Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity

Abstract

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

Applications of Zebrafish Embryo Models to Predict Developmental Toxicity for Agrochemical Product Development

“…The comparatively lower sensitivity results was due to the absence of treatment-related effects observed for phenytoin in the Tg( kdrl:EGFP ) zebrafish line due to precipitation of the compound in fish media, while this compound is known to cause cardiovascular disruption in mammals. , Similar results were also observed in the zebrafish Teratotox assay when exposed to phenytoin. We and others have previously evaluated the combination of Tg( kdrl:EGFP ) fish line and image analysis for the disruption of ISVs as a proxy for vascular disruption , and reviewed in ref . While these studies are based on the structure of the ISVs, a recent study applied video subtraction to detect moving blood cells in the ISVs to also score for their function …”

“…8,37 Similar results were also observed in the zebrafish Teratotox assay when exposed to phenytoin. We and others have previously evaluated the combination of Tg(kdrl:EGFP) fish line and image analysis for the disruption of ISVs as a proxy for vascular disruption 38,39 and reviewed in ref 40. While these studies are based on the structure of the ISVs, a recent study applied video subtraction to detect moving blood cells in the ISVs to also score for their function.…”

“…While these studies are based on the structure of the ISVs, a recent study applied video subtraction to detect moving blood cells in the ISVs to also score for their function. 38 When evaluating the skeletal (bone) assay as a predictive tool for mammalian skeletal defects, accuracy was found to be 66.67% (balanced accuracy = 75.00%) with 100.00% specificity and 50.00% sensitivity. Of the four positive controls evaluated in the bone assay, two (all-trans retinoic acid and methotrexate) were considered positive hits (at least two of the highest exposure concentrations showed significant decrease in cleithrum length), while acitretin and 5-fluorouracil showed no significant decrease in cleithrum length.…”

A practical guide to bioimaging research data management in core facilities

“…They provide cross-institutional personnel capacity for bioimaging-specific data stewards who foster FAIR data sharing in collaboration with core facilities. Example studies supported by NFDI4BIOIMAGE and I3D:bio are Nöth et al 138,160 or Jannasch et al 161 We do not assume that individual core facilities must sustain data steward positions individually. However, data stewards will likely remain key to the research process at large.…”

Molecular signatures of angiogenesis inhibitors: a single-embryo untargeted metabolomics approach in zebrafish

“…Currently, developmental toxicity is mostly assessed using rats and rabbits. Yet, the zebrafish embryo (ZFE) has emerged as a promising alternative model and has shown to induce an antiangiogenic phenotype following exposure to SU4312 (Nöth et al 2024 ), sorafenib (Beedie et al 2016 ), methotrexate (Sun et al 2009 ), and rotenone (McCollum et al 2017 ). A major challenge with alternative models remains the extrapolation of findings to humans, as both exposure scenario and morphology differ between zebrafish and humans.…”