The synthesis of collagen during the development of anal-fin processes in ethisterone-treated females of Oryzias latipes

The synthesis of collagen during the development of anal-fin processes induced by ethisterone was studied by the autoradiographies of tritiated proline (Pro-T) and hydroxyproline (HyPro-T). The rapid incorporation of Pro-T was initiated in the screloblast masses between 48 and 72 hr of ethisterone treatment. The increased incorporation of HyPro-T was also detected in the screloblast masses after 48 hr. Furthermore, the grains of both radioactive amino acids were distributed distinctly in the horny substance secreted by the screloblast masses at and after 72 hr. It is suggested that the rapid increase in incorporation of labelled amino acids is mainly due to the collagen synthesis, and that the synthesis of collagen is initiated in the screloblast masses between 48 and 72 hr of the treatment.     

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka ( Oryzias latipes ) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated ¹⁴C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.     

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.     

Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes

Specific antibodies against the major chorionic glycoproteins (ZI1 -2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozo, play an important role in sperm guidance into the micropyle.     

Seasonal change in the locomotor activity rhythm of the medaka,Oryzias latipes

A group of the medaka,Oryzias latipes (Cyprinodontidae, orange-red variety, 25 males and 25 females), was kept in an aquarium, which was placed outdoors under natural conditions from December 1984 to January 1986. Locomotor activity at three layers (upper, middle, and lower layers) was recorded with a phototransistor system in each season. In summer, the fish showed typical diurnal activity at all three layers and the activity was greater than in other seasons. However, in autumn and winter, the fish became less active and showed relatively high activity at night at the upper or middle layer and diurnal activity at the lower layer. Nocturnal activity seemed to appear when the water temperature was decreased and the photoperiod was shortened. A free-running activity rhythm was also recorded under continuous darkness (DD) in each season; however, the fish showed clear free-running activity rhythms under DD only in summer.     

Ultrastructural changes in micropylar cells and formation of the micropyle during oogenesis in the medaka Oryzias latipes

Ultrastructural changes in differentiating micropylar cells and in the micropyle occur during oogenesis in the medaka, Oryzias latipes. A micropylar cell is not detectable in previtellogenic ovarian follicles. In the early vitellogenic phase, the micropylar cell becomes differentiated from neighboring granulosa cells by its electrondense cytoplasm. The micropylar cell in this phase characteristically displays an increase in rough endoplasmic reticulum, Golgi complexes, and tonofilaments around the nucleus. By the late vitellogenic phase, the enlarged micropylar cell extends a broad cytoplasmic process to the oocyte surface. A conspicuous feature of the process is a large bundle of microtubules oriented perpendicular to the oocyte surface. The inner surface of the micropylar canal has a spiral structure and is covered with the outermost layer of the chorion. In the postvitellogenic phase, the main cell body possesses many tonofilaments, mitochondria, Golgi complexes, and rough endoplasmic reticulum, and the winding cytoplasmic process contains a twisted large bundle of microtubules with a bundle of tonofilaments as its core. The spiral structure of the micropylar vestibule and the micropylar canal reflects the twisting associated with elongation of fibrous bundles of the micropylar cell anchored on the chorion at the animal pole of the oocyte.     

Mutations affecting the formation of posterior lateral line system in Medaka, Oryzias latipes

We performed a systematic screen for mutations affecting the trajectory of axons visualized by immunohistochemical staining of Medaka embryos with anti-acetylated tubulin antibody. Among the mutations identified, yanagi (yan) and kazura (kaz) mutations caused specific defects in projection of the posterior lateral line (PLL) nerve. In yan and kaz mutant embryos, the PLL nerve main bundle was misrouted ventrally and dorsally or anteriorly. Medaka semaphorin3A, sdf1, and cxcr4 cDNA fragments were cloned to allow analysis of these mutants. There were no changes in semaphorin3A or sdf1 expression in mutant embryos, suggesting that the tissues expressing semaphorin3A or sdf1 that are involved in PLL nerve guidance are present in these mutant embryos. Double staining revealed that the mislocated PLL primordium and growth cone of the ectopically projected PLL nerve were always colocalized in both yan and kaz mutant embryos, suggesting that migration of PLL primordia and PLL nerve growth cones are not uncoupled in these mutants. Although homozygous yan larvae showed incomplete migration of the PLL primordium along the anteroposterior axis, ventral proneuromast migration was complete, suggesting that ventral migration of the proneuromast does not require the signaling affected in yan mutants. In addition to the PLL system, the distribution of primordial germ cells (PGCs) was also affected in both yan and kaz mutant embryos, indicating that yan and kaz genes are required for the migration of both PLL primordia and PGCs. Genetic linkage analysis indicated that kaz is linked to cxcr4, but yan is not linked to sdf1 or cxcr4. These mutations will provide genetic clues to investigate the molecular mechanism underlying formation of the PLL system.     

Mutations affecting somite formation in the Medaka (Oryzias latipes)

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.     

Yolk Phosphoprotein Metabolism during Early Development of the Fish, Oryzias latipes

Changes in yolk phosphoproteins of Oryzias latipes embryos during early development were examined by electrophoresis. Four phosphoproteins were identified in the yolk of blastulae by polyacrylamide gel electrophoresis. Two of them were high molecular weight phosphoproteins containing 0.7% (w/w) phosphorus and with similar amino acid compositions to that of vitellogenin. The other two were low molecular weight phosphoproteins, characterized by high contents of phosphorus [12.2% (w/w)] and serine (44.8 mole%) and low contents of aromatic amino acid residues. From these characteristics, together with their behaviors on DEAE-cellulose chromatography and electrophoresis and low stainability with dyes, the latter two were concluded to be phosvitins. These phosvitins were isolated and partially characterized.
The yolk phosphoproteins, especially the phosvitins, were degraded, their amounts in embryos decreased throughout early development. Studies on the mechanism of endogenous phosphoprotein degradation strongly suggested the participation of some protease(s) that was precipitated on centrifugation of the egg homogenate at 14,000 × g for 10 min.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

Development of the endoderm and gut in medaka, Oryzias latipes

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants.     

Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka, Oryzias latipes

Medaka is an attractive model to study epimorphic regeneration. The fins have remarkable regenerative capacity and are replaced about 14 days after amputation. The formation of blastema, a mass of undifferentiated cells, is essential for regeneration; however, the molecular mechanisms are incompletely defined. To identify the genes required for fin regeneration, especially for blastema formation, we constructed cDNA libraries from fin regenerates at 3 days postamputation and 10 days postamputation. A total of 16,866 expression sequence tags (ESTs) were sequenced and subjected to BLASTX analysis. The result revealed that about 60% of them showed strong matches to previously identified proteins, and major signaling molecules related to development, including FGF, BMP, Wnt, Notch/Delta, and Ephrin/Eph signaling pathways were isolated. To identify novel genes that showed specific expression during fin regeneration, cDNA microarray was generated based on 2900 independent ESTs from each library which had no sequence similarity to known proteins. We obtained 6 candidate genes associated with blastema formation by gene expression pattern screening in competitive hybridization analyses and in situ hybridization. Olrfe16d23 and olrfe14k04 were expressed only in early regenerating stages when blastema formation was induced. The expression of olrf5n23, which encodes a novel signal peptide, was detected in wound epidermis throughout regeneration. Olrfe23l22, olrfe20n22, and olrfe24i02 were expressed notably in the blastema region. Our study has thus identified the gene expression profiles and some novel candidate genes to facilitate elucidation of the molecular mechanisms of fin regeneration.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.     

Changes in Adrenergic Innervation to Chromatophores During Prolonged Background Adaptation in the Medaka,Oryzias latipes

The pattern of adrenergic innervation to scale chromatophores of the wild-type medaka, Oryzias latipes, was examined by autoradiography with ³H-norepinephrine and found for the first time to be changed reversibly during prolonged background adaptation. In scales of the medaka, which was adapted to a black background for 10-15 days, a great number of melanophores and dense networks of varicose fibers were observed: many fibers built up a radial plexus around each melanophore. However, the dense distribution of varicose fibers disappeared with a decrease in the number of melanophores during long-term adaptation to a white background. As to the changes in the innervation pattern to amelanotic melanophores of the medaka, orange-red variety, a similar result was obtained. Although the increase in the number of leucophores was observed in the medaka adapted to a white background, no exact plexuses of labeled fibers were confirmed around leucophores. From these results, it is concluded that the density of chromatic nerve fibers changes in parallel with the variation of the number of melanophores during prolonged background adaptation.