Technologies bringing young Zebrafish from a niche field to the limelight

Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context

Recent Advances in Bioimage Analysis Methods for Detecting Skeletal Deformities in Biomedical and Aquaculture Fish Species

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Optimization of the spontaneous tail coiling test for fast assessment of neurotoxic effects in the zebrafish embryo using an automated workflow in KNIME®

Neuroactive chemicals are frequently detected in the environment. At sufficiently high concentrations or within mixtures, they could provoke neurotoxic effects and neurological diseases to organisms and humans. Fast identification of such neuroactive compounds in the environment could help in hazard assessment and risk mitigation. Behavior change is considered as an important endpoint and might be directly or indirectly connected to a neuroactive mode of action. For a fast evaluation of environmental samples and pure substances, we optimized the measurement of a behavioral endpoint in zebrafish embryos - the spontaneous tail coiling (STC). Evaluation of results is automated via the use of a workflow established with the KNIME® software. Analysis duration and developmental stage were optimized to 1 min and 25 ± 1 hpf respectively during measurement. Exposing the embryos in a group of 10 or 20 and acclimatizing for 30 min at room temperature proved to be reliable. The optimized method was used to investigate neurotoxic effects of 18 substances with different modes of action (MoA). The STC test accurately detected the effect of 8 out of 11 neuroactive substances (chlorpyrifos, chlorpyrifos-oxon, diazinon, paraoxon-methyl, abamectin, carbamazepine, propafenone and diazepam). Aldicarb and nicotine showed subtle effects which were considered to be conditional and imidacloprid showed no effect. For substances with unknown neuroactive MoA, 3 substances did not provoke any effect on the STC (pyraclostrobin, diuron and daunorubicin-hydrochloride) while 4 other substances provoked an increased STC (hexaconazole, aniline, dimethyl-sulfoxide and 3,4-dichloroaniline). Such unexpected effects indicate possible neuroactive side effects or unknown mechanisms of action that impact on the STC. In conclusion, the optimized STC parameters and the automated analysis in KNIME® indicate opportunities for the harmonization of the STC test and further development for prospective and diagnostic testing

Adverse effects in the fish embryo acute toxicity (FET) test : a catalogue of unspecific morphological changes versus more specific effects in zebrafish (Danio rerio) embryos

Funding Open Access funding enabled and organized by Projekt DEAL. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grand agreement No 681102.Publisher PD

Pattern Recognition in High-Throughput Zebrafish Imaging

High Throughput (HT) methods are high volume experimental approaches that are common in the fields of the life-sciences. The instrumentation for these methods differs per application. We will focus on the HT methods that are concerned with imaging. The aim of this thesis is to find robust methods for object extraction and analysis. We focus on the Computer Science aspects of such analysis, namely pattern recognition. Pattern Recognition can be seen in the context of object recognition and data mining. Both aspects will be described in this thesis. We present a framework for segmenting and recognizing the objects of interest based on Template Matching. This approach was designed for an application in the HT screening of zebrafish embryos. All proposed methods are fully automated. We further elaborate on the segmentation algorithms to apply these in software that can be used in a HT context to derive measurements. Then we apply the software on a real life problem involving zebrafish infected with Mycobacterium marinum.SmartmixComputer Systems, Imagery and Medi

HCT116 colorectal and MCF7 brest cancer cell lines xenografted into zebrafish embryos gives insight into the importance of microenviroment in tumor growth and metastasis for a future use of the model in clinical research

Zebrafish has emerged as one of the best models to characterize different human diseases due to his genetic similarity (75% of orthologous genes), and more specific, cancer. For this reason, zebrafish has been used in cancer research by means of the xenograft technique (injection of human cancer cells in embryos or adults of this model organism). The objective of this technique is simulate a human-like microenvironment inside a model organism in order to study the disease development with a fastest and accurate approach. As long as the xenograft technique has been implemented in zebrafish, mentioned before, there has been improvements to this technique and the conditions surrounding it. Nevertheless, there are some bottle necks with the necessity of being addressed with the objective of getting a more robust and informative technique in order to establish it in a near future in the personalized medicine field

Validation of the biological responses of reference drugs in the zebrafish embryo by electrocardiographic analysis and by novel phenotyping tools

Drug toxicities represent a major problem in drug discovery and development; therefore there is a push to develop new technologies to detect these early on. In this thesis I investigated the utility of zebrafish embryos and larvae in evaluating the biological activity of novel compounds and developed new methods for assaying the potential toxic effects of drugs $in$ $vivo$. An electrocardiogram (ECG) recording set-up for zebrafish embryos and larvae was developed to assay drug-induced cardiotoxicity. The set-up was validated by testing drugs known to induce cardiotoxicity in humans in zebrafish larvae. The results obtained were in agreement with those documented in humans demonstrating the utility of the zebrafish larva in detecting drug-induced cardiotoxicity. The zebrafish embryo was also found to be a useful model for probing the biological activity of novel and marketed compounds providing an insight into the relationship between chemical properties and biological effects. Additionally, the assessment of the anti-inflammatory activity of a set of reference drugs revealed that the zebrafish larva also presents a promising model for therapeutic drug screens. Overall, the results described in this thesis show that the zebrafish presents an effective, reliable and rapid model for assessing the biological activity of drugs $in$ $vivo$

Reinventing microinjection : new microfluidic methods for cell biology

Regulatory processes are responsible for the organization, division and death of cells in multicellular organisms such as humans. Additionally, cells are highly regulated internally, able to survive and respond in vastly different micro-environments. Many types of interactions of cells with their environment can be distinguished, and need to be controlled in experiments aimed at unravelling and predicting cellular behavior in vivo. The in vivo microenvironment is mimicked by exposing cells to complex and changing environments. To describe the stochastic differences between cells and the local experimental conditions in sufficient detail and to obtain statistically relevant results, high-throughput experimentation is required. In this thesis four new research methods are developed, aimed at a deeper understanding of cellular regulation in vivo.Märzhäuser, ZF-screens BV, Eppendorf, Life Science Methods BVUBL - phd migration 201

Hybrid Microfluidic Devices For On-Demand Manipulation and Screening of Neurons and Organs of Small Model Organisms

Caenorhabditis elegans and Drosophila melanogaster are widely used model organisms for neurological and cardiac studies due to their simple neuronal and cardiac systems, genome similarity to humans, and ease of maintenance in laboratories. However, their 50m-1mm sizes and continuous mobility impede their precise spatiotemporal manipulation, thereby, reducing the throughput of biological assays. By integrating glass capillaries into microfluidic devices and using 3D-printed fixtures for precise control, we have developed hybrid lab-on-a-chip devices to facilitate the processes of animal manipulation and stimuli control, using modules for single-organism selection, orientation, imaging and chemical stimulation. These microdevices enabled us to manipulate organisms individually and to orient them at any desired direction for imaging purposes. The applications of these hybrid microdevices were demonstrated in the optical and fluorescent imaging of C. elegans cells as well as cardiac screening of Drosophila larvae. This technique can be applied in fundamental biology, toxicology, and drug discovery

Identification of novel targets in prostate cancer progression

We have developed novel fluorescence bio-imaging based automated models to screen for novel candidate targets involved in prostate cancer metastasis. Utilizing these models and adopting a functional genomics based approach; we identified SYK as a novel regulator of prostate cancer progression. We also identified functional involvement of MST1R in regulating the progression of prostate cancer. For both of these targets, there is supporting human clinical data to validate our results in prostate cancer.UBL - phd migration 201