Materials and Reagents

1. 150 mm dish (Corning, catalog number: 430599)

2. 24-well flat bottom plastic plates (Corning, catalog number: CLS3473)

3. Wild-type zebrafish strain (AB line)

4. Tg (kdrl:eGFP) zebrafish (Provided by Dr. Stainier)

5. FDA-, EMA-, and other agencies-approved chemical libraries (Prestwick Chemical, Illkirch, France)

6. Niclosamide (Sigma-Aldrich, catalog number: N3510)

7. DMSO (Sigma-Aldrich, catalog number: D8418)

8. Anti-E-cadherin antibody (Cell Signaling Technology, catalog number: 14472)

9. Anti-vimentin antibody (Cell Signaling Technology, catalog number: 5741)

10. Anti-HTR2C antibody (Abcam, catalog number: ab133570)

11. Anti-GAPDH antibody (Cell Signaling Technology, catalog number: 2118)

12. Human breast cancer cell line MCF7 (ATCC, catalog number: HTB-22)

13. NaCl (Sigma-Aldrich, catalog number: S3014)

14. KCl (Sigma-Aldrich, catalog number: P9541)

15. MgSO₄·7H₂O (Sigma-Aldrich, catalog number: M2773)

16. E3 medium (see Recipes)

Equipment

1. External tank, a part of zebrafish breeding tank (Tecniplast, catalog number: ZB10BTE)

2. Perforated internal tank, a part of zebrafish breeding tank (Tecniplast, catalog number: ZB10BTI)

3. Polycarbonate divider, a part of zebrafish breeding tank (Tecniplast, catalog number: ZB10BTD)

4. Polycarbonate lid (Tecniplast, catalog number: ZB10BTL)

5. Incubator (AQUALYTIC, catalog number: 2418210)

6. Stereomicroscope (Leica, catalog number: MZ75)

Procedure

Zebrafish mating setup (Day 0); 10 min

1. At the night before collecting embryos, arrange pairs of male and female zebrafish separated by a divider (Figure 5).

   
   

   
   Figure 5. Zebrafish breeding tank with a divider. Male and female zebrafish are separated by a divider which prevents contact for mating. The images are viewed from overhead angle (left) and from the side (right). The divider is then removed, which allows male zebrafish to contact female zebrafish for mating.

   
   

   Note: Young adult zebrafish (3–9 months old) should be used for the crossing. The quality of zebrafish embryos affects screening efficiency (critical step). Water temperature is 27 °C. Duration of sunshine is set from 7:00 to 19:00.

   
   

Embryo collection and distribution (Day 1); 80 min

2. Remove the divider to allow the fish to spawn in the morning (7:00–10:00).

3. Allow crossing for 10 min to obtain zebrafish embryos of the same developmental stage. For example, if more than 20 chemicals are tested, the crossing might be performed three times at three different time points (Group A: 8:30, Group B: 9:00, and Group C: 9:30).

   For example, a 60-drug screening would require approximately 1,300 embryos if each drug needs to be tested at one concentration:

   Group A: 20 (embryos) × 1 (concentration) × 30 (drugs) + 20 (vehicle control) + 20 (positive control).

   Group B: 20 (embryos) × 1 (concentration) × 30 (drugs) + 20 (vehicle control) + 20 (positive control).

   Similarly, if each drug needs to be tested at two concentrations, approximately 2,600 embryos would be required:

   Group A: 20 (embryos) × 2 (concentrations) × 30 (drugs) + 40 (vehicle controls) + 20 (positive control).

   Group B: 20 (embryos) × 2 (concentrations) × 30 (drugs) + 40 (vehicle controls) + 20 (positive control).

4. After 10 min, replace the divider to prevent the zebrafish from spawning.

   Note: This screen measures the suppressor effect of each chemical on the progression of epiboly in live zebrafish embryos. Therefore, epiboly proceeds while the researcher is measuring the effect under a stereoscopic microscope. If more than 20 chemicals need to be tested, the screening should be divided into more than two sessions, and each of the sessions should start at a different time point. For example, if 60 chemicals need to be tested, zebrafish should be crossed at three different time points that are over 30 min apart. That allows 30 min for measuring the effects (critical step).

5. Collect the embryos in 150 mm dishes containing E3 medium and remove dead embryos.

6. Incubate the embryos at 27 °C for 20 min.

7. Collect two-cell stage embryos under the stereoscopic microscope.

   Note: This step has limited throughput. The collection of zebrafish embryos should be completed during the two-cell stage. Thus, 900–1,200 embryos at the two-cell stage would be the upper limit for one researcher.

8. Array approximately 20 embryos into each well of a 24-well plate.

9. Remove E3 medium from each well by using a pipet.

10. Add 900 µL of E3 medium to each well.

    
    

Embryo development to the sphere stage; 4 h

11. Incubate the embryos at 27 °C until they develop to the sphere stage

    Note: The temperature of the E3 medium affects the development rate of zebrafish embryos. Higher temperatures accelerate the development rate; conversely, lower temperatures slow it down (Urushibata et al., 2021). Therefore, a non-uniform temperature in each well of a 24-well plate can cause false positives (critical step).

    
    

Addition of chemicals; 30 min

12. Prepare a 10-fold concentration of each chemical in E3 medium 30 min before adding the chemicals to the embryos.

    Note: After preparing the respective 10-fold concentration of the media, the medium should be stored at 27 °C.

13. When the embryos develop to the sphere stage, add 100 µL of the respective 10-fold media to the wells.

    For example, a 60-drug screening is divided into three groups:

    1. The first set of 20 test chemicals, as well as niclosamide and DMSO as positive and negative controls, are added into group A when embryos from group A develop to the sphere stage.

    2. The second set of 20 test chemicals, niclosamide, and DMSO are added into group B when embryos from group B develop to the sphere stage.

    3. The last set of 20 test chemicals, niclosamide, and DMSO are added into group C when embryos from group C develop to the sphere stage.

       
       

Development of DMSO-treated embryos to the 90% epiboly stage; 5 h

14. After adding the test chemicals, incubate the embryos at 27 °C for approximately 5 h.

    Note: The temperature of the E3 medium affects the development rate of zebrafish embryos. Non-uniform temperature in each well of a 24-well plate would cause false positives (critical step).

    
    

Measuring the inhibition effects of each chemical; 30 min

15. Compare the epiboly progression of chemical-treated embryos from group A and DMSO-treated embryos under the stereoscopic microscope, when the DMSO-treated embryos develop to the 90% epiboly stage.

    Note: This step has limited throughput. Comparisons should be completed before DMSO-treated embryos at the 90% epiboly stage develop to the next development stage. Thus, 20–30 chemicals would be the upper limit for one researcher.

16. Compare the epiboly progression of chemical-treated embryos from group B and DMSO-treated embryos under the stereoscopic microscope, when the DMSO-treated embryos develop to the 90% epiboly stage.

17. Compare the epiboly progression of chemical-treated embryos from group C and DMSO-treated embryos under the stereoscopic microscope, when the DMSO-treated embryos develop to the 90% epiboly stage.

    Note: Epiboly proceeds while a researcher is comparing the epiboly progression of chemical-treated and DMSO-treated embryos under the stereoscopic microscope. Therefore, measuring the effect should be completed in 30 mins (critical step). To confirm the reproducibility of whether 'hit' chemicals could interrupt the epiboly progression of zebrafish embryos, further testing on subsequent day is advised (critical step).

Data analysis

This screen measures the suppressor effect of chemicals based on the epiboly progression of zebrafish embryos. Niclosamide and DMSO can be used as positive and negative controls, respectively. Epiboly progression of chemical-treated embryos is compared with that of DMSO-treated embryos under a stereoscopic microscope, allowing positive ‘hit’ chemicals that interrupt epiboly progression to be selected (Figure 2) (Nakayama et al., 2021b, 2022).

Limitations

Throughput in steps 7 and 15, determine how many chemicals can be tested in one screening session. In step 7, the collection of zebrafish embryos should be completed during the two-cell stage and before the four-cell stage. Thus, 900–1,200 embryos at the two-cell stage would be the upper limit for one researcher. In step 15, comparing epiboly progression of each chemical-treated embryo with DMSO-treated embryos under the stereoscopic microscope should be completed before DMSO-treated embryos at the 90% epiboly stage develop to the next stage. Thus, 20–30 chemicals would be the upper limit for one researcher.

In addition, there are some limitations regarding chemical deliverance to zebrafish embryos. These are surrounded by the acellular chorion, which is known to be approximately 1.5–2.5 µm thick and to consist of three layers pierced by pore canals. The pore allows passage of water, ions, and chemicals. A study reported that molecules larger than 3–4 KDa fail to pass through the chorion. Therefore, this screen may not be able to measure the suppressor effect of molecules larger than 3–4 KDa (Pelka et al., 2017).

Troubleshooting

The quality of zebrafish embryos affects screening efficiency. For example, low-quality embryos show high frequencies of asymmetric cell cleavage, and their development is arrested at early cleavage stages (Yilmaz et al., 2017). If a screen used low-quality zebrafish embryos, it would generate false ‘hit’ chemicals since the suppressor effect of a test chemical is measured by observing the epiboly progression of chemical-treated embryos. If the number of zebrafish embryos showing morphological abnormalities correlate with the final concentration of a test chemical, those embryo abnormalities may result from the effect of the test chemical.

Anticipated results

Suppressor effects of a tested chemical on the epiboly progression of zebrafish embryos are significantly affected by the final concentration of the chemical. A previous study subjected 1,280 FDA-approved drugs to this screen and showed that 6% (78/1,280) of the tested drugs affected epiboly progression of the embryos treated with 10 μM. Out of these 78 epiboly-interrupting drugs, 25% of the drugs succeeded in suppressing cell motility and invasion of highly metastatic human cancer cells in a Boyden chamber assay. In contrast, epiboly progression was affected more severely when the embryos were treated at 50 μM, by 10.3% (132/1,280) of the tested drugs. From these, 85% (112/132) failed to suppress cell motility and invasion of highly metastatic human cancer cells in a Boyden chamber assay (Nakayama et al., 2021b).

Recipes

1. E3 medium

   5.0 mM NaCl, 0.17 mM KCl, 0.33 mM MgSO₄

   Reagent	Final concentration	Amount
   NaCl	5.0 mM
   KCl	0.17 mM
   MgSO4	0.33 mM
   H2O	n/a	1,000 mL

Acknowledgments

Figure 1 was drawn by Ami Inoue (Kyoto University of the Arts). Pictures of zebrafish mating tank with a divider were taken by Dr. Li Yan (National University of Singapore). This study was funded by grants from National Medical Research Council of Singapore (R-154000547511) and Ministry of Education of Singapore (R-154000A23112 and R154000B88112) to ZG. Experiments using mice were supported in part by research funds from the Yamagata prefectural government and the City of Tsuruoka. This screen protocol was used in previous research (Nakayama et al., 2021a, 2021b, 2022).

Competing interests

J.N., H.M., and Z.G. declare no conflict of interest.

Ethics

The study protocol using zebrafish was approved by the Institutional Animal Care and Use Committee of the National University of Singapore (protocol number: R16-1068). The study protocol using mice (protocol number: BRC IACUC #110612) was approved by A*STAR (Agency for Science, Technology and Research, Singapore).

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