Abstract
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.
Highlights
Besides its role as a classic model in developmental biology, the zebrafish embryo is being increasingly used in biomedical research to decipher disease-associated biological processes, with a high potential for subsequent drug discovery applications [1,2,3]
Zebrafish models for human cystic kidney diseases are characterised by large glomerular cysts in the developing pronephros that can be efficiently scored; this is often combined with other gross morphological alterations of the larval body, which can challenge the consistent data acquisition in automated imaging assays [28]
Loss of function phenotypes in zebrafish recapitulate the severe human phenotype [25,29,30]. We exploited this to generate a zebrafish model for cystic kidney disease using splice-morpholinos-based knockdown of Ift172 in the Tg(wt1b:EGFP) transgenic line, resulting in readily scorable glomerular cysts (Figure 1) [18,25]
Summary
Besides its role as a classic model in developmental biology, the zebrafish embryo is being increasingly used in biomedical research to decipher disease-associated biological processes, with a high potential for subsequent drug discovery applications [1,2,3]. The availability of thousands of transgenic and mutant lines [4] provides a compelling resource for visualising and analysing healthy and pathological biological processes in the context of a live vertebrate animal This is complemented by a vast toolbox of genetic and biochemical techniques allowing for rapid modelling of various human disease phenotypes, including, for instance, disease-associated genetic variants [5], cancer [6], infectious diseases [7] or metabolic diseases [8]. As available screening technologies are usually tailored for cell-based assays, the execution of high-throughput and high-content screening experiments employing automated microscopy remains challenging [11,12,13] This hampers the more widespread usage of zebrafish drug discovery pipelines in pre-clinical research settings
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
