The authors are supported by projects ZeEBRA (R&#38;I-2019-018), a&#160;Technology Development Programme (TDP) fund, STRONG (R&#38;I-2024-007L), a&#160;TDPLite fund, NASDAC (SINO-MALTA-2022-08), a Science and Technology Cooperation grant, and DEMONSTRATE (R&#38;I-2018-007A), a Go2Market grant, all funded by the XjenzaMalta for and on behalf of the Foundation for Science and Technology. Figure 1 was created using BioRender.

Zebrafish were handled according to EU Directive 2010/63/EU. All experiments were approved by the Faculty Research Ethics Committee (FREC) and the Joint FREC Animal Research Sectoral Subcommittee (JFARSS) of the University of Malta. A flowchart of the proposed protocol can be found in Figure 1.
1. Egg production, collection, and maintenance
<ol>
	<li>Set up the fish in breeding tanks the day before, at least an hour after the last feed, with dividers in place separating the males and females. For better egg production results, use a higher ratio of females to males. Set the fish up in a room equipped with a 14 h:10 h light/dark cycle and at 27-28 &#176;C ambient temperature. 
	NOTE: A 1 L breeding tank can hold three adult fish, whereas a 1.7 L breeding tank can hold five adult fish.</li>
	<li>The following morning, following the onset of light, replace the water in the breeding tanks and remove the dividers. In case the breeding tank is not readily sloped, place it in a sloping position to ensure that shallow water is available to promote mating. In case of slow egg production and mating activity, replace the water in the breeding tanks every 45 min. Leave the fish to mate for up to 3 h or as required.</li>
	<li>Once mating is complete, gently squeeze any females that did not produce eggs to prevent long-term egg binding. Place the fish back into their respective tanks.</li>
	<li>Collect any eggs in the breeding tanks using a mesh strainer, rinse with embryo water (E3), and place in a Petri dish with E3. To prepare E3, mix&#160;16.67 mL of 60x E3 (300 mM NaCl; 10 mM KCl; 20 mM CaCl2.2H2O; 20 mM MgSO4.7H2O) with&#160;1 mL of 0.01% methylene blue and 982 mL of deionized water. Remove any unfertilized eggs or debris. Make sure the Petri dishes are not crowded, with 50 eggs per 100 mm x 15 mm Petri dish and 120 eggs per 150 mm x 15 mm Petri dish.</li>
	<li>Keep the eggs in an incubator set to 28.5 &#176;C and equipped with a 14 h:10 h day/night cycle. At 1 dpf, refresh the E3 and remove non-viable eggs to limit fungal growth.</li>
</ol>
2. Test plate preparation
<ol>
	<li>At 3 dpf, transfer one healthy-looking, hatched embryo to each well in a 96-well plate using a 1,000 &#181;L micropipette set to a 100 &#181;L volume. 
	NOTE: Non-hatched embryos should not be used, as this may result in variable compound uptake. Using embryos that appear healthy also limits bias during testing. The size of the pipette tip allows for easy collection of the zebrafish embryo without causing any physical damage. In addition, a micropipette allows for better control of the volume being dispensed into the well as opposed to a transfer pipette.</li>
	<li>In a 12-well plate, prepare the desired concentration(s) for each test chemical in separate wells (here, Bosutinib at a concentration range from 100 &#181;M to 6.25 &#181;M). In each well, add the necessary volume of test chemical using micropipettes and make up to 2.4 mL using E3. 
	NOTE: A literature search should be carried out to determine the adequate concentrations to use for the relevant test chemicals. If no information is available, several concentrations can be trialed until a suitable concentration range is determined. A minimum of five concentrations should be used, with the highest concentration causing 100% mortality and the lowest concentration causing no observable effect7.</li>
	<li>Prepare a vehicle control depending on the solvent used to dissolve the stock test chemical. 
	NOTE: Many test compounds are not water soluble and for this reason, they need to be dissolved in specific solvents (&#34;vehicles&#34;) such as ethanol or dimethyl sulfoxide (DMSO) to generate an aqueous solution. DMSO should preferably not exceed a concentration of 1% when used as a vehicle control, which is the concentration found to be safe in zebrafish14.</li>
	<li>Remove any E3 solution present in the 96-well plate using a 200 &#181;L micropipette and replace it with 100 &#181;L of the corresponding medium being tested. Be sure to test a total of 24 larvae per concentration. Use a single or 8-channel 200 &#181;L micropipette to transfer the test chemicals to the corresponding test plate wells. Include controls (e.g., E3, chemical vehicle, positive controls, etc). 
	NOTE: The plate setup accommodates up to four different test chemicals or four different concentrations of the same chemical. An example of the test plates set up for the investigation of five concentrations of the same chemical (e.g., Bosutinib) accompanied by controls is shown in Figure 1.</li>
	<li>At 24 h post exposure, perform morphological scoring (as described in section 3) and a halfmedium change, removing 50 &#181;L of medium and replacing it with freshly prepared medium. Remove and record any dead larvae in the process. Keep the larvae in an incubator set to 28.5 &#176;C and equipped with a 14 h:10 h day/night cycle. Repeat the process until 7 dpf.</li>
</ol>
3. Morphological assessment
<ol>
	<li>Using a stereo light microscope, observe the individual test larvae at an appropriate magnification. 
	NOTE: Examples of morphological defects that can be assessed are provided in Table 1.</li>
	<li>Have the morphology assessed by two independent scorers, with a third one being consulted in case of disagreements between the two scorers.</li>
	<li>Score the morphology using a binary method (i.e., present/absent), where &#39;absent&#39; indicates normal morphology (scored as &#39;0&#39;) and &#39;present&#39; indicates the presence of morphological abnormality (scored as &#39;1&#39;). Figure 2 shows examples of zebrafish at 5 dpf after 2 days of drug exposure, including an unaffected zebrafish (Figure 2A), a zebrafish with mild pericardial edema (Figure 2B), severe pericardial edema with yolk hemorrhage and yolk extension (Figure 2C), delayed development with reduced pigmentation, swim bladder and total length (Figure 2D), swollen yolk and a kinked caudal fin (Figure 2E), absent caudal fin (Figure 2F), curved notochord (Figure 2G), truncation (Figure 2H) and death with necrosis (Figure 2I). Score all abnormal phenotypes as &#39;1&#39; and the unaffected zebrafish as &#39;0&#39;.</li>
</ol>
4. LC50 determination
<ol>
	<li>Following morphology assessment, plot the cumulative percentage mortality at 7 dpf against concentration. An example is shown in Figure 3. Use the concentration curve to estimate the concentration that causes 50% mortality.</li>
	<li>Once the LC50 concentration is estimated, repeat the experiment described above by exposing 24 replicate larvae in a 96-well plate to this single estimated LC50 concentration.</li>
</ol>
5. Technical replicates
<ol>
	<li>Repeat the entire procedure using the selected five concentrations of the same chemical at least two more times on two separate occasions to ensure that the results are reproducible.</li>
</ol>

<ol>
	<li>Teame, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+(Danio+rerio)+as+biomedical+models.">The use of zebrafish (Danio rerio) as biomedical models.</a> Anim Front. 9 (3), 68-77 (2019).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Patton, E. E., Zon, L. I., Langenau, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+disease+models+in+drug+discovery:+From+preclinical+modelling+to+clinical+trials.">Zebrafish disease models in drug discovery: From preclinical modelling to clinical trials.</a> Nat Rev Drug Discov. 20 (8), 611-628 (2021).</li><li>Clift, D., Richendrfer, H., Thorn, R. J., Colwill, R. M., Creton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+behavior+in+zebrafish+larvae:+Effects+of+feeding.">High-throughput analysis of behavior in zebrafish larvae: Effects of feeding.</a> Zebrafish. 11 (5), 455-461 (2014).</li><li>Keenan, S. R., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+phases+of+zebrafish+myogenesis.">The developmental phases of zebrafish myogenesis.</a> J Dev Biol. 7 (2), 12 (2019).</li><li>Tonelli, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+resourceful+vertebrate+model+to+investigate+skeletal+disorders.">Zebrafish: A resourceful vertebrate model to investigate skeletal disorders.</a> Front Endocrinol (Lausanne). 11, 489 (2020).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Test no. 236: Fish embryo acute toxicity (FET). , (2013).</li><li>Chen, J. R., Lai, Y. H., Tsai, J. J., Hsiao, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+fluorescent+staining+platform+for+drug-screening+and+mechanism-analysis+in+zebrafish+for+bone+mineralization.">Live fluorescent staining platform for drug-screening and mechanism-analysis in zebrafish for bone mineralization.</a> Molecules. 22 (12), 2068 (2017).</li><li>He, H., Wang, C., Tang, Q., Yang, F., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+mechanisms+of+prednisolone-induced+osteoporosis+in+zebrafish+larva.">Possible mechanisms of prednisolone-induced osteoporosis in zebrafish larva.</a> Biomed Pharmacother. 101, 981-987 (2018).</li><li>Huang, H. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+bone+transgenic+zebrafish+in+anti-osteoporosis+chemical+screening.">Application of bone transgenic zebrafish in anti-osteoporosis chemical screening.</a> Animal Model Exp Med. 1 (1), 53-61 (2018).</li><li>Haney, M. G., Moore, L. H., Blackburn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+of+primary+patient+derived+tumor+xenografts+in+zebrafish.">Drug screening of primary patient derived tumor xenografts in zebrafish.</a> J Vis Exp. (158), e60996 (2020).</li><li>Nishimura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+zebrafish+in+systems+toxicology+for+developmental+toxicity+testing.">Using zebrafish in systems toxicology for developmental toxicity testing.</a> Congenit Anom (Kyoto). 56 (1), 18-27 (2016).</li><li>Hayot, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+toxicity+of+chemicals+using+a+zebrafish+vibration+startle+response+screening+system.">Evaluating toxicity of chemicals using a zebrafish vibration startle response screening system.</a> J Vis Exp. (203), e66153 (2024).</li><li>Hoyberghs, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DMSO+concentrations+up+to+1%+are+safe+to+be+used+in+the+zebrafish+embryo+developmental+toxicity+assay.">DMSO concentrations up to 1% are safe to be used in the zebrafish embryo developmental toxicity assay.</a> Front Toxicol. 3, 804033 (2021).</li><li>Lipton, J. H., Br&#252;mmendorf, T. H., Sweet, K., Apperley, J. F., Cortes, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+considerations+in+the+management+of+patients+treated+with+bosutinib+for+chronic+myeloid+leukemia.">Practical considerations in the management of patients treated with bosutinib for chronic myeloid leukemia.</a> Ann Hematol. 103 (9), 3429-3442 (2024).</li></ol>

The authors declare no conflicts of interest.

Zebrafish have become an important model for drug screening. When trying to determine a methodology to carry out toxicity testing, we found several contrasting methods from different laboratories8,9,10,11,12,13. The methods described use larvae at different ages, variable experimental timelines, different rates of medium changes, and different morphology scoring systems. Through trial and error, we established the described methodology, which has provided reliable and reproducible results.
Choosing a plate size 
Initially, experiments were performed in a 24-well plate to ensure that the larvae had enough space to move and grow. The plates were arranged with 10 larvae per well, two wells per concentration, and 1 mL of drug solution per well. This arrangement did not provide reproducible results, leading to the inference that the larvae were not absorbing equal amounts of drug from the well. Trials were then carried out using a 96-well and 48-well plate with one larva per well, which showed that larvae in 96-well plates displayed no issues with growth. Hence, experiments were continued in a 96-well plate utilizing less space and drug volumes.
Daily medium change 
Since zebrafish are absorbing the drug from the medium, we ascertained that a degree of medium change is needed to keep a consistent concentration of the drug in the medium. Moreover, a medium change removes potential waste material accumulated in the well, which can provide a toxic environment and lead to biased results. A trial was carried out with Bosutinib, where three plates were set up for the same drug, one without a daily medium change, one with a daily half medium change, and one with a full daily medium change. While the plate with no medium change exhibited increased mortality rates, the plates with half and full medium changes resulted in the same outcome by 7 dpf. Thus, a daily half medium change was chosen. This choice ensures less consumption of the expensive and limited test material, which could be required for further downstream experiments.
Exposure duration 
The age of test larvae used and drug exposure duration selected in this protocol was driven by our phenotype of interest being bone mineralization. Since bone formation commences at ~4 dpf, we chose to start with zebrafish at 3 dpf to determine whether test chemicals affected the onset of bone development. Subsequently, we ended the drug exposure studies at 7 dpf since a high degree of mortality was observed from 8 dpf onwards in view of the lack of feeding that might bias the results. Nevertheless, timeframes may be adjusted depending on the nature of the study and phenotype of interest.
Limitations 
This methodology has some limitations. Transferring 3 dpf zebrafish to multi-well test plates is time-consuming. Removing the medium from the wells without picking up the larvae is equally laborious. The medium needs to be removed from each well slowly using a single-channel pipette, ensuring that the larvae are not damaged in the process. Moreover, while a daily half medium change replenishes the test compound and ensures the removal of waste material from the well, it does not guarantee that a stable drug concentration can be maintained in the well throughout the 5 days of exposure. While high-performance liquid chromatography analysis could give further insight into the actual amount of drug being absorbed by each larva, the test&#160;would no longer qualify as a high-throughput screening method.
In conclusion, this methodology aims to target the gap in toxicity testing using zebrafish beyond 4 dpf, taking steps towards a standardized protocol applicable to wild-type, mutant, and transgenic zebrafish lines, and that is on par with the FET at the embryonic stage. This opens avenues for other downstream phenotyping assays that might not be possible at earlier stages.

Zebrafish (Danio rerio) have recently emerged as a powerful vertebrate model organism, becoming increasingly&#160;popular for studies to advance biomedical research1. Around 70% of human protein-coding genes have at least one zebrafish ortholog, with ~82% of human disease-causing genes having at least one orthologous gene in zebrafish2. The genetic conservation, coupled with similar basic organ patterning and morphology, make zebrafish an interesting model for human disease research,allowing for cross-species comparisons at the molecular level1,2,3, which is crucial for drug development.
Zebrafish develop rapidly with the tail bud forming at 10 h post fertilization (hpf) and the first somites appearing at 16 hpf. The embryos emerge from the chorion at ~2-3 days post fertilization (dpf). By 4 dpf, their digestive tract is fully developed and by 5 dpf, their swim bladder has inflated, allowing them to swim around freely and hunt for food. Despite the ability to feed by 5 dpf, larvae that are unfed can survive solely on yolk-derived nutrients until at least 7 dpf, circumventing the need for external feeding that may cause contamination and interfere with drug screening, toxicology, and behavioral testing4. By 7 dpf, larvae also develop a complete body plan with several rudimentary organ systems, including the heart and vasculature, muscle, and bone5,6. Zebrafish embryos are also fertilized ex vivo, developing externally, and are optically transparent in the early life stages, making them highly accessible for gene editing and screening of small molecules3. Additionally, their small size and high fecundity rate allow for the inclusion of more replicates, which increases statistical power, as well as for the use of multi-well plates, which saves on drug volumes and increases throughput.
Screening of novel small molecules in an animal model is important to determine the drugs&#39; ability to produce both beneficial or adverse effects, thus identifying which compounds are viable for further testing and others that might require modification to alter their activity. As part of the drug screening process, the lethal concentration 50 (LC50) of the target compound/s needs to be identified. This provides a working concentration that causes death in 50% of test animals as part of the preliminary toxicity testing.
A standardized protocol exists for toxicity testing in zebrafish embryos up to 96 h post fertilization (hpf), namely the Fish Embryo Acute Toxicity (FET) test (TG236)7. In the FET test, newly fertilized eggs are exposed to five concentrations of a chemical for up to 96 h and observed for signs of lethality, namely (i) egg coagulation, (ii) lack of somite formation, (iii) lack of tail bud detachment from the yolk sac, and (iv) lack of a heartbeat. The test allows for the determination of acute toxicity and the LC50. Furthermore, it also advises on test chamber sizes to use for screening (24-well plates), egg distribution in the test chamber, and housing conditions, amongst others, ensuring valid and reproducible results7.
While the FET test is suitable for toxicity testing at the embryonic stage, it does not go beyond 4 dpf. For phenotypes that start to develop at around this time point (e.g., mineralization of the notochord6), drug screening must proceed further than 4 dpf to enable the morphological assessment of such tissue. Moreover, we recommend that drug exposure commences at least a day before the development of the phenotype of interest. To our knowledge, no standardized protocol similar to the FET exists for drug screening in early-stage zebrafish larvae, specifically in zebrafish aged from 3 to 7 dpf. While several studies have been published describing drug testing in zebrafish larvae beyond 4 dpf, there is a lack of consistency in the methodology utilized, including the size of the test chambers, number of biological and technical replicates, rate of media changes, exposure durations, and morphological defects assessed8,9,10,11,12,13. For this reason, we developed a validated protocol for standardized toxicity testing followed by visual observation of morphological changes in zebrafish larvae at 3-7 dpf. The proposed protocol can subsequently be utilized for other downstream phenotyping assays, such as behavioral analysis, gene expression profiling, and histomorphometry.

<table><tbody><tr><td>1.5 mL microcentrifuge tube</td><td>Starlab</td><td>E1415-1510</td><td></td></tr><tr><td>10 &amp;micro;L micropipette (P10)</td><td>Gilson</td><td>F144802&amp;nbsp;</td><td></td></tr><tr><td>10/20 &amp;micro;L pipette tips</td><td>Starlab</td><td>S1110-3810</td><td></td></tr><tr><td>1000 &amp;micro;L micropipette (P1000)</td><td>Gilson</td><td>F123602&amp;nbsp;</td><td></td></tr><tr><td>1000 &amp;micro;L pipette tips</td><td>Starlab</td><td>S1111-6001</td><td></td></tr><tr><td>12-well plate</td><td>Starlab</td><td>CC7672-7512</td><td>Sterile, single-wrapped, non-treated, with lid</td></tr><tr><td>20 &amp;micro;L micropipette (P20)</td><td>Gilson</td><td>F123600&amp;nbsp;</td><td></td></tr><tr><td>200 &amp;micro;L micropipette (P200)</td><td>Gilson</td><td>F123601&amp;nbsp;</td><td></td></tr><tr><td>200 &amp;micro;L multi-channel pipette&amp;nbsp;</td><td>Gilson</td><td>F81024</td><td></td></tr><tr><td>200 &amp;micro;L pipette tips</td><td>Starlab</td><td>S1113-1006</td><td></td></tr><tr><td>96-well plate</td><td>Starlab</td><td>CC7672-7596</td><td>Sterile, single-wrapped, flat-bottom, non-treated, with lid</td></tr><tr><td>Breeding tanks with inside grid sloping bottom, tank dividers and lids</td><td>Tecniplast</td><td>ZB17BTISLOP, ZB17BTE,&amp;nbsp; ZB17BTL, ZB17BTD</td><td>Sloped inner tank</td></tr><tr><td>Mesh strainer</td><td>/</td><td>/</td><td></td></tr><tr><td>Nitrile gloves</td><td>Mercator</td><td>/</td><td>/</td></tr><tr><td>Petri-dish&amp;nbsp;</td><td>Starlab</td><td>CC7672-3394/CC7672-3359</td><td>100 x 20 mm or 60 x 15 mm</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Leica M205 FCA Fluorescence stereo microscope</td><td>Leica</td><td>10450826</td><td></td></tr><tr><td>Peltier-cooled incubator including 2 shelves with light module cold white</td><td>Memmert</td><td>IPP110ecoplus, T8</td><td>Equipped with day/night cycle</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Biochem Chemopharma</td><td>504341000</td><td>To prepare a 1% solution in E3</td></tr><tr><td>Calcium chlorided dihydrate</td><td>Biochem Chemopharma</td><td>303080500</td><td>To prepare E3</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Biochem Chemopharma</td><td>313060500</td><td>To prepare E3</td></tr><tr><td>Potassium Chloride</td><td>Biochem Chemopharma</td><td>316030500</td><td>To prepare E3</td></tr><tr><td>SCREEN-WELL&amp;nbsp;Wnt Pathway library (Bosutinib)</td><td>Enzo</td><td>BML-2838-0500</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S/3161/53</td><td>To prepare E3</td></tr></tbody></table>

small molecule screening and toxicity testing in early-stage zebrafish larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

The abnormalities can be looked at individually or cumulatively over the experimental timeline until 7 dpf. The maximum morphology score per abnormality can be used to calculate morphological abnormalities as a percentage of the number of larvae used. For example, if on day 7 the pericardial edema morphology scores are at 19 and 20 for scorers 1 and 2, respectively, out of a possible score of 24, meaning 19 or 20 larvae out of 24 display pericardial edema, the percentage score can be calculated as:
<img alt="Equation 1" src="/files/ftp_upload/67759/67759eq1.jpg" />
Supplementary Table S1 shows the morphology scoring results (of both scorers) at 4 dpf following toxicity testing for the compound Bosutinib, a tyrosine kinase inhibitor indicated for the treatment of chronic myeloid leukemia15. The same morphology scoring is repeated for exposed fish aged from 5 dpf to 7 dpf. The compound was tested using a concentration range from 100 &#181;M to 6.25 &#181;M. Three technical replicates were conducted at different time points, and the average cumulative mortality rate at 7 dpf was used to determine LC50, the concentration at which 50% of the larvae survive. The LC50 can be determined at any exposure time point. However, we recommend calculating this at 7 dpf.
The cumulative average mortality at 7 dpf was used to generate an LC50 curve, which can be seen in Figure 3. For Bosutinib, the LC50 concentration was estimated to be 37.95 &#181;M.
NOTE: This estimated LC50 concentration strictly applies to this model system and can then be used for further downstream phenotyping assays.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67759/67759fig1.jpg" /> 
Figure 1: Schematic of the proposed drug screening in larvae at 3-7 dpf. The 96-well test plates are set up for the investigation of five concentrations ranging from 100 &#181;M to 6.25 &#181;M for the compound Bosutinib. Abbreviation: dpf = days post fertilization. <a href="https://www.jove.com/files/ftp_upload/67759/67759fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67759/67759fig2.jpg" /> 
Figure 2: Representative morphological defects observed in zebrafish larvae at 5 dpf exposed to various chemical concentrations (at 2 days post exposure). (A) Control (unaffected) zebrafish, (B) Zebrafish larva showing mild pericardial edema, (C) Zebrafish with severe pericardial edema and yolk hemorrhage, (D) Zebrafish with developmental delay, reduced pigmentation, small swim bladder, (E) Zebrafish with swollen yolk and a kinked caudal fin, (F) Zebrafish with absence of caudal fin, (G) Zebrafish with curved notochord,&#160;(H) Zebrafish with truncated tail, and (I) Dead zebrafish with necrosis. Defects are noted with red arrowheads. Scale bars = 1 mm. <a href="https://www.jove.com/files/ftp_upload/67759/67759fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67759/67759fig3.jpg" /> 
Figure 3: LC50 curve showing concentration against % mortality. The five concentrations tested were 100 &#181;M, 50 &#181;M, 25 &#181;M, 12.5 &#181;M, and 6.25 &#181;M, with % mortality being 100%, 100%, 29.2%, 12.5%, and 12.5%, respectively. <a href="https://www.jove.com/files/ftp_upload/67759/67759fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Morphological Defects</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>Pericardial edema</td>
			<td>Fluid buildup around the heart with a stretched heart and malfunctioning of the blood circulation</td>
		</tr>
		<tr>
			<td>Heart beats (per minute)</td>
			<td>Reduced number of beats per minute&#160;</td>
		</tr>
		<tr>
			<td>Yolk</td>
			<td>Presence or absence of yolk edema or hemorrhage that can occur simultaneously with cardiac defects&#160;</td>
		</tr>
		<tr>
			<td>Yolk extension</td>
			<td>Yolk which is swollen, thin or absent&#160;</td>
		</tr>
		<tr>
			<td>Curved notochord</td>
			<td>Presence of a kinked or undulating notochord, and/or curved tail&#160;</td>
		</tr>
		<tr>
			<td>Swim bladder</td>
			<td>Presence, absence or partial development of the swim bladder</td>
		</tr>
		<tr>
			<td>Pigmentation</td>
			<td>Less or no melanocytes compared to the untreated fish of the same age</td>
		</tr>
		<tr>
			<td>Necrosis</td>
			<td>Presence (mild or severe) or absence of necrosis in any part of the body</td>
		</tr>
		<tr>
			<td>Delayed development</td>
			<td>Delayed development or stalled compared to wild-type controls of the same age.&#160;</td>
		</tr>
		<tr>
			<td>Response to touch</td>
			<td>Delayed or no response following a startle (e.g., using a pipette tip)</td>
		</tr>
		<tr>
			<td>Truncation</td>
			<td>Absence of posterior zebrafish body (i.e. formation of the tail)</td>
		</tr>
		<tr>
			<td>Fins</td>
			<td>Presence or absence of the caudal fin that may be accompanied by thin yolk extension</td>
		</tr>
		<tr>
			<td>Reduced body axis</td>
			<td>Fish that appear normal but when measured are shorter in length</td>
		</tr>
	</tbody>
</table>
Table 1: A list of visual morphological defects that can be observed following drug exposure studies.
Supplementary Table S1: Representative morphological results for Bosutinib at 4 dpf (1 day post exposure).&#160;<a href="https://www.jove.com/files/ftp_upload/67759/SupplementalTable_1.xlsx" target="_blank">Please click here to download this table.</a>

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

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Research

JoVE Journal

Biology

1.1K Views.  University of Malta. Verzamel om te beginnen zebravisembryo's in een vroeg stadium drie dagen na de bevruchting in een petrischaaltje. Met een micropipet van 1000 microliter op een volume van 100 microliter, brengt u een gezond ogend uitgekomen embryo over in elk putje van een plaat met 96 putjes. Piket nu de benodigde hoeveelheid van de teststof in elk putje van een plaat met 12 putjes.Pas vervolgens het totale volume aan op 2.4 milliliter met E3-medium. Bereid de voertui...

Video: Screening van kleine moleculen en toxiciteitstesten bij zebravislarven in een vroeg stadium

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Immunology and Infection

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Cancer Research

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Environment

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Medicine

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Embryo-Based Chemical Toxicity Screen: Assessing Effects on Developing Zebrafish Embryos

Source: Hao J. et al., <a href="https://www.jove.com/v/2243/" target="_blank">Large Scale Zebrafish-Based In vivo Small Molecule Screen</a>. J. Vis. Exp. (2010)
In this article, zebrafish embryos are used for chemical toxicity screening. Chemical toxicity is correlated with phenotype readouts. The sample protocol shows a methodological approach of performing the chemical toxicity screening.

Source: Hao J. et al., Large Scale Zebrafish-Based In vivo Small Molecule Screen. J. Vis. Exp. (2010)
In this article, zebrafish embryos are used for ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

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