Phenotypic analyses of a medaka mutant reveal the importance of bilaterally synchronized expression of isthmic fgf8 for bilaterally symmetric formation of the optic tectum

Developing neural tubes are bilaterally symmetric in all vertebrate embryos, irrespective of the presence of gene networks that generate left-right asymmetry. To explore the mechanisms that underlie the bilaterally symmetric formation of the neural tube, we examined a medaka (Oryzias latipes) dominant mutant, Oot, the neural tube of which transiently lacks normal symmetry in the optic tectum. We found that spatial changes in isthmic fgf8 expression do not occur on one side of the mutant, resulting in a transient desynchronized expression that correlates with tectal asymmetry. The application of exogenous FGF8 on one side of a wild-type embryo mimics the Oot phenotype, indicating that the bilaterally equivalent expression of isthmic fgf8 is crucial for the bilaterally symmetric development of the tectum. These results suggest that tectal symmetry is not a "default" state, but rather is maintained actively by a bilaterally coupled and synchronized regulation of isthmic fgf8 expression.     

Medaka genomics: a bridge between mutant phenotype and gene function

Recent advances in medaka genetics have proven that the medakafish is an excellent model system for developmental and evolutionary biology studies and that it can complement similar studies in zebrafish. Large-scale mutagenesis projects are now being conducted by several groups in Japan and are delivering a vastly expanded pool of medaka mutant stocks. This growing availability of genomic resources will greatly accelerate progress in moving from mutant phenotypes to the elucidation of gene function. This phenotype-driven approach can be expected to lead to the identification and characterization of novel genes and pathways in vertebrate genomes. This review discusses the current state of medaka genomic resources, the state of medaka gene mapping and medaka genome sequencing projects.     

Central connection of the optic, oculomotor, trochlear and abducens nerves in medaka, Oryzias latipes

Medaka (Oryzias latipes) is one of the few vertebrate experimental animals in which inbred lines have been established. It is also a species that has advanced in genetic studies in a manner comparable to zebrafish. This fish is therefore a good model for studying functional organization of the nervous system, but anatomical analysis of its nervous system has been limited to embryonic stages. In the present study, we investigated anatomy of cranial nerves in adult fish focusing on the visual function, using an inbred strain of medaka. Cranial nerves of medaka were labeled using biocytin, revealing a central distribution of retinofugal terminals, retinopetal neurons, and oculomotor, trochlear and abducens motor neurons. The optic nerve of the adult medaka was of a complete decussation type. Retinofugal terminals were located in 8 brain nuclei, the suprachiasmatic nucleus, nucleus pretectalis superficialis, nucleus dorsolateralis thalami, area pretectalis pars dorsalis (APd), area pretectalis pars ventralis (APv), nucleus of the posterior commissure (NPC), accessory optic nucleus, and the tectum opticum. Retinopetal neurons were identified in 6 brain nuclei, the ganglion of the terminal nerve, preoptic retinopetal nucleus, nucleus dorsolateralis thalami, APd, APv, and NPC. The oculomotor neurons were mostly labeled ipsilaterally and were located dorsomedially, abutting the fasciculus longitudinalis medialis in the mesencephalon. The trochlear nucleus was located contralaterally and dorsolaterally adjacent to the fasciculus longitudinalis medialis in the mesencephalon. The abducens nucleus was located ipsilaterally in a ventrolateral part of the rhombencephalic reticular formation. These results, generally similar to those in other teleosts, provide the basis for future behavioral and genetic studies in medaka.     

The rice field eel as a model system for vertebrate sexual development

Complex developmental mechanisms of vertebrates are unraveled using comparative genomic approaches. Several teleosts, such as zebrafish, medaka and pufferfish, are used as genetic model systems because they are amenable to studies of gene function. The rice field eel, a freshwater fish, is emerging as a specific model system for studies of vertebrate sexual development because of its small genome size and naturally occurring sex reversal. Data presented here support the use of the rice field eel as another important fish model for comparative genome studies, especially in vertebrate sexual development. This model system is complementary rather than redundant.     

Optic nerve input‐dependent regulation of neural stem cell proliferation in the optic tectum of adult zebrafish

Adult neurogenesis attracts broad attention as a possible cure for neurological disorders. However, its regulatory mechanism is still unclear. Therefore, they have been studying the cell proliferation mechanisms of neural stem cells (NSCs) using zebrafish, which have high regenerative potential in the adult brain. The presence of neuroepithelial‐type NSCs in the optic tectum of adult zebrafish has been previously reported. In the present study, it was first confirmed that NSCs in the optic tectum decrease or increase in proportion to projection of the optic nerves from the retina. At 4 days after optic nerve crush (ONC), BrdU‐positive cells decreased in the optic tectum's operation side. In contrast, at 3 weeks after ONC, BrdU‐positive cells increased in the optic tectum's operation side. To study the regulatory mechanisms, they focused on the BDNF/TrkB system as a regulatory factor in the ONC model. It was found that bdnf was mainly expressed in the periventricular gray zone (PGZ) of the optic tectum by using in situ hybridization. Interestingly, expression level of bdnf significantly decreased in the optic tectum at 4 days after ONC, and its expression level tended to increase at 3 weeks after ONC. They conducted rescue experiments using a TrkB agonist and confirmed that decrease of NSC proliferation in the optic tectum by ONC was rescued by TrkB signal activation, suggesting stimuli‐dependent regulation of NSC proliferation in the optic tectum of adult zebrafish. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Differential expression of calretinin in the developing and regenerating zebrafish visual system

Calretinin is a calcium-binding protein which participates in a variety of functions including calcium buffering and neuronal protection. It also serves as a developmental marker of retinal ganglion cells (RGCs). In order to study the role of calretinin in the development and regeneration of RGCs, we have studied its pattern of expression in the retina at different developmental stages, as well as during optic nerve regeneration by means of immunohistochemistry. During development, calretinin is found for the first time in RGCs when they connect with the optic tectum. Optic nerves from adult zebrafish were crushed and after different survival times, calretinin expression in the retina, optic nerve tract and optic tectum was studied. From the day of crushing to 10 days later, calretinin expression was found to be downregulated within RGCs and their axons, as was also observed during the early developmental stages of RGCs, when they are not committed to a definite cell phenotype. Moreover, 13 days after lesion, when the regenerating axons arrived at the optic tectum, a recovery of calretinin immunoreactivity within the RGCs was observed. These results indicate that calretinin may play an important role during optic nerve regeneration, Thus, the down-regulation of Calretinin during the growth of the RGC axons towards the target during development as well as during their regeneration after injury, indicates that an increase the availability of cytosolic calcium is integral to axon outgrowth thus recapitulating the pattern observed during development.     

Functional characterization of visual opsin repertoire in Medaka (Oryzias latipes)

A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.     

A call to fins! Zebrafish as a gerontological model

Among the wide variety of model organisms commonly used for studies on aging, such as worms, flies and rodents, a wide research gap exists between the invertebrate and vertebrate model systems. In developmental biology, a similar gap has been filled by the zebrafish (Danio rerio). We propose that the zebrafish is uniquely suited to serve as a bridge model for gerontology. With high fecundity and economical husbandry requirements, large populations of zebrafish may be generated quickly and cheaply, facilitating large-scale approaches including demographic studies and mutagenesis screens. A variety of mutants identified in such screens have led to modelling of human disease, including cardiac disorders and cancer. While zebrafish longevity is at least 50% longer than in commonly used mouse strains, as an ectothermic fish species, its life span may be readily modulated by caloric intake, ambient temperature and reproductive activity. These features, coupled with a growing abundance of biological resources, including an ongoing genome sequencing project, make the zebrafish a compelling model organism for studies on aging.     

Rapid adaptation of molecular resources from zebrafish and medaka to develop an estuarine/marine model

Many estuary and coastal waters are highly threatened by heavy anthropogenic pollutants. Oryzias melastigma, also called O. dancena, a marine medaka that showed sensitive response to hypoxia and estrogenic endocrine disruptors in previous studies, is becoming a sentinel species for marine ecotoxicology studies. However, the lack of strong molecular foundation and knowledge of early developmental stages hampers its practical applications. Combining our research strength on zebrafish embryos, this study revealed both morphological and molecular (at mRNA and protein levels) development of embryos of this emergent model. Whole mount immunostaining technique specific for O. melastigma was successfully developed based on zebrafish standard protocols. We demonstrated that 17 out of 61 primary antibodies, which were previously tested in zebrafish, showed specific immunoreactivity with O. melastigma. These antibodies clearly illustrated the embryonic development of target tissues (principally neurons) in this medaka. Additionally, partial cDNA fragments of 11 organ-specific marker genes were isolated according to genomic resources of zebrafish, Japanese medaka and other fishes. Of the 11 genes, 8 are widely used as organ markers and their expression patterns were remarkably similar to their homologues in zebrafish and Japanese medaka. The expression profiles of the remaining 3 genes in fish are reported for the first time. These molecular markers (17 antibodies and 11 mRNA probes) can be used as responsive indicators in environmental toxicity evaluation. Moreover, this study brought forward and demonstrated the advantage of transferring techniques and resources from one model to another to hasten the research of interest.     

A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes)

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.     

Involvement of L1.1 in memory consolidation after active avoidance conditioning in zebrafish

To investigate the involvement of the cell adhesion molecules L1.1, L1.2, NCAM, and tenascin-C in memory formation, zebrafish (Brachydanio rerio) were trained in an active avoidance paradigm to cross a hurdle to avoid mild electric shocks after a light signal. Application of [(14)C]deoxyglucose prior to the training session revealed an increased energy demand in the optic tectum during acquisition of the active avoidance response compared with untrained fish and with fish not learning the task (nonlearners). In situ hybridization with digoxigenin-labeled cRNA probes directed against zebrafish L1.1, L1.2, NCAM, and tenascin-C revealed an enhanced expression of L1.1 and NCAM mRNA in the optic tectum of learners 3 h after acquisition of the task compared with untrained fish, nonlearners, overtrained fish, and learners decapitated 1 or 6 h after acquisition. Levels of L1.2 mRNA were not significantly increased in the tectum 3 h after learning. Tenascin-C was neither expressed in the optic tectum of untrained fish nor in the tectum of learners. To test for a possible involvement of L1.1 in memory consolidation, antibodies were injected intracerebroventricularly 1 h after the last training trial. Two days later, injected zebrafish were tested for recall and evaluated by a retention score (RS), ranging from 1.0 for immediate recall to 0.0 indicating no savings. The average retention score of L1.1 antibody-injected fish (RS = 0. 29) was different from that of tenascin-C antibody-injected (RS = 0. 71) or uninjected fish (RS = 0.78), indicating a pivotal function of L1.1 in long-term memory formation in zebrafish.     

Effective generation of transgenic reporter and gene trap lines of the medaka (Oryzias latipes) using the Ac/Ds transposon system

In model teleost fishes like the medaka and the zebrafish many genes which have been identified in genome sequencing projects await their functional characterization. Techniques for the effective generation of transgenic animals are a prerequisite for this challenging task, and, due to their transparency, fish offer the possibility to combine the use of fluorescent proteins and developmental analysis in vivo. Here we describe the application of the Ac/Ds transposon system to generate transgenic medaka reporter and gene trap lines. We determined a germline transmission rate of 30% in our experiments using constructs ranging in size from 1.8 to 6 kilobase pairs. The genomic integration site of the Ds-elements can be easily identified which is an important feature for gene trap mutagenesis experiments and similar approaches. We constructed gene trap vectors with functional elements of medaka sequences that produce in frame fusions of the endogenous sequence to EGFP. These vectors mimic endogenous expression of the trapped allele in transgenic animals and are capable to interfere with the expression of the wild type allele in the homozygous individuals.     

Maternal-zygotic medaka mutants for fgfr1 reveal its essential role in the migration of the axial mesoderm but not the lateral mesoderm

The medaka fish (Oryzias latipes) is an emerging model organism for which a variety of unique developmental mutants have now been generated. Our recent mutagenesis screening of the medaka identified headfish (hdf), a null mutant for fgf receptor 1 (fgfr1), which fails to develop structures in the trunk and tail. Despite its crucial role in early development, the functions of Fgfr1-mediated signaling have not yet been well characterized due to the complexity of the underlying ligand-receptor interactions. In our present study, we further elucidate the roles of this pathway in the medaka using the hdf (fgfr1) mutant. Because Fgfr1 is maternally supplied in fish, we first generated maternal-zygotic (MZ) mutants by transplanting homozygous hdf germ cells into sterile interspecific hybrids. Interestingly, the host hybrid fish recovered their fertility and produced donor-derived mutant progeny. The resulting MZ mutants also exhibited severe defects in their anterior head structures that are never observed in the corresponding zygotic mutants. A series of detailed analyses subsequently revealed that Fgfr1 is required for the anterior migration of the axial mesoderm, particularly the prechordal plate, in a cell-autonomous manner, but is not required for convergence movement of the lateral mesoderm. Furthermore, fgfr1 was found to be dispensable for initial mesoderm induction. The MZ hdf medaka mutant was thus found to be a valuable model system to analyze the precise role of fgfr1-mediated signaling in vertebrate early development.