Mosaic gene expression analysis of semaphorin-plexin interactions in Caenorhabditis elegans using the IR-LEGO single-cell gene induction system.

Genetic mosaic analysis is a powerful means of addressing the sites of gene action in multicellular organisms. In conventional genetic analysis, the generation of desired mosaic patterns is difficult to control due to the randomness of generating the genetic mosaic which often renders the analysis laborious and time consuming. The infrared laser-evoked gene operator (IR-LEGO) microscope system facilitates genetic mosaic analysis by enabling gene induction in targeted single cells in a living organism. However, the level of gene induction is not controllable due to the usage of a heat-shock promoter. Here, we applied IR-LEGO to examine the cell-cell interactions mediated by semaphoring-plexin signaling in Caenorhabditis elegans by inducing wild-type semaphorin/plexin in single cells within the population of mutant cells lacking the relevant proteins. We found that the cell contact-dependent termination of the extension of vulval precursor cells is elicited by the forward signaling mediated by the semaphorin receptor, PLX-1, but not by the reverse signaling via the transmembrane semaphorin, SMP-1. By utilizing Cre/loxP recombination coupled with the IR-LEGO system to induce SMP-1 at a physiological level, we found that SMP-1 interacts with PLX-1 only in trans upon contact between vulval precursor cells. In contrast, when overexpressed, SMP-1 exhibits the ability to cis-interact with PLX-1 on a single cell. These results indicate that mosaic analysis with IR-LEGO, especially when combined with an in vivo recombination system, efficiently complements conventional methods.

Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

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.

Therapy-related Ph+ leukemia after both bone marrow and mesenchymal stem cell transplantation for hypophosphatasia.

Bone marrow (BM) transplantation (BMT) is one of the treatment strategies for congenital metabolic disease, but leukemia secondary to intensive cytoreductive treatment is a major concern. Besides BM cells, mesenchymal stem cells (MSC) are also used for transplantation. An 8-month-old girl with hypophosphatasia underwent transplantation of haploidentical BM cells followed by two transplants of MSC obtained from her father to facilitate osteogenesis. Fludarabine(Flu)/cyclophosphamide (CPA)/anti-thymocyte globulin were used for myeloablative conditioning, but the patient developed therapy-related leukemia harboring t(9;22)(q34;q11.2); minor BCR-ABL (t-leukemia with Ph) at the age of 32 months. At the age of 40 months she underwent a second BM and third MSC transplant from the same donor. Thereafter, she achieved complete histological and molecular remission. The present case suggests that the combination of cytotoxic agents (Flu/CPA) and MSC led to t-leukemia with Ph as a consequence of chromosome instability and suppression of host anti-tumor immunity.

Analysis of medaka GAP43 gene promoter activity in transgenic lines.

We produced transgenic medaka fish lines that mimicked the expression of the GAP43 gene. Fish lines with the proximal 2-kilobase (kb) 5'-untranslated region (UTR) as the expression promoter specifically expressed enhanced green fluorescent protein (EGFP) in neural tissues, such as the brain, spinal cord, and peripheral nerves, and its expression decreased with growth, but persisted until adulthood. A functional analysis of the promoter using partially deleted UTRs revealed that functions related to neural tissue-specific promoter activity were widely distributed in the region upstream of the proximal 400-b. Furthermore, the distal half of the 2-kb UTR contributed to expression throughout the brain, while the region 400-b upstream of the proximal 600-b was strongly associated with expression in specific areas, such as the telencephalon. In addition, a region from 957 to 557b upstream of the translation initiation site was important for the long-term maintenance of promoter activity into adulthood. Among the transcription factors with recognition sequences in this region, Sp1 and CREB1 have been suggested to play important roles in the GAP43 promoter expression characteristics, such as strong expression in the telencephalon and long-term maintenance of expression.

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.

Induction of pluripotent stem cells from human third molar mesenchymal stromal cells.

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

Physical properties of mesenchymal stem cells are coordinated by the perinuclear actin cap.

Mesenchymal stem cells (MSCs) have been extensively investigated for their applications in regenerative medicine. Successful use of MSCs in cell-based therapies will rely on the ability to effectively identify their properties and functions with a relatively non-destructive methodology. In this study, we measured the surface stiffness and thickness of rat MSCs with atomic force microscopy and clarified their relation at a single-cell level. The role of the perinuclear actin cap in regulating the thickness, stiffness, and proliferative activity of these cells was also determined by using several actin cytoskeleton-modifying reagents. This study has helped elucidate a possible link between the physical properties and the physiological function of the MSCs, and the corresponding regulatory role of the actin cytoskeleton.

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.

Generation of Xeroderma Pigmentosum-A Patient-Derived Induced Pluripotent Stem Cell Line for Use As Future Disease Model.

Xeroderma pigmentosum group A (XP-A) is a genetic disorder in which there is an abnormality in nucleotide excision repair that causes hypersensitivity to sunlight and multiple skin cancers. The development of central and peripheral neurological disorders not correlated to ultraviolet light exposure is associated with XP-A. The genes responsible for XP-A have been identified and a XPA knockout mouse has been generated. These knockout mice exhibit cutaneous symptoms, but they do not show neurological disorders. The mechanism of pathogenesis of neurological disorders is still unclear and therapeutic methods have not been established. Therefore, we generated XP-A patient-derived human induced pluripotent stem cells (XPA-iPSCs) to produce in vitro models of neurological disorders. We obtained iPSC lines from fibroblasts of two patients carrying different mutations. Drugs screened using XPA-iPSC lines can be helpful for treating XP-A patients in Japan. Additionally, we revealed that these iPSCs have the potential to differentiate into neural lineage cells, including dopaminergic neurons, which decrease in XP-A patients. Our results indicate that expression of the normal XPA gene without mutations is not required for generation of iPSCs and differentiation of iPSCs into neural lineage cells. XPA-iPSCs may become useful models that clarify our understanding of neurological pathogenesis and help to establish therapeutic methods.

Ex Vivo Expanded Allogeneic Mesenchymal Stem Cells With Bone Marrow Transplantation Improved Osteogenesis in Infants With Severe Hypophosphatasia.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

Genomic organization and chromosomal localization of the importin alpha1 gene in the mouse.

Importin alpha1 (also referred to as NPI1 or importin alphaS1) gene encodes a member of the cytosolic receptor protein superfamily that recognizes classical monopartite and bipartite basic type nuclear localization signals and mediates nuclear protein import via an importin beta-dependent pathway. Here we report on the organization of the importin alpha1 locus in the mouse genome. The gene is approximately 40 kb in length from the translation initiation codon to the poly(A) additional site. The translated region of the gene is comprised of 13 coding exons and the exon-intron boundaries conform to the GT/AG rule. Importin alpha1 was mapped to a middle region of mouse chromosome 16 by fluorescence in situ hybridization analysis. Moreover, it was found by reverse transcriptase polymerase chain reaction analysis that importin alpha1 is widely expressed in various tissues in adult mice and at various stages during embyogenesis. This study is the first example that provides detailed genomic information on nuclear transport factors such as importins and exportins and provides a basis for further studies such as the generation of mutants in mice for purposes of investigating the role of importin alpha1 in development and differentiation.

Non-neuronal acetylcholine as an endogenous regulator of proliferation and differentiation of Lgr5-positive stem cells in mice.

Non-neuronal acetylcholine (ACh) is predicted to function as a local cell signaling molecule. However, the physiological significance of the synthesis of non-neuronal ACh in the intestine remains unclear. Here, experiments using crypt-villus organoids that lack nerve and immune cells in culture led us to suggest that endogenous ACh is synthesized in the intestinal epithelium to evoke growth and differentiation of the organoids through activation of muscarinic ACh receptors (mAChRs). The extracts of the cultured organoids showed a noticeable capacity for ACh synthesis that was sensitive to a potent inhibitor of choline acetyltransferase. Imaging MS revealed endogenous ACh localized in the epithelial layer in mouse small intestinal epithelium in vivo, suggesting that there are non-neuronal resources of ACh. Treatment of organoids with carbachol downregulated the growth of organoids and the expression of marker genes for epithelial cells. On the other hand, antagonists for mAChRs enhanced the growth and differentiation of organoids, indicating the involvement of mAChRs in regulating the proliferation and differentiation of Lgr5-positive stem cells. Collectively, our data provide evidence that endogenous ACh released from intestinal epithelium maintains homeostasis of intestinal epithelial cell growth and differentiation via mAChRs in mice.

A neural mechanism underlying mating preferences for familiar individuals in medaka fish.

Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.