Intraperitoneal dye injection method for visualizing the functioning lymphatic vascular system in zebrafish and medaka.

A hierarchically organized lymphatic vascular system extends throughout the vertebrate body for tissue fluid homeostasis, immune trafficking, and the absorption of dietary fats. Intralymphatic dye injection and serial sectioning have been the main tools for visualizing lymphatic vessels. Specific markers for identifying the lymphatic vasculature in zebrafish and medaka have appeared as new tools that enable the study of lymphangiogenesis using genetic and experimental manipulation. Transgenic fishes have become excellent organisms for visualizing the lymphatic vasculature in living embryos, but this method has limited usefulness, especially in later developmental stages. The functional lymphatic endothelium predominantly takes up foreign particles in zebrafish and medaka. We utilized this physiological activity and lymph flow to label lymphatic vessels. Intraperitoneal injection of trypan blue is useful for temporal observations of the lymphatic ducts, which are essential for large-scale genetic screening, while cinnabar (HgS) injection allows identification of the lymphatic endothelium under electron microscopy, avoiding artefactual damage. This injection method, which is not high in cost and does not require high skill or special devices, is applicable to any live fish with functioning lymphatic vessels, even mutants, with high reproducibility for visualizing the entire lymphatic vascular system.

Photothermic regulation of gene expression triggered by laser-induced carbon nanohorns.

The development of optical methods to control cellular functions is important for various biological applications. In particular, heat shock promoter-mediated gene expression systems by laser light are attractive targets for controlling cellular functions. However, previous approaches have considerable technical limitations related to their use of UV, short-wavelength visible (vis), and infrared (IR) laser light, which have poor penetration into biological tissue. Biological tissue is relatively transparent to light inside the diagnostic window at wavelengths of 650-1,100 nm. Here we present a unique optical biotechnological method using carbon nanohorn (CNH) that transforms energy from diagnostic window laser light to heat to control the expression of various genes. We report that with this method, laser irradiation within the diagnostic window resulted in effective heat generation and thus caused heat shock promoter-mediated gene expression. This study provides an important step forward in the development of light-manipulated gene expression technologies.

Application of infrared laser to the zebrafish vascular system: gene induction, tracing, and ablation of single endothelial cells.

OBJECTIVE: Infrared laser-evoked gene operator is a new microscopic method optimized to heat cells in living organisms without causing photochemical damage. By combining the promoter system for the heat shock response, infrared laser-evoked gene operator enables laser-mediated gene induction in targeted cells. We applied this method to the vascular system in zebrafish embryos and demonstrated its usability to investigate mechanisms of vascular morphogenesis in vivo. APPROACH AND RESULTS: We used double-transgenic zebrafish with fli1:nEGFP to identify the endothelial cells, and with hsp:mCherry to carry out single-cell labeling. Optimizing the irradiation conditions, we finally succeeded in inducing the expression of the mCherry gene in single targeted endothelial cells, at a maximum efficiency rate of 60%. In addition, we indicated that this system could be used for laser ablation under certain conditions. To evaluate infrared laser-evoked gene operator, we applied this system to the endothelial cells of the first intersegmental arteries, and captured images of the connection between the vascular systems of the brain and spinal cord. CONCLUSIONS: Our results suggest that the infrared laser-evoked gene operator system will contribute to the elucidation of the mechanisms underlying vascular morphogenesis by controlling spatiotemporal gene activation in single endothelial cells, by labeling or deleting individual vessels in living embryos.

Collagen patches releasing phosphatidylserine liposomes guide M1-to-M2 macrophage polarization and accelerate simultaneous bone and muscle healing.

Bilateral communication between bones and muscles is essential for healing composite bone-muscle injuries from orthopedic surgeries and trauma. However, these injuries are often characterized by exaggerated inflammation, which can disrupt bone-muscle crosstalk, thereby seriously delaying the healing of either tissue. Existing approaches are largely effective at healing single tissues. However, simultaneous healing of multiple tissues remains challenging, with little research conducted to date. Here we introduce collagen patches that overcome this overlooked issue by harnessing the plasticity of macrophage phenotypes. Phosphatidylserine liposomes (PSLs) capable of shifting the macrophage phenotype from inflammatory M1 into anti-inflammatory/prohealing M2 were coated on collagen patches via a layer-by-layer method. Original collagen patches failed to improve tissue healing under inflammatory conditions coordinated by M1 macrophages. In contrast, PSL-coated collagen patches succeeded in accelerating bone and muscle healing by inducing a microenvironment dominated by M2 macrophages. In cell experiments, differentiation of preosteoblasts and myoblasts was completely inhibited by secretions of M1 macrophages but unaffected by those of M2 macrophages. RNA-seq analysis revealed that type I interferon and interleukin-6 signaling pathways were commonly upregulated in preosteoblasts and myoblasts upon stimulation with M1 macrophage secretions, thereby compromising their differentiation. This study demonstrates the benefit of PSL-mediated M1-to-M2 macrophage polarization for simultaneous bone and muscle healing, offering a potential strategy toward simultaneous regeneration of multiple tissues. STATEMENT OF SIGNIFICANCE: Existing approaches for tissue regeneration, which primarily utilize growth factors, have been largely effective at healing single tissues. However, simultaneous healing of multiple tissues remains challenging and has been little studied. Here we demonstrate that collagen patches releasing phosphatidylserine liposomes (PSLs) promote M1-to-M2 macrophage polarization and are effective for simultaneous healing of bone and muscle. Transcriptome analysis using next-generation sequencing reveals that differentiation of preosteoblasts and myoblasts is inhibited by the secretions of M1 macrophages but promoted by those of M2 macrophages, highlighting the importance of timely regulation of M1-to-M2 polarization in tissue regeneration. These findings provide new insight to tissue healing of multiple tissues.

In vivo visualization of the lymphatic vessels in pFLT4-EGFP transgenic medaka.

Feline McDonough Sarcoma (FMS)-like tyrosine kinase 4 (FLT4) is a marker for lymphatic vessels and some high endothelial venules in human adult tissues. We generated a transgenic medaka fish in which the lymphatic vessels and some blood vessels are visible in vivo by transferring the promoter of medaka flt4 driving the expression of enhanced green fluorescent protein (EGFP) using a see-through medaka line. To do this, we identified and cloned medaka flt4 and generated a construct in which the promoter was the 4-kb region upstream of the translation initiation site. The fluorescent signal of EGFP could be observed with little background, and the expression pattern correlated well with that of flt4 determined by whole-mount RNA in situ hybridization. Because a see-through medaka line is transparent until adult, the model is useful for visualizing the lymphatic vessels not only in embryo and fry but also in adult. This model will be a useful tool for analyzing lymphatic development.

Identification and developmental expression of leucine-rich repeat-containing G protein-coupled receptor 6 (lgr6) in the medaka fish, Oryzias latipes.

G protein-coupled receptors are critical regulators of diverse developmental processes such as oocyte maturation, fertilization, gastrulation, and organogenesis. To further study the molecular mechanisms underlying these processes, we cloned and characterized the orphan leucine-rich repeat-containing G protein-coupled receptor 6 (LGR6), a stem cell marker in mammalian hair follicles, in medaka fish, Oryzias latipes. To examine the expression pattern of lgr6, we performed whole-mount in situ hybridization (WISH) during embryogenesis. The expression of lgr6 was first detected as a band in the anterior part of the posterior brain vesicle in 0.5-1 day post fertilization (dpf) embryos. This band disappeared by 2 dpf, but new signals appeared in the otic vesicles bordering the original band and also detected in the nasal placode and posterior lateral line primordia. At later stages (3-5 dpf), lgr6 was widely expressed in the brain, otic vesicle, neuromasts, root of the pectoral fin, cranial cartilage, and gut. Then, we conducted more detailed expression analysis of lgr6 in adult gut using WISH and immunohistochemical staining. Lgr6-positive cells were detected in the crypt-like proliferative zone and in parts of the villus. We also performed RT-PCR of mRNAs from different tissues. The lgr6 mRNA was found highest in the kidney and gill. The transcript was also present in the brain, heart, liver, spleen, intestine, skeletal muscle, testis, and ovary, similar to that of mammalian LGR6. These results suggest that medaka lgr6 plays an important role in organ development during embryogenesis and serves as a good molecular marker for future studies of postembryonic organ-specific development in mammals.

Infrared laser-mediated gene induction in targeted single cells in vivo.

We developed infrared laser-evoked gene operator (IR-LEGO), a microscope system optimized for heating cells without photochemical damage. Infrared irradiation causes reproducible temperature shifts of the in vitro microenvironment in a power-dependent manner. When applied to living Caenorhabditis elegans, IR-LEGO induced heat shock-mediated expression of transgenes in targeted single cells in a more efficient and less deleterious manner than a 440-nm dye laser and elicited physiologically relevant phenotypic responses.

Gfap transgenic medaka as a novel reporter line for neural stem cells.

Radial glial cells (RGCs) play an essential role in developing, maintaining, and repairing the central nervous system (CNS). However, a specific reporter line of RGCs is limited in medaka. Glial fibrillary acid protein (GFAP) is abundant in teleost CNS, including the brain and spinal cord, and is a possible candidate for a marker for RGCs in medaka CNS. We generated a transgenic medaka in which enhanced green fluorescent protein (EGFP) expression is regulated under putative medaka gfap regulatory elements. We observed EGFP expression in the CNS of live larval and juvenile medaka through the transparent body of the See-through medaka strain. Histological analysis for juvenile and adult Tg(gfap:EGFP) medaka showed that EGFP was expressed in GFAP-positive cells in the telencephalon, optic tectum, retina, and spinal cord. We further found another EGFP expressing cells in the optic tectum and retina. These cells are possibly neuroepithelial-like stem cells, deducing from the distribution of these EGFP-positive cells. We concluded that this reporter line would be valuable in the investigation of neural stem cell function during the development and regeneration of medaka CNS visualizing two types of neural stem cells, RGCs and neuroepithelial-like stem cells.

High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library.

BACKGROUND: During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective. RESULTS: In this study, we developed a high resolution melting (HRM) assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. CONCLUSIONS: These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

Identification of a functional medaka heat shock promoter and characterization of its ability to induce exogenous gene expression in medaka in vitro and in vivo.

Heat shock protein promoters (hsp promoters) are powerful tools for investigating gene functions, as the expression of targeted genes can be controlled simply by heating. However, there have been no reports of the utilization of an endogeneous medaka (Oryzias latipes) hsp promoter to induce exogenous gene expression in medaka. We identified and cloned a functional medaka hsp promoter (olphsp70.1) and verified its ability to act as an inducible promoter both in vitro and in vivo. The hsp promoter efficiently induced exogenous gene expression in cultured cells, developing embryos, and also in adult fishes. When used to control the expression of Venus, a variant of yellow fluorescent protein, in transgenic medaka, the hsp promoter was functional in all tissues except for the gonads of adults. These results indicate that the medaka hsp promoter can be a powerful tool for inducing exogenous gene expression and investigating gene functions both in vitro and in vivo in medaka.

Molecular cloning and gene expression of the prox1a and prox1b genes in the medaka, Oryzias latipes.

Prox1 is a prospero-related homeobox gene. Prox1 is expressed in various internal organs and is related to those differentiations. Small fishes such as the zebrafish and the medaka are useful model animals in the clarification of the mechanism of development. The zebrafish prox1 is also identified, and it contributes to clarifying the function of prox1. However, it is necessary to note that many genes are duplicated in teleost fishes. In this study, we identified the orthologs of the mammalian prox1 gene in the medaka. The gene was also duplicated in the medaka, and we named it prox1a and prox1b. In silico analysis from the perspective of synteny indicated that medaka prox1a was similar to the prox1 gene of other vertebrates. Medaka prox1a was expressed in all internal organs that we have examined by RT-PCR. In contrast, medaka prox1b expression was limited to the brain, heart, liver, kidney, thymus, gill, testis, and ovary. This suggests that the two prox1 genes do not have a complementary relationship. In addition, we examined their expression patterns during embryonic development using whole-mount in situ hybridization. The expression pattern of prox1a showed a pattern similar to that of zebrafish prox1. In contrast, medaka prox1b was expressed asymmetrically in part of the central nervous system, especially strongly in the right side of the habenula.

Expression patterns of the Egr1 and Egr3 genes during medaka embryonic development.

Egr1 and Egr3 are zinc finger-type transcription factors and known as synaptic activity-inducible immediate-early genes. Egr1 also plays important roles in many aspects of vertebrate development. Egr3 is known as the gene that is related to biological rhythm and muscular development, but its behavior in the central nervous system during development is not clear. We cloned the cDNA of the egr1 and egr3 orthologs in medaka, and examined their expression patterns during embryonic development using whole-mount in situ hybridization. Medaka egr3 was the first cloned egr3 gene in fish. The expression of egr1 mRNA was first detected at 1day post-fertilization (dpf). It was expressed in the whole embryonic body. At 3dpf, the egr1 mRNA was strongly expressed in the telencephalon, diencephalon, hypothalamus, optic tectum, dorsal medulla oblongata, retina, heart, pharynx, and pectoral fin. The expression of egr3 mRNA was first detected at 3dpf. It was expressed in the telencephalon, hypothalamus, optic tectum, and pharynx. By sectioning the whole-mount specimens, expression of both the egr1 and egr3 mRNAs were observed in the telencephalon, hypothalamus, and optic tectum. However, the positions at which the genes were expressed were different.

Functional and comparative genomics analyses of pmp22 in medaka fish.

BACKGROUND: Pmp22, a member of the junction protein family Claudin/EMP/PMP22, plays an important role in myelin formation. Increase of pmp22 transcription causes peripheral neuropathy, Charcot-Marie-Tooth disease type1A (CMT1A). The pathophysiological phenotype of CMT1A is aberrant axonal myelination which induces a reduction in nerve conduction velocity (NCV). Several CMT1A model rodents have been established by overexpressing pmp22. Thus, it is thought that pmp22 expression must be tightly regulated for correct myelin formation in mammals. Interestingly, the myelin sheath is also present in other jawed vertebrates. The purpose of this study is to analyze the evolutionary conservation of the association between pmp22 transcription level and vertebrate myelin formation, and to find the conserved non-coding sequences for pmp22 regulation by comparative genomics analyses between jawed fishes and mammals. RESULTS: A transgenic pmp22 over-expression medaka fish line was established. The transgenic fish had approximately one fifth the peripheral NCV values of controls, and aberrant myelination of transgenic fish in the peripheral nerve system (PNS) was observed. We successfully confirmed that medaka fish pmp22 has the same exon-intron structure as mammals, and identified some known conserved regulatory motifs. Furthermore, we found novel conserved sequences in the first intron and 3'UTR. CONCLUSION: Medaka fish undergo abnormalities in the PNS when pmp22 transcription increases. This result indicates that an adequate pmp22 transcription level is necessary for correct myelination of jawed vertebrates. Comparison of pmp22 orthologs between distantly related species identifies evolutionary conserved sequences that contribute to precise regulation of pmp22 expression.

Infrared laser-mediated local gene induction in medaka, zebrafish and Arabidopsis thaliana.

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single-cell gene induction using an infrared laser-evoked gene operator (IR-LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR-LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR-LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.

Valves Are a Conserved Feature of the Zebrafish Lymphatic System.

The lymphatic system comprises blind-ended tubes that collect interstitial fluid and return it to the circulatory system. In mammals, unidirectional lymphatic flow is driven by muscle contraction working in conjunction with valves. Accordingly, defective lymphatic valve morphogenesis results in backflow leading to edema. In fish species, studies dating to the 18th century failed to identify lymphatic valves, a precedent that currently persists, raising the question of whether the zebrafish could be used to study the development of these structures. Here, we provide functional and morphological evidence of valves in the zebrafish lymphatic system. Electron microscopy revealed valve ultrastructure similar to mammals, while live imaging using transgenic lines identified the developmental origins of lymphatic valve progenitors. Zebrafish embryos bearing mutations in genes required for mammalian valve morphogenesis show defective lymphatic valve formation and edema. Together, our observations provide a foundation from which to further investigate lymphatic valve formation in zebrafish.

Abnormal nuclear morphology is independent of longevity in a zmpste24-deficient fish model of Hutchinson-Gilford progeria syndrome (HGPS).

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.

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 retinotectal axonal pathfinding in Medaka, Oryzias latipes.

We screened for mutations affecting retinotectal axonal projection in Medaka, Oryzias latipes. In wild-type Medaka embryos, all the axons of retinal ganglion cells (RGCs) project to the contralateral tectum, such that the topological relationship of the retinal field is maintained. We labeled RGC axons using DiI/DiO at the nasodorsal and temporoventral positions of the retina, and screened for mutations affecting the pattern of stereotypic projections to the tectum. By screening 184 mutagenized haploid genomes, seven mutations in five genes causing defects in axonal pathfinding were identified, whereas mutations affecting the topographic projection of RGC axons were not found. The mutants were grouped into two classes according to their phenotypes. In mutants of Class I, a subpopulation of the RGC axons branched out either immediately after leaving the eye or after reaching the midline, and this axonal subpopulation projected to the ipsilateral tectum. In mutants of Class II, subpopulations of RGC axons branched out after crossing the midline and projected aberrantly. These mutants will provide clues to understanding the functions of genes essential for axonal pathfinding, which may be conserved or partly divergent among vertebrates.

Mutations affecting thymus organogenesis in Medaka, Oryzias latipes.

The thymus is an organ for T lymphocyte maturation and is indispensable for the establishment of a highly developed immune system in vertebrates. In order to genetically dissect thymus organogenesis, we carried out a large-scale mutagenesis screening for Medaka mutations affecting recombination activating gene 1 (rag1) expression in the developing thymus. We identified 24 mutations, defining at least 13 genes, which led to a marked reduction of rag1 expression in the thymus. As thymus development depends on pharyngeal arches, we classified those mutations into three classes according to the defects in the pharyngeal arches. Class 1 mutants had no or slight morphological abnormalities in the pharyngeal arches, implying that the mutations may include defects in such thymus-specific events as lymphocyte development and thymic epithelial cell maturation. Class 2 mutants had abnormally shaped pharyngeal arches. Class 3 mutants showed severely attenuated pharyngeal arch development. In Class 2 and Class 3 mutants, the defects in thymus development may be due to abnormal pharyngeal arch development. Those mutations are expected to be useful for identifying the molecular mechanisms underlying thymus organogenesis.

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo.

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.