Medaka Mutant Research Articles

Modular development of the teleost trunk along the dorsoventral axis and zic1/zic4 as selector genes in the dorsal module

Teleost fish exhibit remarkable diversity in morphology, such as fins and coloration, particularly on the dorsal side. These structures are evolutionary adaptive because their back is highly visible to other individuals. However, owing to the late phenotypic appearance (from larva to adult) and lack of appropriate mutants, the genetic mechanisms that regulate these dorsoventrally asymmetric external patterns are largely unknown. To address this, we have analyzed the spontaneous medaka mutant Double anal fin (Da), which exhibits a mirror-image duplication of the ventral half across the lateral midline from larva to adult. Da is an enhancer mutant for zic1 and zic4 in which their expression in dorsal somites is lost. We show that the dorsoventral polarity in Da somites is lost and then demonstrate using transplantation techniques that somites and their derived tissues globally determine the multiple dorsal-specific characteristics of the body (fin morphology and pigmentation) from embryo to adult. Intriguingly, the zic1/zic4 expression in the wild type persists throughout life in the dorsal parts of somite derivatives, i.e. the myotome, dermis and vertebrae, forming a broad dorsal domain in the trunk. Comparative analysis further implies a central role for zic1/zic4 in morphological diversification of the teleost body. Taken together, we propose that the teleost trunk consists of dorsal/ventral developmental modules and that zic1/zic4 in somites function as selector genes in the dorsal module to regulate multiple dorsal morphologies.

A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.

Reassessment of the function of somatolactin alpha in lipid metabolism using medaka mutant and transgenic strains

BackgroundSomatolactin alpha (SLa) is a fish-specific peptide hormone secreted from the pituitary. In medaka, SLa functions to darken the skin color and lack of SLa makes it pale. Transcription of SLa is enhanced or suppressed when fish are kept in dark or bright conditions, respectively, indicating SLa’s important role in background acclimation of the skin color. Bizarrely, however, the lack of SLa seems to cause the additional defect of increased triglycerides in organs, which could not be rescued (decreased) by its overexpression.ResultsTo assess this enigmatic result, we investigated genetic (the SLa, Slc45a2, r, and Y genes) and nongenetic (age, fasting, water temperature, and background color) effects on hepatic triglycerides. These experiments found that percent hepatic triglycerides quickly change in response to external/internal environments. Effects of SLa seemed to be much less obvious, although it may increase the proportion of hepatic triglycerides at least during certain breeding conditions or under certain genetic backgrounds.ConclusionsThe present results do not exclude the possibility that SLa takes part in lipid metabolism or other physiological processes. However, we suggest that skin-color regulation is the only definite role of SLa so far demonstrated in this species.

Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.

The Medaka zic1/zic4 Mutant Provides Molecular Insights into Teleost Caudal Fin Evolution

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.

SEC23-SEC31 the Interface Plays Critical Role for Export of Procollagen from the Endoplasmic Reticulum

COPII proteins are essential for exporting most cargo molecules from the endoplasmic reticulum. The membrane-facing surface of the COPII proteins (especially SEC23-SEC24) interacts directly or indirectly with the cargo molecules destined for exit. As we characterized the SEC23A mutations at the SEC31 binding site identified from patients with cranio-lenticulo-sutural dysplasia, we discovered that the SEC23-SEC31 interface can also influence cargo selection. Remarkably, M702V SEC23A does not compromise COPII assembly, vesicle size, and packaging of cargo molecules into COPII vesicles that we have tested but induces accumulation of procollagen in the endoplasmic reticulum when expressed in normal fibroblasts. We observed that M702V SEC23A activates SAR1B GTPase more than wild-type SEC23A when SEC13-SEC31 is present, indicating that M702V SEC23A causes premature dissociation of COPII from the membrane. Our results indicate that a longer stay of COPII proteins on the membrane is required to cargo procollagen than other molecules and suggest that the SEC23-SEC31 interface plays a critical role in capturing various cargo molecules.

Analysis of Medaka sox9 Orthologue Reveals a Conserved Role in Germ Cell Maintenance

The sex determining gene is divergent among different animal species. However, sox9 is up-regulated in the male gonads in a number of species in which it is the essential regulator of testis determination. It is therefore often discussed that the sex determining gene-sox9 axis functions in several vertebrates. In our current study, we show that sox9b in the medaka (Oryzias latipes) is one of the orthologues of mammalian Sox9 at syntenic and expression levels. Medaka sox9b affects the organization of extracellular matrices, which represents a conserved role of sox9, but does not directly regulate testis determination. We made this determination via gene expression and phenotype analyses of medaka with different copy numbers of sox9b. Sox9b is involved in promoting cellular associations and is indispensible for the proper proliferation and survival of germ cells in both female and male medaka gonads. Medaka mutants that lack sox9b function exhibit a seemingly paradoxical phenotype of sex reversal to male. This is explained by a reduction in the germ cell number associated with aberrant extracellular matrices. Together with its identified roles in other vertebrate gonads, a testis-determining role for Sox9 in mammals is likely to have been neofunctionalized and appended to its conserved role in germ cell maintenance.

Ciliary motility: The components and cytoplasmic preassembly mechanisms of the axonemal dyneins

Motile cilia and flagella are organelles, which function in cell motility and in the transport of fluids over the surface of cells. Motility defects often result in a rare human disease, primary ciliary dyskinesia (PCD). Cell motility depends on axonemal dynein, a molecular motor that drives the beating of cilia and flagella. The dyneins are composed of multiple subunits, which are thought to be preassembled in the cytoplasm before they are transported into cilia and flagella. Axonemal dyneins have been extensively studied in Chlamydomonas. In addition, analyses of human PCDs over the past decade, together with studies in other model animals, have identified the conserved components required for dynein assembly. Recently also, the first cytoplasmic component of dynein assembly, kintoun (ktu), was elucidated through the analysis of a medaka mutant in combination with human genetics and cell biology and biochemical studies of Chlamydomonas. The components of dynein and the proteins involved in its cytoplasmic assembly process are discussed.

Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro

Filamin C is an actin-crosslinking protein that is specifically expressed in cardiac and skeletal muscles. Although mutations in the filamin C gene cause human myopathy with cardiac involvement, the function of filamin C in vivo is not yet fully understood. Here we report a medaka mutant, zacro ( zac), that displayed an enlarged heart, caused by rupture of the myocardiac wall, and progressive skeletal muscle degeneration in late embryonic stages. We identified zac to be a homozygous nonsense mutation in the filamin C ( flnc) gene. The medaka filamin C protein was found to be localized at myotendinous junctions, sarcolemma, and Z-disks in skeletal muscle, and at intercalated disks in the heart. zac embryos showed prominent myofibrillar degeneration at myotendinous junctions, detachment of myofibrils from sarcolemma and intercalated disks, and focal Z-disk destruction. Importantly, the expression of γ-actin, which we observed to have a strong subcellular localization at myotendinous junctions, was specifically reduced in zac mutant myotomes. Inhibition of muscle contraction by anesthesia alleviated muscle degeneration in the zac mutant. These results suggest that filamin C plays an indispensable role in the maintenance of the structural integrity of cardiac and skeletal muscles for support against mechanical stress.

Scale and tooth phenotypes in medaka with a mutated ectodysplasin-A receptor: implications for the evolutionary origin of oral and pharyngeal teeth

Ectodermal contribution to the induction of pharyngeal teeth that form in the endodermal territory of the oropharyngeal cavity in some teleost fishes has been a matter of considerable debate. To determine the role of ectodermal cell signaling in scale and tooth formation and thereby to gain insights in evolutionary origin of teeth, we analyzed scales and teeth in rs-3 medaka mutants characterized by reduced scale numbers due to aberrant splicing of the ectodysplasin-A receptor (edar). Current data show that, in addition to a loss of scales (83% reduction), a drastic loss of teeth occurred in both oral (43.5% reduction) and pharyngeal (73.5% reduction) dentitions in rs-3. The remaining scales of rs-3 were irregular in shape and nearly 3 times larger in size relative to those of the wild-type. In contrast, there was no abnormality in size and shape in the remaining teeth of rs-3. In wild-type medaka embryos, there was a direct contact between the surface ectoderm and rostral endoderm in pharyngeal regions before the onset of pharyngeal tooth formation. However, there was no sign of ectodermal cell migration in the pharyngeal endoderm and hence no direct evidence of any ectodermal contribution to pharyngeal odontogenesis. These data suggest differential roles for Eda-Edar signaling in the induction and growth of scales and teeth and support the intrinsic odontogenic competence of the rostral endoderm in medaka.

2N1436 MR顕微鏡によるp53遺伝子変異型メダカの3次元画像解析(バイオイメージング2,第49回日本生物物理学会年会)

2N1436 MR顕微鏡によるp53遺伝子変異型メダカの3次元画像解析(バイオイメージング2,第49回日本生物物理学会年会)

Dynein axonemal intermediate chain 2 is required for formation of the left–right body axis and kidney in medaka

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.

M-marker 2009, a Marker set for Mapping Medaka Mutants Using PCR Length Polymorphisms with an Automated Microchip Gel Electrophoresis System

We have developed a highly effective marker set for assigning medaka mutants to specific linkage groups (LG) in bulk segregation analysis by PCR product length polymorphisms (PLPs). The marker set permits analysis using an automated microchip electrophoresis system as well as conventional agarose gel electrophoresis.

Bef medaka mutant reveals the essential role of c-myb in both primitive and definitive hematopoiesis

Vertebrate hematopoiesis is characterized by two evolutionally conserved phases of development, i.e., primitive hematopoiesis, which is a transient phenomenon in the early embryo, and definitive hematopoiesis, which takes place in the later stages. Beni fuji (bef) was originally isolated as a medaka mutant that has an apparently reduced number of erythrocytes in its peripheral blood. Positional cloning revealed that the bef mutant has a nonsense mutation in the c-myb gene. Previous studies have shown that c-myb is essential for definitive hematopoiesis, and c-myb is now widely used as a marker gene for the onset of definitive hematopoiesis. To analyze the phenotypes of the bef mutant, we performed whole-mount in situ hybridization with gene markers of hematopoietic cells. The bef embryos showed decreased expression of α-globin and l-plastin, and a complete loss of mpo1 and rag1 expression, suggesting that the bef embryos had defects not only in erythrocytes but also in other myeloid cells, which indicates that their definitive hematopoiesis was aberrant. Interestingly, we observed a diminution in the number of primitive erythrocytes and a delay in the emergence of primitive macrophages in the bef embryos. These results suggest that c-myb also functions in the primitive hematopoiesis, potentially demonstrating a link between primitive and definitive hematopoiesis.

Production of Wnt4b by floor plate cells is essential for the segmental patterning of the vertebral column in medaka

The floor plate is a key organizer that controls the specification of neurons in the central nervous system. Here, we show a new role of the floor plate: segmental pattern formation of the vertebral column. Analysis of a spontaneous medaka mutant, fused centrum (fsc), which exhibits fused centra and the absence of the intervertebral ligaments, revealed that fsc encodes wnt4b, which was expressed exclusively in the floor plate. In fsc mutants, we found that wnt4b expression was completely lost in the floor plate and that abnormal conversion of the intervertebral ligament cells into osteoblasts appeared to cause a defect of the intervertebral ligaments. The establishment of the transgenic rescue lines and mosaic analyses allowed the conclusion to be drawn that production of wnt4b by floor plate cells is essential for the segmental patterning of the vertebral column. Our findings provide a novel perspective on the mechanism of vertebrate development.

Sec24d encoding a component of COPII is essential for vertebra formation, revealed by the analysis of the medaka mutant, vbi

We characterized a medaka mutant, vertebra imperfecta (vbi), that displays skeletal defects such as craniofacial malformation and delay of vertebra formation. Positional cloning analysis revealed a nonsense mutation in sec24d encoding a component of the COPII coat that plays a role in anterograde protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Immunofluorescence analysis revealed the accumulation of type II collagen in the cytoplasm of craniofacial chondrocytes, notochord cells, and the cells on the myoseptal boundary in vbi mutants. Electron microscopy analysis revealed dilation of the ER and defective secretion of ECM components from cells in both the craniofacial cartilage and notochord in vbi. The higher vertebrates have at least 4 sec24 paralogs; however, the function of each paralog in development remains unknown. sec24d is highly expressed in the tissues that are rich in extracellular matrix and is essential for the secretion of ECM component molecules leading to the formation of craniofacial cartilage and vertebra.

FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium

Introduction During the morphogenesis of the neural tube, neural progenitors continuously divide, and post mitotic neurons then migrate to form complex neural networks. It is widely conserved among vertebrates that this mitotic process takes place near the lumen, the apical side of the neural tube (Gotz and Huttner, 2005). When viewed at the cellular level, this apically localized mitosis is the region of output for the cell-cycle-dependent oscillation of the nuclei within the neuroepithelium. Mitotic (M phase) nuclei are located in close proximity to the apical surfaces, whereas nuclei undergoing DNA synthesis (S phase) are displaced more basally. This characteristic movement of nuclei is known as interkinetic nuclear migration (INM) (Fujita, 1964; Sauer, 1935) and is thought to be important for maintaining a neural progenitor pool during neurogenesis (Baye and Link, 2007; Del Bene et al., 2008; Murciano et al., 2002; Xie et al., 2007). Furthermore, planar cell divisions near the apical surface are thought to be important to maintain the progenitor pool (Gotz and Huttner, 2005). Indeed, recent works suggest that most divisions during both proliferative and neurogenic stages are planar (that is, parallel to the apical surface), whereas vertical or oblique divisions are rarely seen, and daughter cells that inherit both the apical and basal components are suggested to remain in the progenitor pool (Konno et al., 2008). Although INM and planar cell divisions have been regarded as hallmarks of vertebrate neural progenitors, their cellular and molecular mechanisms are just beginning to be elucidated. Previous studies have suggested the involvement of several intracellular components such as the actin and microtubule cytoskeletons, molecules involved in adherens junctions, polarity complex and the centrosome (Cappello et al., 2006; Imai et al., 2006; Lien et al., 2006; Murciano et al., 2002; Siller and Doe, 2009; Xie et al., 2007). However, it remains unclear whether extracellular molecules, such as components of the extracellular matrix (ECM), have any role in INM and mitotic orientation of neural progenitors, and if so, how they interact with the intracellular machinery that drives this process. Indeed, the ECM has long been shown to participate in neurogenesis (see below), but there are no reports of vertebrate ECM mutants with defective INM and mitotic orientation. In this context, the medaka mutant tacobo (tab) used in the present study provides a unique framework. The medaka fish, Oryzias latipes, is an emerging vertebrate model and now has a high quality draft genome and a FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium

Proliferation zones in adult medaka ( Oryzias latipes) brain

Cell proliferation in the adult mammalian brain is maintained at a low rate, but cell proliferation in the adult fish brain is prominent. To compare the distribution of proliferating cells among fish species, mutants, and under different growing environments, we mapped the zones of cell proliferation in the adult medaka ( Oryzias latipes) brain and identified 17 proliferation zones in both male and female brains. These zones were distributed in the telencephalon (4 zones), preoptic area (2 zones), pineal body (1 zone), hypophysis (1 zone), habenular nucleus (1 zone), optic tectum (2 zones), third ventricular zone (1 zone), ventromedial nucleus (1 zone), hypothalamus (1 zone), and cerebellum (3 zones). Of the 17 zones, 16 corresponded to brain regions where cells proliferate in the zebrafish brain, suggesting that the persistence of the generation of new cells, at least in these zones, might be conserved among some fish species. We then compared the distribution of proliferation zones using two body-color mutant medaka, the T5 and Quintet, the latter of which is an albino mutant that completely lacks pigmentation. There was no apparent difference in the distribution pattern among these mutant strains. Finally, we compared these proliferation zones in the brains of isolated- and group-reared fish and detected no significant difference between the two groups. These findings demonstrate that there is persistent cell proliferation in at least these 16 zones of the adult medaka brain, irrespective of sex, body-color, and growth environment, suggesting that proliferation capacity in the 16 zones is maintained robustly in the adult medaka brain.

FAK-mediated extracellular signals are essential for interkinetic nuclear migration and planar divisions in the neuroepithelium

During the development of the vertebrate nervous system, mitosis of neural progenitor cells takes place near the lumen, the apical side of the neural tube, through a characteristic movement of nuclei known as interkinetic nuclear migration (INM). Furthermore, during the proliferative period, neural progenitor cells exhibit planar cell divisions to produce equivalent daughter cells. Here, we examine the potential role of extracellular signals in INM and planar divisions using the medaka mutant tacobo (tab). This tab mutant shows pleiotropic phenotypes, including neurogenesis, and positional cloning identified tab as laminin gamma1 (lamc1), providing a unique framework to study the role of extracellular signals in neurogenesis. In tab mutant neural tubes, a number of nuclei exhibit abnormal patterns of migration leading to basally mislocalized mitosis. Furthermore, the orientation of cell division near the apical surface is randomized. Probably because of these defects, neurogenesis is accelerated in the tab neural tube. Detailed analyses demonstrate that extracellular signals mediated by the FAK pathway regulate INM and planar divisions in the neuroepithelium, possibly through interaction with the intracellular dynein-motor system.

Cell Lines Derived from a Medaka Radiation-Sensitive Mutant have Defects in DNA Double-Strand Break Responses

It was reported that the radiation-sensitive Medaka mutant "ric1" has a defect in the repair of DNA double-strand breaks (DSBs) induced by gamma-rays during early embryogenesis. To study the cellular response of a ric1 mutant to ionizing radiation (IR), we established the mutant embryonic cell lines RIC1-e9, RIC1-e42, RIC1-e43. Following exposure to gamma-irradiation, the DSBs in wild-type cells were repaired within 1 h, while those in RIC1 cells were not rejoined even after 2 h. Cell death was induced in the wild-type cells with cell fragmentation, but only a small proportion of the RIC1 cells underwent cell death, and without cell fragmentation. Although both wild-type and RIC1 cells showed mitotic inhibition immediately after gamma-irradiation, cell division was much slower to resume in the wild-type cells (20 h versus 12 h). In both wild-type and RIC1 cells, Ser139 phosphorylated H2AX (gammaH2AX) foci were formed after gamma-irradiation, however, the gammaH2AX foci disappeared more quickly in the RIC1 cell lines. These results suggest that the instability of gammaH2AX foci in RIC1 cells cause an aberration of the DNA damage response. As RIC1 cultured cells showed similar defective DNA repair as ric1 embryos and RIC1 cells revealed defective cell death and cell cycle checkpoint, they are useful for investigating DNA damage responses in vitro.