Morphometric and Quantitative Behavioral Analysis of Inbred Medaka Lines

With advances in genotyping and cost-effective sequencing technologies, Genome-wide association studies (GWAS) have emerged as approaches to study the genetics of natural variation. GWAS are particularly useful when inbred lines are available (as once they are genotyped, these lines can be phenotyped multiple times) and also with the availability of automated image acquisition and analysis systems for rapid phenotyping. The objective of this thesis is to identify a variety of phenotypic traits from the inbred lines of the teleost fish Medaka (Oryzias latipes) which will then assist in the investigation of the genetic basis for such a variety. Medaka is chosen as the model organism because of the presence of still free living wild populations in Japan and East Asia and for the ability to generate new inbred strains from these wild fish. Moreover, Quantitative Trait Loci (QTL) analysis done so far on craniofacial traits in adult Medaka shows that a substantial genetic component underlies the variance seen between two inbred strains. In this study different southern and northern Japanese Medaka hatchlings at 10 days post fertilization (dpf) and 20 dpf were characterized. The focus is on the two elements that essentially define an organism: morphology and behavior. Gross morphological features were extracted and quantified using custom developed algorithms. In addition, behavioral patterns of the different inbred lines are studied since behavior provides a link and a perspective of how an organism relates to its environment. Specifically, locomotion, feeding, and prey capture behavior were analyzed and quantified. To our knowledge, this is the first characterization of prey capture behavior in Medaka. This behavior reveals interesting prey capture strategies and a comparison with a related teleost fish, the zebrafish, suggests that prey capture is not necessarily conserved. This combination of morphometric and behavioral features provides a large phenotype parameter set that will be used as a basis for genotyping to study the degree of polymorphism and to eventually establish a phenotype-genotype map for the inbred lines

Quantification Platform for Touch Response of Zebrafish Larvae using Machine Learning

A touch-evoked response of zebrafish larvae provides information of the mechanism of the gene functional expressions. Recently, an automated system has been developed for precise and repeated touch-response experimentation with minor human intervention. The data collected by the system are analyzed with regard to an automated quantification pipeline for scientific conclusions, including five quantification criteria: latency time, C-Bend curvature maximum, C-Bend peak time, response time, and moving distance. To quantify these criteria, we propose a larva tracking based automatic quantification pipeline by using a U-Net for initialization of tracking, a particle filter as tracking strategy, and region growing for the segmentation of larvae. Experimental data with different treatments are analyzed by using the proposed quantification platform for demonstration, and the result proves that this platform can generate comparable touch-response behavior quantification readouts in an efficient and automatic way. This platform provides an alternative to automatically screening the drugs for knowledge discovery according to the pattern of the touch-response behaviors of zebrafish larvae mutated by chemicals

Higginsianins A and B, two fungal diterpenoid α-pyrones with cytotoxic activity against human cancer cells

Two new diterpenoid α-pyrones, named higginsianins A and B, were isolated from the mycelium of the microbial fungus Colletotrichum higginsianum grown in liquid culture. In previous studies, we have shown that both compounds reduce viability of different types of cancer cells in culture. Here, we extend our previous observations and explore, at a deeper level, the cellular effects of higginsianins treatment. Higginisianins A and B reduce viability of A431, HeLa and H1299 cancer cells. Both compounds increase the level of the cell cycle inhibitor p21WAF and reduce the rate of cell proliferation. Cell cycle analyses reveal that higginsianins arrest cancer cells in S-phase. Furthermore, cells incubated with higginsianins reveal discrete γ-H2AX positive nuclear foci indicating the occurrence of DNA lesions. At longer incubation times, higginsianins induce massive cell detachment and non-apoptotic cell death. Human primary keratinocytes and spontaneously immortalized Hacat cells, a preneoplastic cell line model, are less sensitive to higginsianins effects. These findings suggest that higginsianins exhibit considerable cytotoxicity against a wide spectrum of malignant cells and may be considered as promising anticancer agents

An automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos

The knowledge on environmentally relevant chemicals that may interfere with thyroid signaling is scarce. Here, we present a method for the screening of goitrogens, compounds that disrupt the thyroid gland function, based on the automatic orientation of zebrafish in a glass capillary and a subsequent imaging of reporter gene fluorescence in the thyroid gland of embryos of the transgenic zebrafish line tg(tg:mCherry). The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to a negative control compound (3,4-dichloroaniline), or a concentration series of known goitrogenic compounds (resorcinol, methimazole, potassium perchlorate, 6-propyl-2-thiouracil, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on mortality and/or solubility. Exposure to 3,4-dichloroaniline decreased the fluorescence signal. All goitrogenic compounds exhibited clear concentration-dependent inductions of reporter fluorescence 1.4 to 2.6 fold above control levels. Concentration-response modelling was used to calculate goitrogenic potencies based on EC50 values. The new automated method offers an efficient screening approach for goitrogenic activity.</div

Droplet Microarray Based Screening Identifies Proteins for Maintaining Pluripotency of hiPSCs

Human induced pluripotent stem cells (hiPSCs) are crucial for disease modeling, drug discovery, and personalized medicine. Animal-derived materials hinderapplications of hiPSCs in medical fields. Thus, novel and well-defined substrate coatings capable of maintaining hiPSC pluripotency are important for advancing biomedical applications of hiPSCs. Here a miniaturized droplet microarray (DMA) platform to investigate 11 well-defined proteins, their 55 binary and 165 ternary combinations for their ability to maintainpluripotency of hiPSCs when applied as a surface coating, is used. Using this screening approach, ten protein group coatings are identified, which promote significantly higher NANOG expression of hiPSCs in comparison with Matrigel coating. With two of the identified coatings, long-term pluripotency maintenance of hiPSCs and subsequent differentiation into three germ layers are achieved. Compared with conventional high-throughput screening (HTS) in 96-well plates, the DMA platform uses only 83 µL of protein solution (0.83 µg total protein) and only ≈2.8 × 10$^5$ cells, decreasing the amount of proteins and cells ≈860 and 25-fold, respectively. The identified proteins will be essential for research and applications using hiPSCs, while the DMA platform demonstrates great potential for miniaturized HTS of scarce cells or expensive materials such as recombinant proteins

Delay in development and behavioural abnormalities in the absence of p53 in zebrafish

p53 is well-known for its tumour-suppressive activity. However, in the past decade it became clear that p53 is also involved in other processes including stem cell proliferation, differentiation and animal development. To investigate the role of p53 in early embryonic development, we targeted p53 by CRISPR/Cas9 to make a p53 knock-out zebrafish (Danio rerio). Our data show developmental and behavioural effects in p53-deficient zebrafish embryos and larvae. Specifically, we found that early development of zebrafish was clearly delayed in the absence of p53. However, after 1 day (1 dpf), the p53-deficient embryos appeared to recover, as evidenced by a similar level of pigmentation at 26 hpf, similar size of the eye at 4 dpf and only a minor difference in body size at 4 dpf compared to p53 wild-type siblings. The recovery of development after 1 dpf in p53-deficient embryos could be due to a compensatory mechanism involving other p53 family members. p63 and p73 were found over-expressed with respect to wild-type siblings. However, despite this adaptation, the hatching time remained delayed in p53$^{-/-}$ zebrafish. In addition to differences in development, p53-null zebrafish embryos also showed differences in behaviour. We observed an overall reduced activity and a reduced travel distance under non-stressed conditions and after exposing the larvae to vibration. We also observed a longer latency until the larvae started to move after touching with a needle. Overall, these data indicate that p53 is involved in early development and locomotion activities

In Vitro and In Vivo Toxicity Evaluation of Natural Products with Potential Applications as Biopesticides

The use of natural products in agriculture as pesticides has been strongly advocated. However, it is necessary to assess their toxicity to ensure their safe use. In the present study, mammalian cell lines and fish models of the zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used to investigate the toxic effects of ten natural products which have potential applications as biopesticides. The fungal metabolites cavoxin, epi-epoformin, papyracillic acid, seiridin and sphaeropsidone, together with the plant compounds inuloxins A and C and ungeremine, showed no toxic effects in mammalian cells and zebrafish embryos. Conversely, cyclopaldic and α-costic acids, produced by Seiridium cupressi and Dittrichia viscosa, respectively, caused significant mortality in zebrafish and medaka embryos as a result of yolk coagulation. However, both compounds showed little effect in zebrafish or mammalian cell lines in culture, thus highlighting the importance of the fish embryotoxicity test in the assessment of environmental impact. Given the embryotoxicity of α-costic acid and cyclopaldic acid, their use as biopesticides is not recommended. Further ecotoxicological studies are needed to evaluate the potential applications of the other compounds

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes