Zebrafish embryos and Larvae : a new generation of disease model and drug screens
Ali, S.
Citation
Ali, S. (2011, December 7). Zebrafish embryos and Larvae : a new generation of disease model and drug screens. Retrieved from https://hdl.handle.net/1887/18191
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/18191
Note: To cite this publication please use the final published version (if applicable).
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Summary of thesis
The zebrafish (Danio rerio) is small, cheap to maintain, rapid to develop and has high fecundity.
Its early-stages embryos have a transparent body, making it relatively easy to assemble abundant datapoints using high-quality imaging. Annual maintenance expenditure for adult zebrafish are to some extent lower than those for rodents. However, this cost benefit is enormously multiplied when the test animal is a zebrafish embryo, because a female zebrafish can lay as many as 10,000 eggs per annum. For these and other reasons, zebrafish embryos have been projected as an in vitro animal model which could bridge the gap between simple assays based on cell or tissue culture, and biological validation in whole animals such as rodents. The zebrafish embryo has emerged as a powerful tool to address the unmet need in biomedical research for low-cost, high-throughput whole-animal assays and models. In vitro assays offer the advantages of low cost, of being less prone to permissible and ethical limitations and of having the capability to be scaled-up. By contrast, whole-animal assays make available the data that are more easily extrapolated to humans and allow complex organismal functions (e.g. behavior and development) to be studied. We argued in this thesis that zebrafish embryos and early larvae can serve as invaluable screening tools in the pre-regulatory, preclinical phase of drug discovery and drug safety in humans, and therefore zebrafish disease models have been developed. They can be used as a kind of sieve that reduces the number of compounds passing through to testing on the much more high-priced rodent models.
We established that our employ of acute, stage-specific exposure of embryos to ethanol allowed stage-dependent and stage-independent effects to be identified and allowed sensitive periods to be detected. The effects imitate key aspects of fetal alcohol syndrome (FAS) including
174 craniofacial abnormality, microphthalmia, growth retardation and behavioral impairment. We also identified a critical time window (prim-6 and prim-16) for ethanol sensitivity and implicate cell death in the postmigratory cranial neural crest as a possible mechanism. This is in contrast with other studies that implicate the migratory crest. Furthermore, our detection of a wide phenotypic spectrum is reminiscent of human FAS, and may provide a useful model for studying disease resilience. In the future, our large scale approach could also make it potential to identify candidate genes conferring protection against ethanol effects in the minority of individuals that show resilience.
A custom lab-on-a-chip for zebrafish embryos was developed by us and manufactured by Micronit Microfluidics. We used micro particle imaging velocimetry to characterize the flow patterns of nutrient buffer in the chip. We further used thermal imaging to determine the temperature stability across the chip when in use. Phenotypic teratology screening was used to determine whether or not the embryos developed normally in the biochip. An acute ethanol exposure test was used to see if embryos developing within the chip could be used to replicate drug or chemical assays used in traditional 96-well embryo cultures.The ability to culture vertebrate embryos in a lab-on-chip is the holy grail of next-generation
microfluidics, because current techniques — Petri dishes and microtitre plates — were
developed for cell culture, not for the in vitro culture of embryos. Our discovery of microfluidic embryo culture could revolutionize human IVF, drug discovery and biomedical research, and could allow rats and mice to be replaced in many areas of research with zebrafish embryos.
We demonstrated for the first time that an animal embryo could develop in a glass microfluidic flow-through environment. Zebrafish embryos grown in the biochip commonly showed minor phenotypic effects — including the possible stress phenotype of dispersed melanocytes.
However, there was no increase in gross malformations. Our scoring of the former as ‘minor’
175 defects is of course subjective, and does not rule out the possibility of significant but undetected effects. There was a strong, non-linear relationship between buffer flow-rate and 5 day embryo survival in the biochip, such that the most favourable flow rate was in the range 2.0-4.0
µL/well/min. Survival rates at 5 day reached 100% in two chip runs at 2.0 µL/well/min. These results could lead to a new generation of assays for the pharmaceutical industry based on the low-cost, microfluidic culture of zebrafish embryos.
We calculated zebrafish LC50 of 60 compounds demonstrating a range of chemical classes and toxicological mechanisms and their statistical comparison with published data on rodent LD50
(log mmol/kg). The data sets were found to be strongly correlated (using Kendall’s rank
correlation tau and Pearson’s product-moment correlation). The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly
influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa..
Our findings showed that the zebrafish embryo is a tool that offers potential in the evaluation of drug safety. However, we showed that the predictivity varies between the class of compound studied. These findings could be useful in relation to the use of zebrafish in possible predictive model in toxicity testing and toxicity screening
The zebrafish embryo/larva has been shown to be applicable to the high-throughput behavioral screening of compound libraries. We have analysed 60 water-soluble toxic compounds covering a range of common drugs, toxins and chemicals, and representing various pharmacological mechanisms. We used the visual motor response test, in which movement of individual larvae was analysed using automated video-tracking. For all compounds, LC50 values were found to decrease as the embryo developed. The majority of compounds (57/60) produced an effect in both the basal and challenge phases. These effects were either (i) suppression of locomotor
176 activity (monotonic concentration-response); (ii) stimulation then suppression (biphasic
response); (iii) stimulation (monotonic response). We conclude that behavioural assays with zebrafish embryos could be useful for pharmaceutical efficacy and toxicity screening. The precise readout obtained with behavioural assay varies with compound class. Our findings show that behavioural recording on zebrafish embryo can provide a sensitive evaluation of toxicity of a wide range of compounds. It is also possible that this type of assay could be useful for
assessing efficacy, that is, possible therapeutic effects. Thus it could, in principle, also provide aid in the discovery of new drugs for the human disease treatment. The methodology is complementary to traditional toxicity assays and offers a improvement of the traditional endpoint method relying on mortality at a distinct life stage. The addition of a physiology-based approach increases the sensitivity of our toxicity assays by detecting potential neurotoxicity at dosage that could not be detected with traditional LC50 studies. This assay is amenable to high- throughput capability and can be implemented early in the drug discovery pipeline for early evaluation of drug safety.
