Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells.

Gametogenesis is the process through which germ cells differentiate into sexually dimorphic gametes, eggs and sperm. In the teleost fish medaka (Oryzias latipes), a germ cell-intrinsic sex determinant, foxl3, triggers germline feminization by activating two genetic pathways that regulate folliculogenesis and meiosis. Here, we identified a pathway involving a dome-shaped microtubule structure that may be the basis of oocyte polarity. This structure was first established in primordial germ cells in both sexes, but was maintained only during oogenesis and was destabilized in differentiating spermatogonia under the influence of Sertoli cells expressing dmrt1. Although foxl3 was dispensable for this pathway, dazl was involved in the persistence of the microtubule dome at the time of gonocyte development. In addition, disruption of the microtubule dome caused dispersal of bucky ball RNA, suggesting the structure may be prerequisite for the Balbiani body. Collectively, the present findings provide mechanistic insight into the establishment of sex-specific polarity through the formation of a microtubule structure in germ cells, as well as clarifying the genetic pathways implementing oocyte-specific characteristics.

Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

De novo PAM generation to reach initially inaccessible target sites for base editing.

Base editing by CRISPR crucially depends on the presence of a protospacer adjacent motif (PAM) at the correct distance from the editing site. Here, we present and validate an efficient one-shot approach termed 'inception' that expands the editing range. This is achieved by sequential, combinatorial base editing: de novo generated synonymous, non-synonymous or intronic PAM sites facilitate subsequent base editing at nucleotide positions that were initially inaccessible, further opening the targeting range of highly precise editing approaches. We demonstrate the applicability of the inception concept in medaka (Oryzias latipes) in three settings: loss of function, by introducing a pre-termination STOP codon in the open reading frame of oca2; locally confined multi-codon changes to generate allelic variants with different phenotypic severity in kcnh6a; and the removal of a splice acceptor site by targeting intronic sequences of rx3. Using sequentially acting base editors in the described combinatorial approach expands the number of accessible target sites by 65% on average. This allows the use of well-established tools with NGG PAM recognition for the establishment of thus far unreachable disease models, for hypomorphic allele studies and for efficient targeted mechanistic investigations in a precise and predictable manner.

Identifying Universal Fish Biomarker Genes in Response to PCB126 Exposure by Comparative Transcriptomic Analyses.

Water pollution remains a major environmental concern, with increased toxic by-products being released into water bodies. Many of these chemical contaminants persist in the environment and bio-accumulate in aquatic organisms. At present, toxicological tests are mostly based on laboratory tests, and effective methods for monitoring wild aquatic environments remain lacking. In the present study, we used a well-characterized toxic chemical, 3,3',4,4',5-polychlorinated biphenyl (PCB126), as an example to try to identify common biomarker genes to be used for predictive toxicity of this toxic substance. First, we used two laboratory fish models, the zebrafish (Danio rerio) and medaka (Oryzias latipes), to expose PCB126 to obtain liver transcriptomic data by RNA-seq. Comparative transcriptomic analyses indicated generally conserved and concerted changes from the two species, thus validating the transcriptomic data for biomarker gene selection. Based on the common up- and downregulated genes in the two species, we selected nine biomarker genes to further test in other fish species. The first validation experiment was carried out using the third fish species, Mozambique tilapia (Oreochromis mossambicus), and essentially, all these biomarker genes were validated for consistent responses with the two laboratory fish models. Finally, to develop universal PCR primers suitable for potentially all teleost fish species, we designed degenerate primers and tested them in the three fish species as well as in another fish species without a genomic sequence available: guppy (Poecilia reticulata). We found all the biomarker genes showed consistent response to PCB126 exposure in at least 50% of the species. Thus, our study provides a promising strategy to identify common biomarker genes to be used for teleost fish analyses. By using degenerate PCR primers and analyzing multiple biomarker genes, it is possible to develop diagnostic PCR arrays to predict water contamination from any wild fish species sampled in different water bodies.

Protective Potential of a Botanical-Based Supplement Ingredient against the Impact of Environmental Pollution on Cutaneous and Cardiopulmonary Systems: Preclinical Study.

Air pollution is a growing threat to human health. Airborne pollution effects on respiratory, cardiovascular and skin health are well-established. The main mechanisms of air-pollution-induced health effects involve oxidative stress and inflammation. The present study evaluates the potential of a polyphenol-enriched food supplement ingredient comprising Lippia citriodora, Olea europaea, Rosmarinus officinalis, and Sophora japonica extracts in mitigating the adverse effects of environmental pollution on skin and cardiopulmonary systems. Both in vitro and ex vivo studies were used to assess the blend's effects against pollution-induced damage. In these studies, the botanical blend was found to reduce lipid peroxidation, inflammation (by reducing IL-1α), and metabolic alterations (by regulating MT-1H, AhR, and Nrf2 expression) in human skin explants exposed to a mixture of pollutants. Similar results were also observed in keratinocytes exposed to urban dust. Moreover, the ingredient significantly reduced pollutant-induced ROS production in human endothelial cells and lung fibroblasts, while downregulating the expression of apoptotic genes (bcl-2 and bax) in lung fibroblasts. Additionally, the blend counteracted the effect of urban dust on the heart rate in zebrafish embryos. These results support the potential use of this supplement as an adjuvant method to reduce the impact of environmental pollution on the skin, lungs, and cardiovascular tissues.

Gsdf is not indispensable for male differentiation in the medaka species Oryzias hubbsi.

Sex determination mechanisms differ widely among vertebrates, particularly in fish species, where diverse sex chromosomes and sex-determining genes have evolved. However, the sex-differentiation pathways activated by these sex-determining genes appear to be conserved. Gonadal soma-derived growth factor (Gsdf) is one of the genes conserved across teleost fish, especially in medaka fishes of the genus Oryzias, and is implicated in testis differentiation and germ cell proliferation. However, its role in sex differentiation remains unclear. In this study, we investigated Gsdf function in Oryzias hubbsi, a species with a ZW sex-determination system. We confirmed its male-dominant expression, as in other species. However, histological analyses revealed no male-to-female sex reversal in Gsdf-knockout fish, contrary to findings in other medaka species. Genetic sex determination remained intact without Gsdf function, indicating a Gsdf-independent sex-differentiation pathway in O. hubbsi. Instead, Gsdf loss led to germ cell overproliferation in both sexes and accelerated onset of meiosis in testes, suggesting a role in germ cell proliferation. Notably, the feminizing effect of germ cells observed in O. latipes was absent, suggesting diverse germ cell-somatic cell relationships in Oryzias gonad development. Our study highlights species-specific variations in the molecular pathways governing sex determination and differentiation, emphasizing the need for further exploration to elucidate the complexities of sexual development.

Igf signaling couples retina growth with body growth by modulating progenitor cell division.

How the body and organs balance their relative growth is of key importance for coordinating size and function. This is of particular relevance in organisms, which continue to grow over their entire life span. We addressed this issue in the neuroretina of medaka fish (Oryzias latipes), a well-studied system with which to address vertebrate organ growth. We reveal that a central growth regulator, Igf1 receptor (Igf1r), is necessary and sufficient for proliferation control in the postembryonic retinal stem cell niche: the ciliary marginal zone (CMZ). Targeted activation of Igf1r signaling in the CMZ uncouples neuroretina growth from body size control, and we demonstrate that Igf1r operates on progenitor cells, stimulating their proliferation. Activation of Igf1r signaling increases retinal size while preserving its structural integrity, revealing a modular organization in which progenitor differentiation and neurogenesis are self-organized and highly regulated. Our findings position Igf signaling as a key module for controlling retinal size and composition, with important evolutionary implications.

A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Modeling familial and sporadic Parkinson's disease in small fishes.

The establishment of animal models for Parkinson's disease (PD) has been challenging. Nevertheless, once established, they will serve as valuable tools for elucidating the causes and pathogenesis of PD, as well as for developing new strategies for its treatment. Following the recent discovery of a series of PD causative genes in familial cases, teleost fishes, including zebrafish and medaka, have often been used to establish genetic PD models because of their ease of breeding and gene manipulation, as well as the high conservation of gene orthologs. Some of the fish lines can recapitulate PD phenotypes, which are often more pronounced than those in rodent genetic models. In addition, a new experimental teleost fish, turquoise killifish, can be used as a sporadic PD model, because it spontaneously manifests age-dependent PD phenotypes. Several PD fish models have already made significant contributions to the discovery of novel PD pathological features, such as cytosolic leakage of mitochondrial DNA and pathogenic phosphorylation in α-synuclein. Therefore, utilizing various PD fish models with distinct degenerative phenotypes will be an effective strategy for identifying emerging facets of PD pathogenesis and therapeutic modalities.

Japanese medaka (Oryzias latipes) Nectin4 plays an important role against red spotted grouper nervous necrosis virus infection.

Nectins are adhesion molecules that play a crucial role in the organization of epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus. However, the role of Nectin4 remains poorly understood in fish. In this study, nectin4 gene was cloned from medaka (OlNectin4). OlNectin4 was located on chromosome 18 and contained 11 exons, with a total genome length of 25754 bp, coding sequences of 1689 bp, coding 562 amino acids and a molecular weight of 65.5 kDa. OlNectin4 contained four regions, including an Immunoglobulin region, an Immunoglobulin C-2 Type region, a Transmembrane region and a Coiled coil region. OlNectin4 shared 47.18 % and 25.00 % identity to Paralichthys olivaceus and Mus musculus, respectively. In adult medaka, the transcript of nectin4 was predominantly detected in gill. During red spotted grouper nervous necrosis virus (RGNNV) infection, overexpression of OlNectin4 in GE cells significantly increased viral gene transcriptions. Meanwhile, Two mutants named OlNectin4△4 (+4 bp) and OlNectin4△7 (-7 bp) medaka were established using CRISPR-Cas9 system. Nectin4-KO medaka had higher mortality than WT after infected with RGNNV. Moreover, the expression of RGNNV RNA2 gene in different tissues of the Nectin4-KO were higher than WT medaka after challenged with RGNNV. The brain and eye of Nectin4-KO medaka which RGNNV mainly enriched, exhibited significantly higher expression of interferon signaling genes than in WT. Taken together, the OlNectin4 plays a complex role against RGNNV infection by inducing interferon responses for viral clearance.

Medaka Terb1 Mutant Displays Defects of Synaptonemal Complex Formation and Sexual Difference in Gametogenesis.

Formation of the synaptonemal complex (SC) is a prerequisite for proper recombination and chromosomal segregation during meiotic prophase I. One mechanism that ensures SC formation is chromosomal movement, which is driven by the force derived from cytoskeletal motors. Here, we report the phenotype of medaka mutants lacking the telomere repeat binding bouquet formation protein 1 (TERB1), which, in combination with the SUN/KASH protein, mediates chromosomal movement by connecting telomeres and cytoskeletal motors. Mutations in the terb1 gene exhibit defects in SC formation in medaka. Although SC formation was initiated, as seen by the punctate lateral elements and fragmented transverse filaments, it was not completed in the terb1 mutant meiocytes. The mutant phenotype further revealed that the introduction of double strand breaks was independent of synapsis completion. In association with these phenotypes, meiocytes in both the ovaries and testes exhibited an aberrant arrangement of homologous chromosomes. Interestingly, although oogenesis halted at the zygotene-like stage in terb1 mutant, testes continued to produce sperm-like cells with aberrant DNA content. This indicates that the mechanism of meiotic checkpoint is sexually different in medaka, similar to the mammalian checkpoint in which oogenesis proceeds while spermatogenesis is arrested. Moreover, our results suggest that spermatogenesis is mechanistically dissociable from meiosis.

Loss of function in somatostatin receptor 5 has no impact on the growth of medaka fish due to compensation by the other paralogs.

Somatic growth in vertebrates is regulated endocrinologically by the somatotropic axis, headed by the growth hormone (GH) and the insulin growth factor-I (IGF-I). Somatostatin (Sst), a peptide hormone synthesized in the hypothalamus, modulates GH actions through its receptors (Sstr). Four Sstr subtypes (Sstr 1-3 and 5) have been identified in teleosts. However, little is known about whether they have a specific function or tissue expression. The aim of this study was to determine the role of sstr2 and sstr5 in the growth of the medaka (Oryzias latipes). The assessed expression pattern across diverse tissues highlighted greater prevalence of sstr1 and sstr3 in brain, intestine and muscle than in pituitary or liver. The expression of sstr2 was high in all the tissues tested, while sstr5 was predominantly expressed in the pituitary gland. A CRISPR/Cas9 sstr5 mutant with loss of function (sstr5-/-) was produced. Assessment of sstr5-/- indicated no significant difference with the wild type regarding growth parameters such as standard length, body depth, or peduncle depth. Furthermore, the functional loss of sstr5 had no impact on the response to a nutritional challenge. The fact that several sstr subtypes were upregulated in different tissues in sstr5-/- medaka suggests that in the mutant fish, there may be a compensatory effect on the different tissues, predominantly by sstr1 in the liver, brain and pituitary, with sstr2 being upregulated in pituitary and liver, and sstr3 only presenting differential expression in the brain. Analysis of the sstr subtype and the sstr5-/- fish showed that sstr5 was not the only somatostatin receptor responsible for Sst-mediated Gh regulation.

Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

Single and mixture toxicity of cadmium and copper to swim bladder in early life stages of Japanese medaka (Oryzias latipes).

Toxicity observed in aquatic ecosystems often cannot be explained by the action of a single pollutant. Likewise, evaluation standards formulated by a single effect cannot truly reflect the environmental quality requirements. The study of mixtures is needed to provide environmental relevance and knowledge of combined toxicity. In this study, the embryos of Japanese medaka (Oryzias latipes) were treated with individual and binary mixture of copper (Cu) and cadmium (Cd) until 12 days post-fertilization (dpf). Hatching, mortality, development, histology and gene expression were assessed. Our results showed that the highest concentration mixture of Cd (10 mg/L) and Cu (1 mg/L) affected survival, hatching time and hatching success. Occurrence of uninflated swim bladder was the highest (value) with exposure to 10 mg/L Cd. Swim bladder was commonly over-inflated in a mixture (0.1 mg/L Cd + 1.0 mg/L Cu) exposure. Individuals exposed to the mixture (0.1 Cd + 1.0 Cu mg/L) showed up to a 7.69% increase in swim bladder area compared to the control group. The mixtures containing 0.1 or 10 mg/L Cd, each with 1.0 mg/L Cu resulted in significantly increased of Pbx1b expression, higher than any Cd or Cu alone (p < 0.01). In the co-exposure group (0.1/10 Cd + 1.0 Cu mg/L), Pbx1b expression was found at 12 dpf but not 7 dpf in controls. Higher concentrations of Cd may progressively reduce Pbx1b expression, potentially explaining why 75% of individuals in the 10 mg/L Cd group failed to inflate their swim bladders. Additionally, the swim bladder proved to be a valuable bio-indicator for biological evaluation.

Local tissue interactions govern pLL patterning in medaka.

Vertebrate organs are arranged in a stereotypic, species-specific position along the animal body plan. Substantial morphological variation exists between related species, especially so in the vastly diversified teleost clade. It is still unclear how tissues, organs and systems can accommodate such diverse scaffolds. Here, we use the distinctive arrangement of neuromasts in the posterior lateral line (pLL) system of medaka fish to address the tissue-interactions defining a pattern. We show that patterning in this peripheral nervous system is established by autonomous organ precursors independent of neuronal wiring. In addition, we target the keratin 15 gene to generate stuck-in-the-midline (siml) mutants, which display epithelial lesions and a disrupted pLL patterning. By using siml/wt chimeras, we determine that the aberrant siml pLL pattern depends on the mutant epithelium, since a wild type epithelium can rescue the siml phenotype. Inducing epithelial lesions by 2-photon laser ablation during pLL morphogenesis phenocopies siml genetic mutants and reveals that epithelial integrity defines the final position of the embryonic pLL neuromasts. Our results using the medaka pLL disentangle intrinsic from extrinsic properties during the establishment of a sensory system. We speculate that intrinsic programs guarantee proper organ morphogenesis, while instructive interactions from surrounding tissues facilitates the accommodation of sensory organs to the diverse body plans found among teleosts.

High-fat diet in early life triggers both reversible and persistent epigenetic changes in the medaka fish (Oryzias latipes).

BACKGROUND: The nutritional status during early life can have enduring effects on an animal's metabolism, although the mechanisms underlying these long-term effects are still unclear. Epigenetic modifications are considered a prime candidate mechanism for encoding early-life nutritional memories during this critical developmental period. However, the extent to which these epigenetic changes occur and persist over time remains uncertain, in part due to challenges associated with directly stimulating the fetus with specific nutrients in viviparous mammalian systems. RESULTS: In this study, we used medaka as an oviparous vertebrate model to establish an early-life high-fat diet (HFD) model. Larvae were fed with HFD from the hatching stages (one week after fertilization) for six weeks, followed by normal chow (NC) for eight weeks until the adult stage. We examined the changes in the transcriptomic and epigenetic state of the liver over this period. We found that HFD induces simple liver steatosis, accompanied by drastic changes in the hepatic transcriptome, chromatin accessibility, and histone modifications, especially in metabolic genes. These changes were largely reversed after the long-term NC, demonstrating the high plasticity of the epigenetic state in hepatocytes. However, we found a certain number of genomic loci showing non-reversible epigenetic changes, especially around genes related to cell signaling, liver fibrosis, and hepatocellular carcinoma, implying persistent changes in the cellular state of the liver triggered by early-life HFD feeding. CONCLUSION: In summary, our data show that early-life HFD feeding triggers both reversible and persistent epigenetic changes in medaka hepatocytes. Our data provide novel insights into the epigenetic mechanism of nutritional programming and a comprehensive atlas of the long-term epigenetic state in an early-life HFD model of non-mammalian vertebrates.

Japanese medaka Olpax6.1 mutant as a potential model for spondylo-ocular syndrome.

pax6 is a canonic master gene for eye formation. Knockout of pax6 affects the development of craniofacial skeleton and eye in mice. Whether pax6 affects the development of spinal bone has not been reported yet. In the present study, we used CRISPR/Cas9 system to generate Olpax6.1 mutant in Japanese medaka. Phenotype analysis showed that ocular mutation caused by the Olpax6.1 mutation occurred in the homozygous mutant. The phenotype of heterozygotes is not significantly different from that of wild-type. In addition, knockout Olpax6.1 resulted in severe curvature of the spine in the homozygous F2 generation. Comparative transcriptome analysis and qRT-PCR revealed that the defective Olpax6.1 protein caused a decrease in the expression level of sp7, col10a1a, and bglap, while the expression level of xylt2 did not change significantly. The functional enrichment of differentially expressed genes (DEGs) using the Kyoto Encyclopedia of Genes and Genomes database showed that the DEGs between Olpax6.1 mutation and wild-type were enriched in p53 signaling pathway, extracellular matrix (ECM) -receptor interaction, et al. Our results indicated that the defective Olpax6.1 protein results in the reduction of sp7 expression level and the activation of p53 signaling pathway, which leads to a decrease in the expression of genes encoding ECM protein, such as collagen protein family and bone gamma-carboxyglutamate protein, which further inhibits bone development. Based on the phenotype and molecular mechanism of ocular mutation and spinal curvature induced by Olpax6.1 knockout, we believe that the Olpax6.1-/- mutant could be a potential model for the study of spondylo-ocular syndrome.

Notch1 deficiency alters the migratory behavior of developing T cells and calcium signaling in the thymus of medaka.

The differentiation of T cells from lymphoid progenitors in the thymus follows sequential developmental stages that constantly require interaction with thymic epithelial cells. Several distinct aspects of early T cell development depend on the activation of Notch receptors on thymocytes, while the selection of thymocytes at later stages are believed to be Notch independent. Using reverse genetic approaches and whole-thymus live imaging in an in vivo teleost model, the medaka, we report that Notch1 signals is required for proliferation and specification of developing T cells as well as involved in their selection in the thymus. We reveal that Notch1 controls the migratory behavior of thymocytes through controlling the chemokine receptor Ccr9b and thereby influence the T cell receptor (TCR) activation. Hence, we propose that, in lower vertebrates, the function of Notch signaling extends to all stages of T cell development, except when thymocytes undergo TCRß rearrangement.

Forced expression of α2,3-sialyltransferase IV rescues impaired heart development in α2,6-sialyltransferase I-deficient medaka.

Sialic acids (Sias) are often linked to galactose (Gal) residues by α2,6- and α2,3-linkages in glycans of glycoproteins. Sias are indispensable for vertebrate development, because organisms deficient in some enzymes in the Sia synthetic pathway are lethal during the development. However, it remains unknown if the difference of Siaα2,6Gal or α2,3Gal linkage has a critical meaning. To find a clue to understand significance of the linkage difference at the organism level, medaka was used as a vertebrate model. In embryos, Siaα2,6Gal epitopes recognized by Sambucus nigra lectin (SNA) and Siaα2,3Gal epitopes recognized by Maackia amurensis lectin (MAA) were enriched in the blastodisc and the yolk sphere, respectively. When these lectins were injected in the perivitelline space, SNA, but not MAA, impaired embryo body formation at 1 day post-fertilization (dpf). Most Siaα2,6Gal epitopes occurred on N-glycans owing to their sensitivity to peptide:N-glycanase. Of knockout-medaka (KO) for either of two ß-galactoside:α2,6-sialyltransferase genes, ST6Gal I and ST6Gal II, only ST6Gal I-KO showed severe cardiac abnormalities at 7-16 dpf, leading to lethality at 14-18 dpf. Interestingly, however, these cardiac abnormalities of ST6Gal I-KO were rescued not only by forced expression of ST6Gal I, but also by that of ST6Gal II and the ß-galactoside:α2,3-sialyltransferase IV gene (ST3Gal IV). Taken together, the Siaα2,6Gal linkage synthesized by ST6Gal I are critical in heart development; however, it can be replaced by the linkages synthesized by ST6Gal II and ST3Gal IV. These data suggest that sialylation itself is more important than its particular linkage for the heart development.