In Situ Hybridization Assay‐Based Small‐Molecule Screening in Zebrafish

Lili Jing1,2, Ellen M. Durand1,2, Catherine Ezzio3, Stephanie M. Pagliuca4, Leonard I. Zon1,5

1 Stem Cell Program and Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, 2 These authors contributed equally to this work., null, null, 3 Muhlenberg College, Allentown, Pennsylvania, 4 Department of Evolutionary Anthropology, Duke University, Durham, North Carolina, 5 Howard Hughes Medical Institute, Chevy Chase, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110236
Online Posting Date:  June, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

In vitro biochemical and cell‐based small‐molecule screens have been widely used to identify compounds that target specific signaling pathways, but the identified compounds frequently fail at the animal testing stage, largely due to the in vivo absorption, metabolism, and toxicity properties of the chemicals. Zebrafish has recently emerged as a vertebrate whole‐organism model for small‐molecule screening. The in vivo bioactivity and specificity of compounds are examined from the very beginning of zebrafish screens. In addition, zebrafish is suitable for large‐scale chemical screens, similar to many cellular assays. This protocol describes an approach for in situ hybridization (ISH)‐based chemical screening in zebrafish, which, in principle, can be used to screen any gene product. The described protocol has been used to identify small molecules affecting specific molecular pathways and biological processes. It can also be adapted to zebrafish screens with different readouts. Curr. Protoc. Chem. Biol. 4:143‐160 © 2012 by John Wiley & Sons, Inc.

Keywords: zebrafish; in situ hybridization; small molecule screen; drug discovery; in vivo

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Zebrafish Embryo Collection
  • Basic Protocol 2: Zebrafish Embryo Preparation and Chemical Treatment
  • Basic Protocol 3: In Situ Hybridization Staining of Chemical‐Treated Embryos
  • Support Protocol: Synthesis of an Antisense RNA Probe
  • Alternate Protocol: Chemical Treatment of Embryos in 96‐Well Mesh Filter Plates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Zebrafish Embryo Collection

 Materials
  • Zebrafish (a wild‐type strain or a specific mutant strain, depending on the purpose of the screen)
  • E3 buffer (see recipe)
  • Breeding vessel (iSPAWN, Tecniplast)
  • Sieve
  • 100‐ or 150‐mm petri dishes

Basic Protocol 2: Zebrafish Embryo Preparation and Chemical Treatment

 Materials
  • Embryos (collected from Basic Protocol 1) plated several hours before the desired stage, considering the timing of transferring and plating embryos
  • E3 buffer (see recipe)
  • Chemical library (e.g., Chembridge DIVERSet)
  • Dimethyl sulfoxide (DMSO)
  • Pronase (Roche, cat. No. 11‐459‐643‐001; dissolve in E3 buffer to 2.5 mg/ml)
  • PBT (see recipe)
  • 4% PFA (see recipe)
  • Methanol
  • Microscope (e.g., Zeiss Stereo Discovery.V8)
  • Transfer pipets (Fisher Scientific, cat. no. 13‐711‐5AM)
  • Multi‐well plates (48‐well flat‐bottom plates, Falcon, cat. no. 353078, or 96‐well flat‐bottom plates, Costar, cat. no. 3596)
  • Multi‐channel vacuum wand (optional, e.g., from V& P Scientific)
  • Multichannel pipets
  • Liquid handling robot, optional
  • Parafilm

Basic Protocol 3: In Situ Hybridization Staining of Chemical‐Treated Embryos

 Materials
  • Embryos, fixed and dehydrated (Basic Protocol 2)
  • Methanol
  • PBT (see recipe)
  • Proteinase K solution (Roche, cat. no. 03115828001; dilute in PBT to 10 µg/ml before use)
  • 4% PFA (see recipe)
  • 25% glutaraldehyde (Sigma, cat. no. G4004)
  • Hybe+ (see recipe)
  • DIG‐labeled RNA probe (Support Protocol)
  • Hybe– (see recipe)
  • 2× SSC (see recipe)
  • 0.2× SSC (see recipe)
  • Blocking solution (see recipe)
  • Anti‐DIG‐AP antibody (Roche, cat. no. 11 093 274 910)
  • Prestain solution (see recipe)
  • Staining buffer (see recipe)
  • Stop solution (see recipe)
  • 96‐well mesh filter plate and 96‐well receiver plate (Millipore, cat. no. MANM10010)
  • 1.5‐cup air‐tight container (Lock n' Lock, 4.3 × 5.9 × 1.8–in.)
  • Transfer pipets (Fisher Scientific, cat. no. 13‐711‐5AM)
  • Belly dance rotator (STOVALL Life Science)
  • 70°C incubator (Thermo Electron)
  • Aluminum foil
  • Spot plates in white porcelain, 12 cavities (Fisher, cat. no. S337241)
  • Parafilm
  • Microscope (Zeiss Stereo Discovery.V8)
  • 96‐well round‐bottomed plate (BD, cat. no. 353227)

Support Protocol: Synthesis of an Antisense RNA Probe

 Materials
  • Vector with the cDNA template for the probe (vector should contain a T3, T7, or SP6 RNA polymerase promoter around the cDNA insert)
  • Restriction enzyme digestion reagents (New England Biolabs)
  • Agarose
  • 1× TBE (see recipe)
  • Phenol:chloroform:isoamyl alcohol (25:24:1; Sigma, cat. no. P2069)
  • Chloroform:isoamyl alcohol (24:1; Sigma, cat. no. C0549)
  • 3 M sodium acetate pH 5.5 (Ambion, cat. no. 9740)
  • Ethanol
  • 70% (v/v) ethanol in DEPC H2O
  • DEPC H2O (see recipe)
  • DIG RNA labeling mixture including:
    • 10× transcription buffer (Roche, cat. no. 14735400)
    • NTP DIG labeling mix labeling mixture (Roche, cat. no. 11277073910)
    • Appropriate RNA polymerase (T3, Promega, cat. no. P207C; T7, Promega, cat. no. P207B; SP6 Promega, cat. no. P207A)
    • RNAse inhibitor (RNasein, Promega, cat. no. N2511, 40 U/µl)
    • RNase‐free DNase I (Roche, cat. no. 04716728001, 10 U/µl)
  • 1 M lithium chloride
  • 1.5‐ml microcentrifuge tubes
  • Electrophoresis apparatus
  • Centrifuge
  • Vortex mixer
  • −20°C freezer
  • NanoDrop spectrophotometer (Thermo Scientific, NanoDrop 2000)
  • 37°C incubator

NOTE: To avoid contamination, be sure to use clean tips and make transfers into clean tubes. Unless they are incubating, keep tubes on ice as much as possible.

Alternate Protocol: Chemical Treatment of Embryos in 96‐Well Mesh Filter Plates

 Additional Materials (also see Basic Protocols 2 and 3)
  • 96‐well mesh filter plate (Pion, cat. no. PN 120655)
  • 96‐well receiving plate (Millipore, cat. no. MATRNPS50)
  • Multiscreen single‐well culture tray (Millipore, cat. no. MAMCS0110)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •  FigureFigure 1. Flow chart showing the procedures involved in the zebrafish chemical screen with in situ hybridization (ISH) as a read‐out. Abbreviations: O/N, overnight; AP, alkaline phosphatase.
    View Image
  •  FigureFigure 2. Schematic describing the operation of the breeding vessel (iSpawn). (A) Insert the mesh spawning platform into the vessel and fill the vessel with water. Place male fish above the spawning platform. (B) Place the mesh separator into the vessel so male fish are confined in the bottom half of the iSpawn. (C) Add female fish above the separator. Allow the fish to prime overnight. (D) The next morning, remove the separator so male and female fish mix in the deep water. (E) Raise the spawning platform so that fish are in shallow water. Allow the fish to spawn for desired time (around 10 min). (F) Remove the spawning platform and the fish, to stop spawning. Place a strainer (or a sieve) at the bottom of the vessel. Open the valve and drain the water into the strainer. Collect the fallen embryos.
    View Image
  •  FigureFigure 3. Representative images of (A) normal embryos, (B) unfertilized embryos, and (C) dead embryos at 6‐hr post‐fertilization.
    View Image
  •  FigureFigure 4. Schema showing 48‐ and 96‐well plate layouts for chemical treatment and ISH. (A) For 48‐well plate, columns 1 and 8 can be treated with vehicle (DMSO), the negative control, or positive controls. The rest of the wells can be filled with test chemicals. (B) When two 48‐well plates are combined into one 96‐well mesh filter plate for ISH, one plate of samples (rotated 90°) can be plated on the left side of the 96‐well plate, and the other plate can be plated on the right side of the 96‐well plate. If a 96‐well plate is used in the chemical treatment, rows A and H can be used for negative and positive controls.
    View Image
  •  FigureFigure 5. ISH of neural crest progenitor marker (crestin) in 24 hpf embryos following chemical treatment. (A) DMSO is used as a control for crestin expression. (B) Treatment with NSC210627 completely abrogated crestin levels. (C) Based on structural similarities, the authors tested leflunomide, which was not found in the original chemical library, but displays robust reduction in crestin expression. Adapted from White et al. (2011).
    View Image

Videos

Literature Cited

Literature Cited
    Adatto, I., Lawrence, C., Thompson, M., and Zon, L.I. 2011. A new system for the rapid collection of large numbers of developmentally staged zebrafish embryos. PLoS One 6:e21715.
    Goessling, W., Allen, R.S., Guan, X., Jin, P., Uchida, N., Dovey, M., Harris, J.M., Metzger, M.E., Bonifacino, A.C., Stroncek, D., Stegner, J., Armant, M., Schlaeger, T., Tisdale, J.F., Zon, L.I., Donahue, R.E., and North, T.E. 2011. Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long‐term safety in preclinical nonhuman primate transplant models. Cell Stem Cell 8:445‐458.
    Kaufman, C.K., White, R.M., and Zon, L. 2009. Chemical genetic screening in the zebrafish embryo. Nat. Protoc. 4:1422‐1432.
    Kokel, D., Bryan, J., Laggner, C., White, R., Cheung, C.Y., Mateus, R., Healey, D., Kim, S., Werdich, A.A., Haggarty, S.J., Macrae, C.A., Shoichet, B., and Peterson, R.T. 2010. Rapid behavior‐based identification of neuroactive small molecules in the zebrafish. Nat. Chem. Biol. 6:231‐237.
    Molina, G., Vogt, A., Bakan, A., Dai, W., Queiroz de Oliveira, P., Znosko, W., Smithgall, T.E., Bahar, I., Lazo, J.S., Day, B.W., and Tsang, M. 2009. Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages. Nat. Chem. Biol. 5:680‐687.
    Murphey, R.D., Stern, H.M., Straub, C.T., and Zon, L.I. 2006. A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chem. Biol. Drug Des. 68:213‐219.
    North, T.E., Goessling, W., Walkley, C.R., Lengerke, C., Kopani, K.R., Lord, A.M., Weber, G.J., Bowman, T.V., Jang, I.H., Grosser, T., Fitzgerald, G.A., Daley, G.Q., Orkin, S.H., and Zon, L.I. 2007. Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447:1007‐1011.
    Patton, E.E. and Zon, L.I. 2001. The art and design of genetic screens: zebrafish. Nat. Rev. Genet. 2:956‐966.
    Peterson, R.T., Shaw, S.Y., Peterson, T.A., Milan, D.J., Zhong, T.P., Schreiber, S.L., MacRae, C.A., and Fishman, M.C. 2004. Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation. Nat. Biotechnol. 22:595‐599.
    Stern, H.M., Murphey, R.D., Shepard, J.L., Amatruda, J.F., Straub, C.T., Pfaff, K.L., Weber, G., Tallarico, J.A., King, R.W., and Zon, L.I. 2005. Small molecules that delay S phase suppress a zebrafish bmyb mutant. Nat. Chem. Biol. 1:366‐370.
    Thisse, C. and Thisse, B. 2008. High‐resolution in situ hybridization to whole‐mount zebrafish embryos. Nat. Protoc. 3:59‐69.
    White, R.M., Cech, J., Ratanasirintrawoot, S., Lin, C.Y., Rahl, P.B., Burke, C.J., Langdon, E., Tomlinson, M.L., Mosher, J., Kaufman, C., Chen, F., Long, H.K., Kramer, M., Datta, S., Neuberg, D., Granter, S., Young, R.A., Morrison, S., Wheeler, G.N., and Zon, L.I. 2011. DHODH modulates transcriptional elongation in the neural crest and melanoma. Nature 471:518‐522.
    Yeh, J.R., Munson, K.M., Elagib, K.E., Goldfarb, A.N., Sweetser, D.A., and Peterson, R.T. 2009. Discovering chemical modifiers of oncogene‐regulated hematopoietic differentiation. Nat. Chem. Biol. 5:236‐243.
    Yu, P.B., Hong, C.C., Sachidanandan, C., Babitt, J.L., Deng, D.Y., Hoyng, S.A., Lin, H.Y., Bloch, K.D., and Peterson, R.T. 2008. Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism. Nat. Chem. Biol. 4:33‐41.
    Zon, L.I. and Peterson, R.T. 2005. In vivo drug discovery in the zebrafish. Nat. Rev. Drug Discov. 4:35‐44.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library