Determination of Gene Expression Patterns by Whole-Mount In Situ Hybridization Sergiy Kyryachenko, Kateryna Kyrylkova, Mark Leid, and Chrissa Kioussi Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA Keywords Whole-mount in situ hybridization, mouse embryos, digoxigenin-labeled probe, alkaline phosphataseantibody conjugate. 1 Summary Whole-mount in situ hybridization is a reliable and specific method to study three-dimensional patterns of gene expression. A labeled nucleic acid probe anneals to a complementary target sequence and is visualized and localized in the embryo. This chapter describes a sensitive method for whole-mount in situ hybridization on mouse embryos, using digoxigenin-labeled RNA probes. The technique can be used for the analysis of gene expression patterns during early stages of odontogenesis and in tooth explants. 1. Introduction Whole-mount in situ hybridization (WISH) is very powerful and widely used tool in developmental biology, because it allows the analysis of complex expression patterns of genes in whole embryos. The use of WISH is very helpful to provide a three-dimensional overview of gene expression pattern, since the three-dimensional reconstruction from serial sections can be cumbersome (1, 2). Nowadays, the method can be used to study expression of more than one gene in the same embryo, allowing spatial and temporal overlaps to be examined (0). The method uses RNA probes – molecules of RNA, which are complimentary to the endogenous mRNA and have been synthesized with a particular label. These probes are hybridized to the target RNA of an embryo, and the label can be visualized by different detecting systems (0, 0). Since the discovery of in situ hybridization, different labels and detection systems became available. For example, radio-, fluorescent-, and antigen-labeled bases are detected by autoradiography, fluorescence microscopy, or immunohistochemistry, respectively (2, 0, 0). In the protocol described here, probes are labeled with digoxigenin-11-uridine-5’-triphosphate (DIG-11-UTP) and visualized by an anti-digoxigenin conjugated to alkaline phosphatase (AP), which catalyzes a chromogenic reaction (8). The method has been adapted to different types of tissues and/or embryos. Gene expression analysis by in situ hybridization can be also used for organ cultures to study the functions of the soluble regulatory 2 molecules, which can be added directly to the culture medium or used to generate growth-factor-soaked beads. It is often informative to section whole-mount embryos or tooth explants after labeling (0, 0). Hence, this is very efficient method to study different stages of odontogenesis. However, it is more preferable to perform in situ hybridization on embryo sections for later stages of mouse development because of probe penetration issue into the whole embryo (0). In this chapter we will present a technique that has proven effective in investigating gene expression pattern during early stages of mouse tooth development. 2. Materials Prepare all solutions using ultrapure deionized water and analytical grade reagents. Store all reagents according to manufacturer’s instructions or, if not applicable, at room temperature (unless indicated otherwise). 2.1. Embryo Preparation 1. 10-cm Petri dish. 2. Dissection microscope. 3. Dissection tools: Watchmaker’s forceps. 4. Shaker. 5. 1.5 ml microfuge tubes. 6. Plastic transfer pipettes. 7. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4 in dH2O, pH 7.4 (with HCl). 8. 4% paraformaldehyde in PBS (see Note 1). 9. PBST: PBS containing 0.1% Tween 20. 10. Methanol. 3 11. 25%, 50%, and 75% methanol in PBST. 2.2. In situ hybridization 1. Rocker. 2. Hybridization oven. 3. 2 ml conical bottom O-ring screw cap tubes. 4. 5 ml polystyrene round-bottom tubes. 5. DEPC-treated dH2O (see Note 2). 6. PBST. 7. 25%, 50%, and 75% methanol in PBST. 8. 6% hydrogen peroxide in PBST. 9. Proteinase K, 10 mg/ml stock in 100 mM Tris-HCl (pH 7.5), 10 mM EDTA; store in aliquots at ˗˗20°C. 10. Glycine. 11. 0.2% glutaraldehyde / 4% paraformaldehyde in PBST; store in aliquots at -20°C. 12. SSC: 20x stock: 3 M NaCl, 0.3 M sodium citrate in DEPC-treated dH2O, pH 4.5. 13. DIG-labeled probe. 14. SDS, 10% stock. 15. Hybridization buffer (make fresh before use): 50% formamide, 5x SSC, 1% SDS, 50 µg/ml yeast tRNA, and 50 µg/ml heparin in DEPC-treated dH2O (see Note 3). 2.3. Posthybridization washes and preabsorption of antibody 1. Rocker. 2. Hybridization oven. 3. Shaker. 4 4. Microcentrifuge. 5. Vortex mixer. 6. 2 ml conical bottom O-ring screw cap tubes. 7. 5 ml polystyrene round-bottom tubes. 8. 1.5 ml microfuge tubes. 9. SSC, 20x stock. 10. SDS, 10% stock. 11. Solution 1 (Sol 1): 50% formamide, 4x SSC (pH 4.5), and 1% SDS in dH2O (see Note 3). 12. 5 M NaCl. 13. 1 M Tris-HCl, pH 8.0. 14. 25% Tween 20. 15. Solution 2 (Sol 2): 0.5 M NaCl, 10 mM Tris-HCl (pH 8.0), and 0.1% Tween 20 in dH2O (see Note 3). 16. RNase A. 17. Solution 3 (Sol 3): 50% formamide, 2x SSC (pH 4.5), and 0.2% SDS, in dH2O (see Note 3 and Note 4). 18. 1 M maleic acid, pH 7.5. 19. MBST: 100 mM maleic acid, 150 mM NaCl, and 0.1% Tween 20. Adjust pH to 7.5 with solid NaOH. 20. Sheep serum (HISS): heat-inactivate at 60°C for 30 min; store in aliquots at -20°C. 21. Blocking buffer: 2% blocking reagent (Roche) in MBST; store in aliquots at -20°C. 22. Anti-digoxigenin-AP, fab fragments (Roche). 23. Embryo powder: 1) homogenize E11.5 – E13.5 mouse embryos in a minimum volume of ice cold PBS; 2) add four volumes of ice-cold acetone, mix and incubate on ice for 30 min; 3) collect the precipitate by centrifugation at 10,000 g for 10 min, remove and discard the supernatant; 4) wash the pellet with ice-cold acetone for 10 min then spin again; 4) transfer the pellet to a clean piece 5 of filter paper, spread, and allow air-drying overnight; 5) grind it into a fine powder using mortar and pestle. Store desiccated at 4°C (see Note 5). 24. Antibody solution: weigh out 3 mg of embryo powder into 1.5 ml microfuge tube. Add 0.5 ml of blocking buffer. Rotate the tube in hybridization oven at 70°C for 30 min. Vortex for 10 min and cool on ice. Add 5 µl of HISS and 1 µl of anti-DIG AP. Shake gently on shaker at 4°C for 1 h. Spin in microcentrifuge at 4°C for 10 min. Take ~350 µl of the supernatant and dilute to 4 ml with 1% HISS in blocking buffer. Store at -20°C. 2.4. Post-antibody washes and staining 1. Rocker. 2. 2 ml conical bottom O-ring screw cap tubes. 3. 5 ml polystyrene round-bottom tubes. 4. 24-well plates. 5. MBST. 6. 2 M levamisole; store in aliquots at -20°C. 7. 1 M MgCl2. 8. 2 M Tris-HCl, pH 9.5. 9. NTTML: 0.15 M NaCl, 0.1 M Tris-HCl (pH 9.5), 0.1% Tween 20, 50 mM MgCl2, and 2 mM levamisole in dH2O (see Note 6). 10. BM Purple AP substrate (Roche). 11. PBST, pH 4.5 (pH with phosphoric acid). 12. 0.1% glutaraldehyde / 4% paraformaldehyde in PBS; store in aliquots at -20°C. 13. PBST. 14. Glycerol. 15. 50%, 75%, and 85% glycerol in PBST. 6 3. Methods Carry out all procedures at room temperature unless otherwise specified. 3.1. Embryo dissection 1. Dissect the embryos in PBS on ice. 2. After all embryos are dissected, transfer them into 1.5 ml microfuge tubes using a plastic transfer pipette (cut the tip to enlarge the opening). 3. Fix in 4% paraformaldehyde in PBS with gentle shaking at 4°C overnight. 4. Wash the embryos 2× with PBST, 10 min each time. 5. Wash the embryos 1× with 25%, 50%, and 75% methanol in PBST, 5 min each time. 6. Wash the embryos 2× with 100% methanol, 10 min each time. You can store embryos in 100% methanol at -20°C for several months. 3.2. In situ RNA hybridization 1. Rehydrate the embryos 1× in 75%, 50%, 25% methanol in PBST, and 2× in PBST, 5 min each time. 2. Transfer the embryos into 5 ml polystyrene round-bottom tubes (see Note 7). 3. Bleach the embryos with 6% hydrogen peroxide in PBST for 1 h on rocker. 4. Wash 3× with PBST, 5 min each time. 5. Treat the embryos with 10 µg/ml of proteinase K in PBST (see Note 8). 6. Rinse with fresh-made, filtered, 2 mg/ml glycine in PBST for 30 sec. 7. Wash 2× with PBST, 5 min each time. 8. Refix the embryos with 0.2% glutaraldehyde / 4% paraformaldehyde in PBST for 20 min. 9. Wash 2× with PBST, 5 min each time. 7 10. Transfer the embryos into 2 ml conical bottom O-ring screw cap tubes and add 1 ml of hybridization buffer, and pre-hybridize for 1 h at 70°C, rotating in hybridization oven. 11. Remove hybridization buffer from the embryos, add 0.5-1 ml of hybridization buffer containing the 4 µg/ml of a probe. Hybridize in hybridization oven at 70°C overnight (see Note 9 and Note 10). 3.3. Posthybridization washes and preabsorption of antibody 1. Remove the probe from the tubes (see Note 11). 2. Add 1 ml of Sol 1 to the tubes and pour the solution with the embryos into 5 ml polystyrene round-bottom tube. Rinse the embryos 1× with Sol 1. 3. Wash 2× with pre-warmed Sol 1 at 70°C, 30 min each time. 4. Wash 1× with pre-warmed mix of Sol 1:Sol 2 (1:1) at 70°C for 10 min. 5. Wash 3× with Sol 2, 5 min each time. 6. Incubate the embryos with 100 µg/ml RNase A in Sol 2 at 37°C for 1 h. 7. Wash 1× with Sol 2 and 1× with Sol 3, 5 min each time. 8. Wash 2× with pre-warmed Sol 3 at 65°C, 30 min each time. 9. Wash 3× with MBST, 5 min each time. 10. Pre-block the embryos with 1-1.5 ml of 10% HISS in the blocking buffer for 3–4 h. Put the tubes upright on a shaker. 11. Remove the blocking solution from the embryos, transfer the embryos into 2 ml conical bottom O-ring screw cap tubes, and add 1 ml of the antibody solution. Incubate on rocker at 4°C overnight (see Note 12). 8 3.4. Post-antibody washes and staining 1. Transfer the embryos into 5 ml polystyrene round-bottom tube. Wash 3× with MBST, 5 min each time. 2. Wash 8× with MBST, 1 hour each time. Leave in MBST at 4°C overnight (see Note 13). 3. Wash the embryos 3× with NTMTL, 5 min each time. 4. Transfer the embryos into 2 ml conical bottom O-ring screw cap tubes. Replace NTMTL with 1 ml of BM purple, wrap tubes in foil, and incubate on a rocker for ~1 h – 2 days. 5. Once a signal is at the desired intensity, wash 3× with PBST, pH 4.5, 5 min each time, keeping in dark. 6. Fix with 0.1% glutaraldehyde / 4% paraformaldehyde in PBS at 4°C for 1 h – overnight. 7. Transfer the embryos into 24-well plates. 8. Clear the embryos in 50%, 75%, and 85% glycerol in PBS series. The embryos should sink in each solution. Store the embryos in 85% glycerol in PBS (see Note 14). 4. Notes 1. Make fresh 4% paraformaldehyde each time. Preparation should be carried out inside a fume hood. Store it at 4°C for up to one week. 2. RNA is easily destroyed by ribonucleases that are extremely stable enzymes and can be present in untreated solutions. Hence, it is essential to avoid contamination of the RNA probe and solutions by RNase before RNA hybridization. It is highly advisable to use DEPC-treated dH2O to inactivate RNase contamination. Add 1 ml of DEPC in 1 l of dH2O or reagent, shake vigorously, leave overnight at room temperature, and autoclave. 3. It is recommended to make fresh hybridization buffer, Solution 1, Solution 2, and Solution 3 each time. 4. Adding of SDS into Sol 3 is optional. 9 5. It is always important to use embryo powder prepared from the species that you are studying. 6. It is advisable to add MgCl2 and fresh aliquot of levamisole directly before use. 7. Up to four E10.5 embryos can be used per tube. 8. Proteinase K solution should be fresh-made each time. Timing is important for treatment with proteinase K and should be adjusted based on the tissue of interest (for example, E7.0 for 2 min; E7.5 for 3 min; E8.5 for 4 min; E9.5 for 15 min; E10.5 for 23 min). Let the tube sit on the table on its side and roll it gently every ~3 min. Do not shake. Embryos are very fragile after Proteinase K digestion before the second fixation. 9. Quantities of the probe needed for the hybridization should be optimized for each probe. 10. Make sure the volume of the hybridization buffer covers the embryos. 11. You can store the probes for recycle and use them once or twice more. 12. You can store the antibody solution for recycle and use them once or twice more. 13. You can repeat step 2. The larger the embryos, the longer the washes should be. The embryos can be left washing at 4°C without apparent loss of signal for up to 3 days. 14. Keep the embryos in 85% glycerol in PBS for a few days before taking pictures. 10 References 1. Rosen B. and Beddington R.S.P. (1993) Whole-mount in situ hybridization in the mouse embryo: gene expression in three dimensions. Trends in Genetics 9, 162-167. 2. Speel E. J. M., Ramaekers F. C. S., and Hopman A. H. N. (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J Histochem Cytochem 45, 1439-1446. 3. Clay H. and Ramakrishnan L. (2005) Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification. Zebrafish 2, 105-111. 4. Hargrave M., Bowles J., and Koopman P. (2006) In situ hybridization of whole-mount embryos. Methods Mol Biol 326, 103-113. 5. Lowe L. A. and Kuehn M. R. (2000) Whole mount in situ hybridization to study gene expression during mouse development. Methods Mol Biol 137, 125-37. 6. Heiskanen M., Peltonen L., and Palotie A. (1996) Visual mapping by high resolution FISH. Trends in Genetics 12, 379-382. 7. Herrington C. S., Graham A. K., and McGee J. O’D. (1991) Interphase cytogenics J Clin Pathol 44, 33-38. 8. Correia K. M. and Conlon R. A. (2001) Whole-mount in situ hybridization to mouse embryos. Methods 23, 335-338. 9. Jowett T., Mancera M., Amores A., and Yan Y. (1996) In situ hybridization to embryo whole mounts and tissue sections: mRNA detection and application to developmental studies. In Situ Hybridization (Clark M., ed.), Chapman and Hall, London, UK, 91-121. 10. Piette D., Hendrickx M., Willems E., Kemp C. R., and Leyns L. (2008) An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies. Nature Protocols 3, 1194-1201. 11