovo nanoinjection of 4-nonylphenol (NP) into embryos of a transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain, which has two genotypic sex markers, and examined the effects on development and sexual differentiation. The transgene consisted of a green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene. Germ cell-specific GFP expression was visualized in the gonad through the transparent body wall of the living fish. The development of each embryo was observed after nanoinjection of 2.0, 10, 50, 125, or 250 ng of NP. NP administration caused significant higher mortality at > or = 50 ng egg(-1) and inhibited embryonic development, including abnormal hatch and swim-up failure in all treatment groups except 10 ng egg(-1) group. However, it did not cause adverse effects on germ cell proliferation by 10d posthatch (dph) or sex differentiation of survivors by 100 dph. We concluded that single-dose in ovo exposure to nonylphenol affected embryonic development in the medaka but not gonadal development by 10 dph or sexual differentiation in adult fish by 100 dph. Although further investigations might be needed to elucidate the usefulness of nanoinjection of embryos of this strain, present study indicated that the nanoinjection model using olvas-GFP/STII-YI strain medaka has potential for use in evaluating the effects of chemicals on early development and sexual differentiation.
Asunto(s)
Desarrollo Embrionario/efectos de los fármacos , Nanotecnología/métodos , Oryzias/embriología , Fenoles/toxicidad , Animales , Animales Modificados Genéticamente , Proliferación Celular , Embrión no Mamífero/efectos de los fármacos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inyecciones , Oryzias/genética , Oryzias/crecimiento & desarrollo , Fenoles/administración & dosificación , Diferenciación Sexual/efectos de los fármacos , Análisis de SupervivenciaRESUMEN
The growth-promoting effects of fish body fluids, such as serum and embryonic extract, on fish cell cultures have been widely demonstrated. The bubble-eye variety of aquarium goldfish is characterized as having a large sac filled with fluid (sac fluid) under each eye. These sacs are believed to contain lymph, which is similar in composition to serum or blood plasma. In order to test whether the sac fluid can be used as an additive for fetal bovine serum (FBS) in growth factor supplements, we compared cell growth in media containing FBS together with different concentrations of sac fluid. A dose-dependent growth-promotion effect was observed in early passage caudal fin cells from both medaka and zebrafish. Cell-growth promotion was also confirmed in early passage medaka blastula cells and in a zebrafish embryonic cell line (ZF4). Replacement of the fluid in the eye sacs of bubble-eyes occurs within a couple of months after the sac fluid has been harvested, and the cell-growth promoting activity of the new fluid is similar to that of the fluid that was tapped initially. These findings suggest that sac fluid can be used as a growth-promoting supplement for fish cell culture. Importantly, the ability of the goldfish to replace the fluid combined with the fact that equipotent fluid can be repeatedly harvested from the eye sacs means that a sustainable source of the fluid can be obtained without needing to sacrifice the fish.
Asunto(s)
Líquidos Corporales/fisiología , Proliferación Celular , Carpa Dorada/fisiología , Animales , Células Cultivadas , Extremidades/fisiología , Oryzias , Pez CebraRESUMEN
Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.
Asunto(s)
Mutación , Enfermedades Renales Poliquísticas/genética , Factores de Transcripción/genética , Dedos de Zinc , Animales , Secuencia de Bases , Proliferación Celular , Cartilla de ADN , Colorantes Fluorescentes , Técnicas de Silenciamiento del Gen , Hibridación in Situ , Oryzias , Páncreas/metabolismo , Enfermedades Renales Poliquísticas/patología , Fracciones Subcelulares/metabolismoRESUMEN
Glis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3(-/-) mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin-producing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Transactivadores/deficiencia , Animales , Animales Recién Nacidos , Secuencia de Bases , Glucemia/metabolismo , Carboxipeptidasas A/metabolismo , Cartilla de ADN/genética , Proteínas de Unión al ADN , Diabetes Mellitus Tipo 1/patología , Modelos Animales de Enfermedad , Humanos , Recién Nacido , Insulina/genética , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Ratones , Ratones Noqueados , Mutación , Páncreas/metabolismo , Páncreas/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Transactivadores/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genéticaRESUMEN
Although renal regeneration is limited to repair of the proximal tubule in mammals, some bony fish are capable of renal regeneration through nephron neogenesis in the event of renal injury. We previously reported that nephron development in the medaka mesonephros is characterized by four histologically distinct stages, generally referred to as condensed mesenchyme, nephrogenic body, relatively small nephron, and the mature nephron. Developing nephrons are positive for wt1 expression during the first three of these stages. In the present study, we examined the regenerative response to renal injury, artificially induced by the administration of sublethal amounts of gentamicin in adult medaka. Similar to previous reports in other animals, the renal tubular epithelium and the glomerulus of the medaka kidney exhibited severe damage after exposure to this agent. However, kidneys showed substantial recovery after gentamicin administration, and a significant number of developing nephrons appeared 14 days after gentamicin administration (P < 0.01). Similarly, the expression of wt1 in developing nephrons also indicated the early stages of nephrogenesis. These findings show that medaka has the ability to regenerate kidney through nephron neogenesis during adulthood and that wt1 is a suitable marker for detecting nephrogenesis.
Asunto(s)
Mesonefro/fisiología , Nefronas/fisiología , Regeneración/fisiología , Células Madre Adultas/fisiología , Animales , Biomarcadores , División Celular , Gentamicinas/toxicidad , Masculino , Mesonefro/química , Mesonefro/efectos de los fármacos , Nefronas/química , Oryzias , Especificidad de la Especie , Proteínas WT1/análisisRESUMEN
DNA-based transposable elements are present in the genomes of various organisms, and generally occur in autonomous and nonautonomous forms, with a good correspondence to complete and defective copies, respectively. In vertebrates, however, the vast majority of DNA-based elements occur only in the nonautonomous form. Until now, the only clear exception known has been the Tol2 element of the medaka fish, which still causes mutations in genes of the host species. Here, we report another exception: the Tol1 element of the same species. This element was thought likely to be a "dead" element like the vast majority of vertebrate elements, but recent identification of an autonomous Tol1 copy in a laboratory medaka strain gave rise to the possibility that the element is still "alive" in medaka natural populations. We examined variation in the structure of Tol1 copies through genomic Southern blot analysis, and revealed that 10 of the 32 fish samples examined contained full-length Tol1 copies in their genomes. The frequency at which these copies occur among Tol1 copies is at most 0.5%, yet some of them still have the ability to produce a functional transposase. The medaka fish thus harbors two active DNA-based elements in its genome, and is in this respect unique among vertebrates.
Asunto(s)
Elementos Transponibles de ADN/fisiología , Genética de Población , Oryzias/genética , Oryzias/metabolismo , Transposasas/metabolismo , Animales , Evolución Biológica , Southern BlottingRESUMEN
We previously reported the generation of fertile diploid adult fish with a donor marker by transfer of adult somatic cell nuclei to recipient diploidized eggs without enucleation in medaka (Oryzias latipes). Although transplants appeared similar to clones of donor fish, the possibility existed that they were chimeras of cells originating from both the donor and recipient nuclei. To clarify the nuclear origin of transplants, the green fluorescent protein gene (GFP) was used as the recipient marker and the DMY/dmrt1bY gene, which directs male differentiation in medaka, was used as the donor marker. The marker genes were examined in the transplants by fluorescence microscopy, polymerase chain reaction assays, and transmission to the progeny. Of the seven adult fish obtained from 974 nuclear transfer procedures, six were analyzed in detail. Three of these exhibited the donor phenotype but did not have the recipient marker, suggesting that they were donor clones. The other three showed GFP expression, with one exhibiting an apparent chimerism in both donor and recipient genetic markers and the other two considered to be parthenogenic. Elucidation of a mechanism capable of eliminating recipient nuclei from nuclear transplants is considered to be key to the establishment of cloning techniques in fish.
Asunto(s)
Animales Modificados Genéticamente/fisiología , Clonación de Organismos/métodos , Técnicas de Transferencia Nuclear , Oryzias/fisiología , Óvulo/fisiología , Quimera por Trasplante/fisiología , Animales , Animales Modificados Genéticamente/genética , Diploidia , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Masculino , Oryzias/genética , Quimera por Trasplante/genéticaRESUMEN
We provide an overview of glomerulogenesis in medaka from the embryo to the adult by means of in situ hybridization with the wt1 gene as a marker as well as histology and three-dimensional images. The pronephric glomus starts to develop in the intermediate mesoderm during early somitogenesis, is completed before hatching, and persists throughout the lifetime of the fish. Within 5 days after hatching, mesonephric glomerulus formation begins in the caudomedial end of the pronephric sinus and duct area. The number of glomeruli reaches approximately 200-300 in each kidney within 2 months after hatching. wt1 expression during nephron maturation served as a marker for the formation of the mesenchymal condensate and the nephrogenic body. Existence of mesenchymal condensates and persistence of wt1 expression in the adult kidney suggest that the mesonephros retains precursor cells that may be capable of contributing to neoglomerulogenesis during adulthood.
Asunto(s)
Glomérulos Renales/embriología , Oryzias/embriología , Animales , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Riñón/embriología , Riñón/metabolismo , Glomérulos Renales/metabolismo , Nefronas/embriología , Nefronas/metabolismo , Oryzias/metabolismo , Proteínas WT1/genéticaRESUMEN
Until recently, the nuclear transfer of adult somatic cell nuclei in fish has been unsuccessful. This is primarily because of chromosomal aberrations in nuclear transplants, which are thought to arise due to asynchrony between the cell cycles of the recipient egg and donor nucleus. We recently succeeded in circumventing this difficulty by using a new nuclear transfer method in medaka fish (Oryzias latipes). Instead of enucleated eggs, the method uses non-enucleated and diploidized eggs, obtained by retention of the second polar body release, as recipients in the nuclear transfer of primary culture cells from the caudal fin of an adult green fluorescent protein gene (GFP)-transgenic strain. We found that 2.7% of the reconstructed embryos grew into diploid and fertile adults exhibiting donor expression characteristics and transmission of the GFP marker gene to progeny. The mechanism underlying the generation of nuclear transplants using this method is unknown at present; however, analyses of donor and recipient nuclei behavior and the cytoskeletal mechanisms involved in the early developmental stages, as well as the special ability of diploidized eggs to facilitate reprogramming of the donor nuclei will result in elucidation of the mechanism.
Asunto(s)
Núcleo Celular/metabolismo , Oryzias/genética , Animales , Trasplante de Células/métodos , Aberraciones Cromosómicas , Citoesqueleto/metabolismo , Diploidia , Femenino , Marcadores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Haploidia , Mosaicismo , Ploidias , Factores de Tiempo , Distribución TisularRESUMEN
Müllerian inhibiting substance (MIS) is a glycoprotein belonging to the TGF-beta superfamily. In mammals, MIS is responsible for the regression of Müllerian ducts in the male fetus. However, the role of MIS in gonadal sex differentiation of teleost fish, which have no Müllerian ducts, has yet to be clarified. In the present study, we examined the expression pattern of mis and mis type 2 receptor (misr2) mRNAs and the function of MIS signaling in early gonadal differentiation in medaka (teleost, Oryzias latipes). In situ hybridization showed that both mis and misr2 mRNAs were expressed in the somatic cells surrounding the germ cells of both sexes during early sex differentiation. Loss-of-function of either MIS or MIS type II receptor (MISRII) in medaka resulted in suppression of germ cell proliferation during sex differentiation. These results were supported by cell proliferation assay using 5-bromo-2'-deoxyuridine labeling analysis. Treatment of tissue fragments containing germ cells with recombinant eel MIS significantly induced germ cell proliferation in both sexes compared with the untreated control. On the other hand, culture of tissue fragments from the MIS- or MISRII-defective embryos inhibited proliferation of germ cells in both sexes. Moreover, treatment with recombinant eel MIS in the MIS-defective embryos dose-dependently increased germ cell number in both sexes, whereas in the MISRII-defective embryos, it did not permit proliferation of germ cells. These results suggest that in medaka, MIS indirectly stimulates germ cell proliferation through MISRII, expressed in the somatic cells immediately after they reach the gonadal primordium.