Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism

Dose-effect analysis of QT and non-QT prolongation inducers.

<p>Four QT prolongation inducers (Terfenadine, Cisapride, Astemizole and Haloperidiol) and 2 non-QT prolongation inducers (Digitoxin and Nitrendipine) were tested on 48 hpf zebrafish embryos. Predominant observed effects are indicated: normal heart rate (N), bradycardia (B), 2∶1 arrhythmia (A), ventricle failure (VF), heart failure (HF).</p

Image analysis of the heart rate.

<p>The figure shows the analysis of the heart rate of a representative control (A), arrhythmic heart (B) and cardiac arrest (C). OF and ED algorithms are applied for the analysis of each video. Only when the OF is above a specific threshold FFT is applied to ED. Control and arrhythmic hearts are classified based on the number of the significant frequencies detected in the FFT, one for control (A) two or more for arrhythmic (B). (C) OF representative of a dead embryo. In FFTs, Periodogram is shown in blue, Original Fourier Transform of the ED signal in black and Heartbeat is shown in red.</p

Two different phenotypes induced by angiogenesis inhibitors.

<p>24 hpf zebrafish embryos were treated with different inhibitors and checked for angiogenesis defects at 48 hpf (A) or at 72 hpf (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036690#s4" target="_blank">material and methods</a> section. (A) Representative fluorescent images of embryos treated with inhibitors that alter the development of ISVs at the level of sprouting and extension (superior panel) or that perturb later stages of maturation and/or DLAV formation (lower panel). Compared with control ones, treated embryos present a lower number of incomplete ISVs in the case of Sorafenib and AG1478 while AG1296 and PD173074 inhibit proper development of the DLAV and the ISVs looks thinner. (B) Representative fluorescent images of embryos treated with the indicated inhibitors showing the presence of thinner ISVs, some of them disrupted (arrows) and interrupted DLAV formation (asterisks).</p

Coefficient of Variation of the angiogenesis assay.

<p>Calculation of the Coefficient of Variation of the indicated parameters calculated from data obtained after 3 independent experiments carried out with the positive compounds shown at one dose close to the IC50 or the effective concentration.</p

Images analysis for ISVs formation.

<p>The figure shows the different steps in the process of image analysis for the determination of the anti-angiogenesis effect. Control embryo (A, B, C and D); embryo treated with KRN633 (A’, B’, C’ and D’). (A) and (A’) are the original images acquired by the microscope; (B) and (B’) are images showing the segmentation between the head and the tail of the embryo to separate the ROI and measure the length; (C) and (C’) are images corresponding to an intermediate step in the filtering process where all the vessels in the tail can be identified; (D) and (D’) are images presenting the area enclosed between ISVs and DLAVs (in white) that are the ones to be quantified.</p

HTS platform layout.

<p>The figure shows all the components of the platform: master PC, embryo sorter, two liquid handlings (LH1 and LH2), plate hotel, incubator and reader. The robotic arm in the central part transports the plate to all the devices.</p

Screening of 34 small bioactive compounds.

<p>The table shows the compounds used for validation, their family and their cardiotoxic effects assessed in humans and zebrafish.TN (true negative), TP (true positive), FN (false negative), FP (false positive).</p