Zebrafish, Danio rerio, have become an important model for developmental studies and have several advantages over other model systems. These advantages include (1) the easy accessibility of zebrafish embryos for direct observation of their development and (2) their suitability for systematic mutagenesis studies for the identification of genes regulating the development of various tissues and organs, including the skeletal system. Recently, it has been reported that glutamate receptors are expressed in many types of bone cells and regulate bone physiological functions. In the present study, we have examined the effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist¡Xdextromethorphan¡Xon the development of the axial skeleton in zebrafish embryos by using calcein stain. Our results revealed that dextromethorphan significantly attenuates the formation of the axial skeleton and that it is inhibited on pretreatment with glutamate. Moreover, immunohistochemical analysis revealed protein level expression of the NMDA subunit NR1 in the axial region of zebrafish. Our results also indicate that attenuation of NMDA receptor activity-induced change in the axial skeleton may be related to heat-shock protein and extracellular signal-regulated kinase (ERK) signalings. In conclusion, we suggest that the NMDA receptor plays an important role in the development of the axial skeleton. However, further studies are required on the cellular mechanisms of glutamate regulated bone formation.

Cyanobacteria, also known as blue-green algae, are known to produce a number of biologically active compounds. Extracts of cultured cyanobacteria isolated from South Florida sources were screened for possible developmental toxins using the zebrafish (Danio rerio) embryo as a model of vertebrate development. A strain of cyanobacteria, Fischerella 52-1, isolated from the Florida Everglades, was found to produce metabolites that caused a consistent developmental dysfunction in embryos exposed to lipophilic extract. Initial chemical characterization of the bioactive fraction identified a series of eight apparent indole-containing compounds. The two main components were purified using the zebrafish embryo model to guide the fractionation. Chemical characterization using 1- and 2-dimensional NMR, HESIMS, HRHESIMS, and IR determined that the two main compounds were the previously identified 12-epi-Hapalindole H Isonitrile, and a novel compound 12-epi-Ambiugine B Nitrile. The major contributor of the developmental defects detected in the zebrafish embryos was 12-epi-Hapalindole H Isonitrile.

The process of a single cell evolving into a complex organism results from a series of coordinated movements of cells and tissues, especially during early embryo development. Although a wealth of morphological data characterises the shapes and movements of cells in embryos, how these movements are driven, patterned and controlled, and how this patterning is related to the mechanical properties of tissues remains unknown. Four-pole electromagnetic tweezers have been developed to probe the mechanical properties of living embryonic tissues that are undergoing active morphogenetic development. The device is capable of generating magnetic forces in the order of nano-Newtons on a grafted magnetic bead. The local passive mechanical properties of the tissues can be characterised by measuring the three-dimensional bead movement and analysing cell shape changes and cell rearrangement in response to this externally applied force. The magnetic device is used to probe the rheology in early zebrafish embryos between high stage (3.3 hpf) and the onset of gastrulation (5.3 hpf) when rapid cell cycles give way to a hollow sphere of cells. The tissue response to the applied force is modelled as linear visco- elastic. The embryo becomes stiffer and more viscous during this period of development, showing that a loose collection of cells becomes cohesive tissues. A computational model is used to explore how cells respond to local or global mechanical perturbations in two systems. First, the model simulates the movement of the bead within an embryo, and the results illustrate the generation, patterning and relaxation of the local cell stress around the bead. Second, the model reproduces the autonomous changes in mitotic cells within a stretched monolayer, and the results show that propensity of cells to divide along their long axis facilitates stress relaxation and contributes to tissue homoeostasis.

The Gram-negative bacterium Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases including bacillary angiomatosis which is characterized by vasoproliferative tumor-like lesions on the skin and internal organs of some immunosuppressed individuals. Several virulence factors associated with Bartonella-induced pathogenesis have been characterized. However, the study of those virulence factors has been limited to in vitro cell culture systems due to the lack of a practical animal model. Therefore, we wanted to investigate whether the zebrafish embryo (Danio rerio) could be used to model human infection with Bh. We investigated if Bh can mount an infection in zebrafish embryos during their early stage of development. Our data showed that Tg(fli1:egfp)y1 zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. This was evident by plating of zebrafish homogenates, quantitative PCR, and confocal microscopy analysis. We assessed the interaction of Bh with EC and the phagocytic cells in live embryos by microscopy. Our data showed that aggregates of Bh interact with the endothelium of the embryo vasculature. Evidence showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. We also wanted to determine the response to infection with Bh. Infected embryos showed evidence of a Bh-induced angiogenic phenotype as well as an increase in expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. A deletion mutant for the entire VirB type IV secretion system (ΔvirB2-11 supported bacterial replication although to a lesser degree compared to the wild type control. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished pro-angiogenic and pro-inflammatory host response compared to wild type Bh, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical animal model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

The world’s growing population is causing an ever increasing demand for clean safe drinking water. In some countries suitable sources of drinking water are becoming scarce and will not be able to satisfy future demand. Consequently, there is a need to find alternative sources of water that can be used for potable supply or to augment current sources. Advanced water treatment methods are now being examined to investigate whether treated domestic sewage effluent can be treated to drinking water standards and discharged upstream of a drinking water abstraction point; a process known as Indirect Potable Reuse (IPR). The aim of this project was to investigate biological activity associated with developmental exposure to IPR water at the various stages of treatment using zebrafish embryos. Embryos reared in water at different stages of the treatment process were observed for developmental abnormalities, and differences in gene expression (compared to an aquarium water control) were used to establish both the nature and persistence of these effects along the treatment process. In addition to the embryo assays, passive sampling devices, Pharmaceutical Polar Organic Integrative Sampler (Pharm-POCIS) were deployed over eight, four week periods to collect composite concentrated samples of some of the contaminants present in the effluent. These concentrated extracts were then used in an in vitro assay; an Enzyme Immunoassay (EIA) to measure the inhibition of prostaglandins (an indirect measure of inhibitors of cyclooxygenase activity). We compared our results of the bioassays with the large body of chemical analysis data recorded over a number of years from each of the treatments. The developmental exposures highlighted a low frequency of consistent abnormalities to the heart and spine, and also a lack of pigmentation. Gene expression analysis demonstrates the developmental stage of the embryo to have the greatest influence on global gene expression as opposed to the treatment. Single genes of interest included the two cytochrome P450s (cyp1a and cyp1b1) and somatolactin beta. Some of the pathways disrupted included steroid synthesis, retinol metabolism, tryptophan metabolism and melanogenesis. The latter was consistent with observations of some embryos devoid of pigment. Along the treatment process reverse osmosis seemed to cause the largest change to the gene expression. The extracts from less treated effluent inhibited prostaglandin production, however following reverse osmosis prostaglandin inhibition was greatly reduced. The chemical contaminantion is greatly reduced as the effluent progresses along the IPR treatment process, this is evident from both the chemical data and the biological assays. Reverse osmosis seems to have the greatest influence on the gene expression. The results have highlighted the importance of an appropriate control, to remove background noise.

Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) provoquent des infections sévères chez les personnes atteintes de mucoviscidose. L'infection peut varier d'une forme asymptomatique à une forme plus aiguë pouvant entraîner une pneumonie nécrosante et une septicémie, connue sous le nom de syndrome cepacia. Afin d'étudier les infections causées par le Bcc, nous avons développé un nouveau modèle in vivo, l'embryon de poisson zèbre. Nous avons montré que B. cenocepacia K56-2 pouvait se répliquer dans les macrophages et causer une infection aiguë mortelle pour les embryons. En revanche, B. stabilis LMG14294 induit une infection persistante chez les embryons. Dans cette étude, nous avons montré que les macrophages jouaient un rôle-clé dans la multiplication de K56-2 et dans l'induction d'une réponse inflammatoire MyD88-dépendante, caractérisée par la surexpression des gènes codant pour Cxcl8 (ou IL-8) et l'IL-1b. En l'absence de macrophages, les bactéries sont incapables de se multiplier durant les premières 24h de l'infection, ce qui donne un avantage pour la survie des embryons. L'absence de MyD88 induit aussi l'augmentation de la survie des embryons infectés par K56-2. Mais de manière paradoxale, les bactéries se multiplient mieux chez les embryons myd88-/- mutants que chez les embryons sauvages. Ceci suggère que ce n'est pas le nombre de bactéries qui est important pour l'infection, mais que c'est la réponse inflammatoire excessive causée par cette infection qui entraîne la mort des embryons. Afin d'avoir une vision globale des changements d'expression des gènes de l'hôte durant l'infection, nous avons effectué une expérience de RNAseq. Comme attendu, l'infection aiguë se caractérise par une importante modulation du transcriptome de l'hôte qui augmente avec le temps. A l'opposé, l'infection persistante n'induit que très peu de changements. La réponse immunitaire innée, et en particulier la voie des TLR, ainsi que l'apoptose sont très fortement activées durant une infection aiguë. Pour sa part, B. stabilis module essentiellement les gènes codant pour le système du complément.Le rôle critique des macrophages lors d'une infection par Bcc chez les poissons zèbre est en accord avec les récentes observations cliniques. Ceci suggère que le stade intracellulaire de B. cenocepacia et la réponse inflammatoire qui s'ensuit peuvent être des cibles pour le développement de nouvelles thérapies permettant de lutter contre cette infection. / Bacteria belonging to the Burkholderia cepacia complex (Bcc) can cause chronic infection with periods of acute exacerbation and sometimes fatal necrotizing pneumonia (“cepacia syndrome”) in individuals with cystic fibrosis (CF), and are associated with poor prognosis. Here, we exploited the exciting possibilities for in vivo non-invasive imaging of Bcc infection in transparent zebrafish embryos, with an innate immune system with remarkable similarity to that of humans, and numerous genetic and genomic tools to study the role of host phagocytes and the innate immune response in the pro-inflammatory character of the infection.We show that macrophages play a critical role in intracellular multiplication of B. cenocepacia K56-2 and induction of a MyD88-dependent fatal inflammatory response, characterised by high levels of cxcl8 and il1b expression. Surprisingly, in sharp contrast to the situation found for infections with other pathogens including Mycobacterium marinum and Staphylococcus aureus, in the absence of macrophages, K56-2 survived but was unable to replicate in the first 24 h, which resulted in a significant pro-survival advantage to the host compared to wild type embryos that died within 2 to 3 days. The Toll-like receptor (TLR) pathway is a major arm of the cell-mediated innate immune response with MyD88 as a key adaptor protein involved in the production of pro-inflammatory cytokines. We found that the absence of MyD88 also provided a pro-survival effect to the embryos after infection with K56-2. Paradoxically, the bacteria replicated better in myd88-/- mutant than wild type embryos, suggesting that it is not bacterial burden per se, but the inflammatory response that kills the embryos. Interestingly, cxcl8 and il1b expression were not significantly induced during the first 7 hours in the myd88-/- mutant while a strong induction was seen in control embryos, suggesting that a Myd88-dependent inflammatory response during early macrophage stages significantly contributes to fatal infection.Next, we performed RNAseq to analyse global changes in host gene expression during acute and persistent infection induced by K56-2 and B. stabilis LMG14294 respectively. Whereas acute infection was characterised by strong modulation of host gene expression increasing over time, persistent infection showed modulation of only a small set of genes. TLR and apoptosis signaling pathways were amongst the strongly activated groups during acute infection, in line with the strong inflammatory character of K56-2. During persistent infection, the major differentially expressed gene set concerned genes encoding complement proteins. The critical role for macrophages in Bcc infection in zebrafish is in agreement with recent clinical observations. We suggest that the intracellular stages of B. cenocepacia and the ensuing inflammatory response are essential targets to explore for the development of new therapies to combat this infection.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22767号 / 工博第4766号 / 新制||工||1745(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 米田 稔, 教授 清水 芳久, 准教授 松井 康人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24222号 / 工博第5050号 / 新制||工||1788(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 米田 稔, 教授 松井 康人, 准教授 松田 知成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.

Mechanical manipulation and characterization of biological cells have wide applications in genetics, reproductive biology, and cell mechanics. This research focuses on (1) the development of enabling microrobotic systems and techniques for automated cell microinjection and in situ mechanical characterization; and (2) the demonstration of molecule efficacy testing and cell quality assessment with the new technologies. Targeting high-speed cell injection for molecule screening, a first-of-its-kind automated microrobotic cell injection system is developed for injecting foreign materials (e.g., DNA, morpholinos, and proteins) into zebrafish embryos (~1.2 millimeter) and mouse oocytes/embryos (~100 micrometers), which overcomes the problems inherent in manual operation, such as long learning curves, human fatigue, and large variations in success rates due to poor reproducibility. Novel cell holding devices are developed for immobilizing a large number of embryos into a regular pattern, greatly facilitating sample preparation and increasing the sample preparation speed. Leveraging motion control and computer vision techniques, the microrobotic system is capable of performing robust cell injection at a high speed with high survival, success, and phenotypic rates. The mouse embryo injection system is applied to molecule testing of recombinant mitochondrial proteins. The efficacy of an anti-apoptotic Bcl-xL (Delta_TM) protein is, for the first time, quantitatively evaluated for enhancing the development competence of mouse embryos. For cell quality assessment, this research develops a vision-based technique for real-time cellular force measurement and in situ mechanical characterization of individual cells during microinjection. A microfabricated elastic device and a sub-pixel computer vision tracking algorithm together resolve cellular forces at the nanonewton level. Experimental results on young and old mouse oocytes demonstrate that the in situ obtained force-deformation data can be used for mechanically distinguishing healthy mouse oocytes from those with cellular dysfunctions. This work represents the first study that quantified the mechanical difference between young and old mouse oocytes, promising a practical way for oocyte quality assessment during microinjection.