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BACKGROUND AND AIMS Focal segmental sclerosis (FSGS) is a severe histopathological condition which often results in end-stage renal disease. Loss of podocytes and severe changes of podocyte foot process morphology accompanied by proteinuria are the hallmarks of FSGS. Since no therapeutic agents are available until now, further efforts must be made to identify new drugs to treat this disease. The zebrafish larva is a widely used kidney research model as the morphology and function of zebrafish and mammalian glomeruli are very similar. Since our group has already established a FSGS-like zebrafish injury model based on the nitroreductase-metronidazole cell ablation system, we have developed a high-throughput based in vivo drug screening assay. METHOD We used a zebrafish screening strain that expresses mCherry and the bacterial enzyme nitroreductase in podocytes. Additionally, a circulating vitamin-D-binding eGFP fusion protein with a size of 78 kDa is expressed and serves as a read-out for proteinuria. Incubation with 80 µM metronidazole for 24 h at 4 days post-fertilization (dpf) induces an FSGS-like phenotype in these larvae. A library of 138 epigenetic drugs were screened and the results are presented herein. Groups of 12 larvae received injury induction as well as drug treatment and were transferred individually into 96-well plates prepared with custom agarose molds. Larvae were oriented laterally and imaged with a high-content screening microscope (Acquifer Imaging Machine). The eGFP signal in the caudal vasculature as well as the glomerular mCherry signal served as readout systems. At 6 dpf, the same larvae and regions were imaged again. Custom written FIJI codes automatically segmented and measured the fluorescence intensities of both readouts and timepoints. For each larva, intensity ratios were determined and the ratios of each treatment group served as the input for statistical analysis. RESULTS Treatment for 24 hours with 80 µM metronidazole resulted in a measurable decline of the eGFP signal in the vasculature and of the podocyte mCherry signal compared to the vehicle (DMSO) group. Simultaneous treatment of metronidazole with the 138 drugs in the epigenetics library resulted in beneficial outcomes in varying degrees (9 drugs were positive, 36 compounds were lethal). CONCLUSION This new in vivo semi-automated high-throughput compatible drug screening assay allows rapid identification of FSGS protective drugs. Here we screened 138 epigenetic drugs, which led to the identification of 9 potential drugs which have had a beneficial effect on podocytes and/or the glomerular filtration barrier.

Chronic kidney disease (CKD) is a major public health burden affecting more than 500 million people worldwide. Podocytopathies are the main cause for the majority of CKD cases due to pathogenic morphological as well as molecular biological alterations of postmitotic podocytes. Podocyte de-differentiation is associated with foot process effacement subsequently leading to proteinuria. Since currently no curative drugs are available, high throughput screening methods using a small number of animals are a promising and essential tool to identify potential drugs against CKD in the near future. Our study presents the implementation of the already established mouse Glom

Quantitative measurements and qualitative description of scientific images are both important to describe the complexity of digital image data. While various software solutions for quantitative measurements in images exist, there is a lack of simple tools for the qualitative description of images in common user-oriented image analysis software. To address this issue, we developed a set of Fiji plugins that facilitate the systematic manual annotation of images or image-regions. From a list of user-defined keywords, these plugins generate an easy-to-use graphical interface with buttons or checkboxes for the assignment of single or multiple pre-defined categories to full images or individual regions of interest. In addition to qualitative annotations, any quantitative measurement from the standard Fiji options can also be automatically reported. Besides the interactive user interface, keyboard shortcuts are available to speed-up the annotation process for larger datasets. The annotations are reported in a Fiji result table that can be exported as a pre-formatted csv file, for further analysis with common spreadsheet software or custom automated pipelines. To illustrate possible use case of the annotations, and facilitate the analysis of the generated annotations, we provide examples of such pipelines, including data-visualization solutions in Fiji and KNIME, as well as a complete workflow for training and application of a deep learning model for image classification in KNIME. Ultimately, the plugins enable standardized routine sample evaluation, classification, or ground-truth category annotation of any digital image data compatible with Fiji.

Despite widespread drug exposure, for example during gestation or in prematurely born children, organ-specific developmental toxicity of most drugs is poorly understood. Developmental and functional abnormalities are a major cause of kidney diseases during childhood; however, the potential causal relationship to exposure with nephrotoxic drugs during nephrogenesis is widely unknown. To identify developmental nephrotoxic drugs in a large scale, we established and performed an automated high-content screen to score for phenotypic renal alterations in theTg(wt1b:EGFP)zebrafish line. During early nephrogenesis, embryos were exposed to a compound library of approved drugs. After treatment, embryos were aligned within microtiter plates using 3D-printed orientation tools enabling the robust acquisition of consistent dorsal views of pronephric kidneys by automated microscopy. To qualitatively and quantitatively score and visualize phenotypes, we developed software tools for the semi-automated analysis, processing and visualization of this large image-based dataset. Using this scoring scheme, we were able to categorize compounds based on their potential developmental nephrotoxic effects. About 10% of tested drugs induced pronephric phenotypes including glomerular and tubular malformations, or overall changes in kidney morphology. Major chemical compound groups identified to cause glomerular and tubular alterations included dihydropyridine derivatives, HMG CoA reductase inhibitors, fibrates, imidazole, benzimidazole and triazole derivatives, corticosteroids, glucocorticoids, acetic acid derivatives and propionic acid derivatives. In conclusion, the presented study demonstrates the large-scale screening of kidney-specific toxicity of approved drugs in a live vertebrate embryo. The associated technology and tool-sets can be easily adapted for other organ systems providing a unique platform forin vivolarge-scale assessment of organ-specific developmental toxicity or other biomedical applications. Ultimately, the presented data and associated visualization and browsing tools provide a resource for potentially nephrotoxic drugs and for further investigations.

Manual annotation of images is considered the gold standard reference for many image-analysis tasks. Compared to automated solutions, manual annotation can be time-consuming and may be less reproducible due to human bias, especially with large datasets. However, for basic tasks like outlining a region-of-interest (ROI) and performing measurements, manual annotation is often faster and more efficient than developing a custom automated pipeline. To facilitate manual annotations of images, we developed a set of 1-click ROI tools for ImageJ/Fiji (Schindelin el al. 2012; Schneider el al. 2012) that can be configured with predefined shapes and trigger simple tasks upon a single mouse click. Using the 1-click tools, a single mouse click on the image generates a ROI of predefined shape and size, centered on the clicked pixel. If selected in the options, the ROI is added to the ImageJ ROI Manager, the features selected in the “Set Measurements” menu are measured for this region and appended to the Results table, and if a stack is used the next image in the stack can be automatically displayed. The dimensions of the ROI as well as the optional actions to be performed can be set in the option window that is accessible by double-clicking the icon of the 1-click ROI tools (Figure 1A, B). The full toolset comprises a line, circle, rectangle and ellipse tool. In addition to setting of ROI dimensions, the line, rectangle and elliptical tool can also be rotated (see options window – Figure 1B). The 1-click ROI tools can be used for many applications, including the quantification of intensity bands in electrophoresis gel (Figure 1C) or the annotation of cells or sub-cellular compartments. The rectangular click tool can also be used to rapidly generate bounding boxes of given dimensions, for instance to generate ground-truth annotations for object-detection algorithms (Figure 1D). The full source code is contained in a single file, entirely written in the ImageJ macro language. Therefore, it can be easily adapted for the development of custom tools. Simple customization can be achieved by providing a user-defined macro command (typically as recorded by the macro-recorder) in the option window of the tool. The custom command will be systematically executed for new ROI generated with any of the click tools. Figure 1D illustrates this option for the automated cropping of the region of interest. Thanks to the identical size of the ROI, the resulting cropped images can directly be joined into a stack and a montage after annotation. More advanced users can also modify the source code at an indicated section, to define more complex command sequences to be executed upon click. For further automation, keyboard shortcuts associated to custom macros can also be defined in the source code. The click tools can thus be used to generate the ROI, while the custom commands are only triggered upon keystroke, e.g. when the shape and position of the ROI is satisfactory. As an example, we defined a custom shortcut (keystroke ‘1’) to generate a stack and montage from the cropped images as in Figure 1D. The example shortcut is automatically functional once the click tools have been installed and selected. The source code contains simple indications to help defining and customizing shortcuts. Together with custom keyboard shortcuts, the ROI 1-click tools have the potential to dramatically reduce the time spent to manually annotate images, while potentially improving the reproducibility of the annotation and processing workflow. The toolset has a broad applicability: from the annotation of cells or nuclei, organs and tissues in model organisms, to regular patterns in material science. Availability The ROI 1-click tools can be installed in Fiji by activating the ROI 1-click tools update site. In ImageJ, the tools can be installed by copying the macro file in the subfolder “macros/toolsets” of ImageJ. Source code and documentation are available on GitHub (https://github.com/LauLauThom/Fiji-RoiClickTools). A video tutorial is available on YouTube (https://youtu.be/ZPS78T_-gUs). The gel image for figure 1C is an ImageJ sample image (credit NIH). The image for figure 1D is available on Zenodo (Gierten and Gehrig 2019).

The zebrafish is being increasingly used in biomedical research and drug discovery to conduct large-scale compound screening. However, there is a lack of accessible methodologies to enable automated imaging and scoring of tissue-specific phenotypes at enhanced resolution. Here, we present the development of an automated imaging pipeline to identify chemical modifiers of glomerular cyst formation in a zebrafish model for human cystic kidney disease. Morpholino-mediated knockdown of intraflagellar transport protein Ift172 in Tg(wt1b:EGFP) embryos was used to induce large glomerular cysts representing a robustly scorable phenotypic readout. Compound-treated embryos were consistently aligned within the cavities of agarose-filled microplates. By interfacing feature detection algorithms with automated microscopy, a smart imaging workflow for detection, centring and zooming in on regions of interests was established, which enabled the automated capturing of standardised higher resolution datasets of pronephric areas. High-content screening datasets were processed and analysed using custom-developed heuristic algorithms implemented in common open-source image analysis software. The workflow enables highly efficient profiling of entire compound libraries and scoring of kidney-specific morphological phenotypes in thousands of zebrafish embryos. The demonstrated toolset covers all the aspects of a complex whole organism screening assay and can be adapted to other organs, specimens or applications.

RationaleAccurate and efficient quantification of heartbeats in small fish models is an important readout to study cardiovascular biology, disease states and pharmacology at large scale. However, dependence on anesthesia, laborious sample orientation or requirement for fluorescent reporters have hampered the establishment of high-throughput heartbeat analysis.ObjectiveTo overcome these limitations, we aimed to develop a high-throughput assay with automated heart rate scoring in medaka (Oryzias latipes) and zebrafish (Danio rerio) embryos under physiological conditions designed for genetic screens and drug discovery and validation.Methods and ResultsWe established an efficient screening assay employing automated label-free heart rate determination of randomly oriented, non-anesthetized specimen in microtiter plates. Automatically acquired bright-field data feeds into an easy-to-useHeartBeatsoftware, a MATLAB algorithm with graphical user interface developed for automated quantification of heart rate and rhythm. Sensitivity of the assay and algorithm was demonstrated by profiling heart rates during entire embryonic development. Our analysis pipeline revealed acute temperature changes triggering rapid adaption of heart rates, which has implications for standardization of experimental layout. The approach is scalable and allows scoring of multiple embryos per well resulting in a throughput of >500 embryos per 96-well plate. In a proof of principle screen for compound testing, our assay captured concentration-dependent effects of nifedipine and terfenadine over time.ConclusionA novel workflow andHeartBeatsoftware provide efficient means for reliable and direct quantification of heart rate and rhythm of small fish in a physiological environment. Importantly, confounding factors such as anesthetics or laborious mounting are eliminated. We provide detailed profiles of embryonic heart rate dynamics in medaka and zebrafish as reference for future assay development. Ease of sample handling, automated imaging, physiological conditions and software-assisted analysis now facilitate various large-scale applications ranging from phenotypic screening, interrogation of gene functions to cardiovascular drug development pipelines.

Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies.

Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long noncoding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies.

Zebrafish embryos offer a unique combination of high-throughput capabilities and the complexity of the vertebrate animal for a variety of phenotypic screening applications. However, there is a need for automation of imaging technologies to exploit the potential of the transparent embryo. Here we report a high-throughput pipeline for registering domain-specific reporter expression in zebrafish embryos with the aim of mapping the interactions between cis-regulatory modules and core promoters. Automated microscopy coupled with custom-built embryo detection and segmentation software allowed the spatial registration of reporter activity for 202 enhancer-promoter combinations, based on images of thousands of embryos. The diversity of promoter-enhancer interaction specificities underscores the importance of the core promoter sequence in cis-regulatory interactions and provides a promoter resource for transgenic reporter studies. The technology described here is also suitable for the spatial analysis of fluorescence readouts in genetic, pharmaceutical or toxicological screens.

Early steps of embryo development are directed by maternal gene products and trace levels of zygotic gene activity in vertebrates. A major activation of zygotic transcription occurs together with degradation of maternal mRNAs during the midblastula transition in several vertebrate systems. How these processes are regulated in preparation for the onset of differentiation in the vertebrate embryo is mostly unknown. Here, we studied the function of TATA-binding protein (TBP) by knock down and DNA microarray analysis of gene expression in early embryo development. We show that a subset of polymerase II-transcribed genes with ontogenic stage-dependent regulation requires TBP for their zygotic activation. TBP is also required for limiting the activation of genes during development. We reveal that TBP plays an important role in the degradation of a specific subset of maternal mRNAs during late blastulation/early gastrulation, which involves targets of the miR-430 pathway. Hence, TBP acts as a specific regulator of the key processes underlying the transition from maternal to zygotic regulation of embryogenesis. These results implicate core promoter recognition as an additional level of differential gene regulation during development.

Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.

Background The zebrafish has been established as the main vertebrate model system for whole organism screening applications. However, the lack of consistent positioning of zebrafish embryos within wells of microtiter plates remains an obstacle for the comparative analysis of images acquired in automated screening assays. While technical solutions to the orientation problem exist, dissemination is often hindered by the lack of simple and inexpensive ways of distributing and duplicating tools. Results Here, we provide a cost effective method for the production of 96-well plate compatible zebrafish orientation tools using a desktop 3D printer. The printed tools enable the positioning and orientation of zebrafish embryos within cavities formed in agarose. Their applicability is demonstrated by acquiring lateral and dorsal views of zebrafish embryos arrayed within microtiter plates using an automated screening microscope. This enables the consistent visualization of morphological phenotypes and reporter gene expression patterns. Conclusions The designs are refined versions of previously demonstrated devices with added functionality and strongly reduced production costs. All corresponding 3D models are freely available and digital design can be easily shared electronically. In combination with the increasingly widespread usage of 3D printers, this provides access to the developed tools to a wide range of zebrafish users. Finally, the design files can serve as templates for other additive and subtractive fabrication methods.