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Hearing Reseurch, 56 (1991) 148-151 (3 1991 Elsevsier Science Publishers B.V. All rights reserved 0.77X-S955/91/$03.50

HEARES 01642

Preservation of mRNA during in situ hybridization in the cochlea Allen F. Ryan t*2,Alan G. Watts ’ and Donna M. Simmons 4 Departments of t Surgery /Otolaryngology and ’ Neurosciences, UCSD School of Medicine and VA Medical Center, .’ Neural Systems Laborutory, The Salk Institrrtefor Biological Studies, La Jo/la and ’ Department of Neurosciences, University of Southern California. Los Angelrs, Cal$orniu, U.S.A.

(Received 26 April 1991; accepted 19 June 1991)

Specific nucleic acid sequences can be identified within cells using in situ hybridization. Hybr~djzation for mRNA can document the distribution and amount of specific gene transcripts. Decalcification protocols used for immunohistochemist~ in the cochlea were evaluated for use with in situ mRNA hybridization. No loss of mRNA was detected following the use of decalcification solutions at 4°C when paraformaldehyde was added to the EDTA solution. The primary determinant of mRNA preservation was paraformaldehyde. Inner ear: Gene expression; mRNA; Hybridization, in situ

Introduction An important step in the expression of genes is the transcription of DNA sequences into mRNA. The measurement of mRNA production is thus an excellent, though by no means complete, indicator of active gene expression. Specific mRNA sequences can be detected by any of several techniques, including both Northern blot and in situ hybridization using labeled complementary DNA fcDNA) probes or complementary RNA (cRNA) probes, Alternatively, mRNA can be detected indirectly, using the polymerase chain reaction (PCR) to amplify cDNA copies of mRNAs (Saiki et al., 1988; Kawasaki, 1990). Gene expression can also be detected by measuring the protein or peptide product using either biochemical or immunochemical assays. In situ mRNA hybridization has certain advantages over methods, such as immunohistochemistry, which detect the final product. One of these is the fact that mRNA is rapidly degraded by RNAses, and thus has a relatively short half-life, measured in hours (Lewin, 1990). This allows better resolution of the temporal aspects of gene activation than does detection of the protein product, which typically persists in the cell for much longer periods. Other advantages include the ability to detect synthesis when a product is rapidly transported from the cell soma, the ability to obtain quantitative data

Correspondence

to: Allen F. Ryan, Division of Otola~ngoio~ 112C, UCSD La Jolla, CA 92093, U.S.A. Fax: (619) 552-7452.

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when radiolabeled probes are used, and access to a different stage of the synthetic process. The advantages must be balanced against the method’s weaknesses. These include the limitation of hybridization to the cell’s synthetic machinery in the soma, not the ultimate site of expression (at the synapse, for example). The method also does not take into account post-transcriptional regulation which occurs between mRNA transcription and generation of the final product. Major regulation takes place at this stage in some synthetic systems, especially polyproteins (Douglas et al., 1984). Northern blot hybridization for mRNA (White and Bancroft, 1982) is readily applicable to the cochlea despite its bony capsule, since tissue must be homogenized prior to RNA extraction, electrophoresis on a sizing gel, transfer to nitrocellulose, and hybridization. However, the complex and heterogeneous structure of the cochlea limits the utility of this method, since the particular inner ear tissue producing the mRNA cannot be identified. In situ mRNA hybridization, which allows the identification of mRNA produced by individual cells, might be more valuable for use in the cochlea. However, the bony capsule and delicate tissues of the cochlea make in situ mRNA hybridization protocols developed for soft tissue difficult to apply to the labyrinth. Wackym et al. (1990) have used a modification of this method in the vestibular ganglion, while we (Ryan and Watts, 1991) have used a modification in the cochlea. The present study was undertaken to test whether in situ mRNA hybridization methods applicable to the inner ear result in loss of mRNA, and to explore factors relevant to mRNA preservation.

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Methods

Cochlear tissue was obtained from young adult (200-300 g) Harlan Sprague-Dawley rats. The rats were deeply anesthetized with pentobarbital (50 mg/kg) and perfused transcardially with room temperature normal saline (pH 7.4) followed by 200 ml of cold (4 o C) 4% paraformaldehyde at pH 6.5 and then 150 ml of 4% paraformaldehyde with 0.1% glutaraldehyde at pH 9.5. The inner ears were dissected from the temporal bones and the cochleas were post-fixed at 4” C for 24 h in 4% paraformaldehyde without glutaraldehyde (pH 9.5) and then decalcified in 8% EDTA with 4% paraformaldehyde (pH 6.6) at 4’ C until soft, about three weeks. Fetal tissue was obtained from fetal mice and rats at a gestational age of 16 days (e16). Pregnant mice or rats were deeply anesthetized with pentobarbital. The fetuses were removed from the uterus and fixed by immersion in 4% paraformaldehyde (pH 9.5) at 4 o C for 24 h. In order to assess the effects of decalcification solutions on mRNA, some fetal specimens were then immersed in 8% EDTA with 4% paraformaldehyde (pH 6.6) at 4 o C for three weeks prior to normal processing. Decalcified cochleas with the stapes removed and fetal tissue were saturated with 30% sucrose containing 4% paraformaldehyde (pH 9.5) overnight. The samples were briefly rinsed in PBS to prevent coagulation of the embedding medium. Cochleas were placed in OCI under vacuum for one hour to remove gas bubbles and then frozen. Fetal tissue was frozen in a 50/50 mixture of OCT and Aquamount (Jones et al., 1986). All samples were sectioned at 20 pm on a cryostat. The sections were mounted on poly-r_-lysine-coated slides, air and vacuum dried, and stored at -20 “C with dessicant until used. In order to estimate the importance of paraformaldehyde and reduced temperature for mRNA preservation, mounted sections of fetal material were exposed to the following combinations of disodium EDTA for one week: 10% EDTA (pH 7.0) in phosphate buffer at 20 “C; 10% EDTA in phosphate buffer at 4°C; 8% EDTA in phosphate buffer (pH 6.6) with 4% paraformaldehyde at 20 ’ C; 8% EDTA in phosphate buffer with 4% paraformaldehyde at 4 o C, prior to hybridization. The protocol utilized for in situ mRNA hybridization has been described in detail elsewhere (Simmons et al., 1989). Briefly, cRNA probes were synthesized from cDNA templates. For testing fetal material, a cRNA probe complementary to mRNA for the POU homeodomain protein brain-3 (Brn-3) (supplied by Dr. Xi He of UCSD) was used. Brn-3 mRNA is expressed in developing sensory neurons, including all sensory ganglia associated with the spinal cord and cranial

nerves (He et al., 1989; Ryan et al., 1991b). For testing adult cochleas, a cRNA probe (supplied by Janet Emanuel and Robert Levenson of Yale University) complementary to mRNA coding for the cu3 isoform of the (Ysubunit of Na/K-ATPase, which is expressed by all neurons (Watts et al., 1991), was used. The antisense strand of each coding sequence was transcribed using SP6 RNA polymerase and 35S-UTP. RNA probes encoding the sense strand of each noncoding sequence were also synthesized for use as controls for nonspecific hybridization. The tissue sections were permeablized with Proteinase K or Triton X-100 (0.5% at 23 o C for 30 min), and dehydrated. Hybridization with 35S-probes (0.5-1.0 X 10’ cpm/ml) was performed at 55-58 ’ C overnight in a solution containing 50% formamide, 0.3 M NaCl, 10 mM Tris (pH 8.01, 1 mM EDTA, 0.5% tRNA, 10 mM dithiothreitol (DTT), 1 X Denhart’s solution and 10% dextran sulfate. After hybridization, sections were treated with ribonuclease A (25 pg/ml, 37 o C, 30 min) and washed in 0.1 X SSC (0.15 M NaCI, 0.015M Na citrate), 1 mM DTT at 60 “C. For initial determination of hybridization, the dehydrated sections were opposed to Amersham Hyperfilm PMax for 72 h. The film was developed in Kodak D-19 (4 min at 20 ’ C) and fixed in Kodak rapid fixer. Densitometric analysis was performed on the Brn-3 films using a Drexel University image analysis system. For each comparison, the sections were prepared, hybridized, exposed and developed together. The density of the image of the trigeminal ganglion and adjacent non-neural tissue was measured in six sections for each condition, film background was subtracted, and the resultant values averaged. The slides were then coated with Kodak NTB-2 liquid autoradiographic emulsion, exposed at 4 ’ C for 2 weeks and then developed in Kodak D-19 (3.5 min at 14” C) and fixed in Kodak rapid fixer. The sections were counterstained through the emulsion using a 0.001% solution of bisbenzimide in KPBS (0.12 Ml. Sections were examined under fluorescence microscopy with UV to identify cochlear structures, and under dark field to identify autoradiographic grains. The sense-strand 35S-cRNA probes showed no evidence of the specific hybridization obtained with the antisense probes. Hybridization for the antisense probes was stable under high-stringency wash conditions (up to at least 85 o C).

Results Hybridization in decalcified cochleas

When frozen sections of decalcified adult inner ears were permeablized with proteinase K, the sections tended to disintegrate during the hybridization protocol. Many sections were lost from the slides, and the

Fig. i. Decalcified frozen sections of adult rat cochlea. a. Toluidine blue stained section. b. Dark field i~~umjnation of a semiadjacent section hybridized with a ““S-labeled riboprobe complemental to mRNA encoding the a3 isoform of the (Y subunit of rat Na,K-ATPase. SV = stria vascularis, OC = organ of Corti, SG = spiral ganglion. (Adapted from Ryan and Watts, 1991).

tissues of the organ of Corti were lost from those sections which remained. Pre-treatment with Triton X-100 produced no apparent damage to cochlear tissues, and this method of permeablization was used in all subsequent cochlear experiments. Sections of adult cochlea which were decalcified in EDTA/paraformaldehyde at 4 * C and permeablized with Triton have now been successfully hybridized with a variety of riboprobes, ranging in length from approximately 400 to more than 1100 bases in length (Crenshaw et al., 1991; Ryan and Watts, 1991; Ryan et al., 1991a,b). An example is illustrated in Fig. 1, in which a riboprobe complementary to mRNA encoding the cu3 isoform of the LYsubunit of the Na,K-ATPase, shows intense hybridization over spiral ganglion neurons. This is consistent with previous studies in other tissues which have shown the (~3 to be almost exclusively the neuronal isoform of the (Ysubunit (Watts et al., 1991). In other cochlear tissues, different (Ysubunit isoforms are found (Ryan and Watts, 1991). Riboprobes longer than about 1000 bases have difficulty penetrating tissue (Simmons et al., 1989). We have used alkaline hydrolysis of riboprobes (Cox et al., 1984) to cleave probes of up to 4500 bases into fragments of approximately 500 bases or less, to allow tissue penetration. With this technique, edge effects and pooling of probe in tissue cavities was sometimes observed, even when the hydrolized probes worked well on brain sections. This problem was especially acute when hydrolysis was extensive, and may be related to the presence of very short probe fragments. Effects of decaici~cation on mRNA

To determine whether mRNA was lost during the decalcification procedure, hybridization for Brn-3 mRNA was compared for fetal tissue which had been

frozen and sectioned immediately after fixation, and for fetal tissue which had been immersed whole in EDTA/paraformaldehyde at 4 * C for three weeks prior to freezing and sectioning. Fig. 2 illustrates the results of a densitometric analysis. Three weeks in the decalcification solution had no significant effect upon either specific hybridization to sensory ganglia, or nonspecific hybridization to non-neuronal tissue in which Brn-3 mRNA would not be expected. Influence of EDTA and temperature on mRNA

To assess the relative roles of paraformaldehyde versus temperature in preserving mRNA during decalcification, fetal tissue sections already mounted on slides were immersed in EDTA with and without

0.5

1

EDTA/para 4-c

Fig. 2. Densitometty of Brn-3 hybridization in e-18 fetal mouse tissue processed for in situ hybridization either immediatefy after normal fixation (Control) or after three weeks imme~ion of intact tissue samples in 8% EDTA with 4% paraformaldehyde at 4* C (EDTA/para 4°C). Solid bars represent specific hybridization in sensory neurons. The hatched bars represent nonspecific hybridization in non-neuronal tissue. Vertical lines represent one standard deviation, Film background was subtracted from all values.

Fig. 3. Autoradiographic images of adjacent e16 fetal mouse sections immersed in EDTA at 20’ C or 4” C, or in EDTA with 4% paraformaldehyde at 20 o C or 4 o C on slides for one week. Control sections were stored at - 20’ C. The sections were then hybridized with a 35S cRNA probe complemental to mRNA encoding a DNA transcription factor, the POU-domain protein Brn-3 (He et al., 1989). Brn-3 is expressed strongly in developing neural tissue, especially sensory ganglia. The arrow on the control section indicates the trigeminal ganglion, used for densitcmetric analysis in Fig. 4.

parafo~aldehyde, at either 4” C or 20 *C. As illustrated in Fig. 3, when sections of fetal tissue were exposed for one week to EDTA at 20 o C, there was a substantial reduction in specific hybridization for Brn-3 mRNA, as well as a reduction in nonspecific background hybridization of the probe to non-neuronal 0.5

0.0

1

COntrDl

EOTA 2o”c

EDTA 4-c

EDTAI part? 20%

EDTA/ para 4°C

Fig. 4. Den&tome&y of Brn-3 hybridization illustrated above. Solid vertical bars represent the optical densities measured in the trigeminal ganglion, indicated by the arrow in Fig. 3. Shaded bars represent the densities measured in nearby non-neural tissue. Each bar represents the mean of six measurements from separate sections. Background optical density measured from film adjacent to each section was subtracted from both neural and non-neural densities. Vertical lines represent one SD.

tissue, when compared to control sections. Exposure to EDTA at 4 o C resulted in a lesser reduction in specific hybridization. In contrast, tissue sections exposed to EDTA plus paraformaldehyde showed an increase in specific hybridization, as well as a decrease in nonspecific hybridization, at both 20 o C and 4 o C. A densitometric analysis of Brn-3 hybridization to sensory nuclei versus non-neural tissue is illustrated in Fig. 4. Immersion of sections in EDTA or EDTA/ paraformaldehyde significantly reduced nonspecific hybridization. Immersion in EDTA alone decreased specific hybridization, with the greatest degree of decrease observed at 20°C. In contrast, immersion in EDTA/ paraformaldehyde increased specific hybridization, with the greatest degree of increase observed at 4 ’ C.

Discussion Our data indicate that in situ hybridization for the detection of mRNA is practical for use in the labyrinth. Inner ear tissue which is well fixed in paraformaldehyde, decalcified in EDTA with parafo~aIdehyde at 4” C, and permeablized after sectioning with Triton X-100, shows excellent hybridization with 3sS-labeled cRNA probes.

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When whole fetal tissue was immersed in EDTA with paraformaldehyde for three weeks at 4 o C, both specific and nonspecific hybridization were comparable to that seen in fetal tissue which was processed immediately after fixation. Thus there appears to be no loss of mRNA due to the decalcification protocol. The results obtained with fetal tissue sections already mounted on slides suggest that the primary determinant of mRNA preservation is the presence of paraformaldehyde in the decalcification solution. A secondary factor appears to be temperature, since maintaining sections at 4” C increased the level of specific hybridization. The experiment on fetal tissue sections also showed that pre-soaking sections on slides in either EDTA or EDTA/paraformaldehyde decreased nonspecific background hybridization, while pre-soaking in EDTA/ paraformaldehyde increased specific hybridization. It should be noted that neither effect was observed with immersion of intact tissue in EDTA/ paraformaldehyde prior to freezing and sectioning. The increase in specific hybridization could result from decreased leaching of mRNA, which is water soluble (Simmons et al., 19891, from tissue. Since paraformaldehyde fixation is reversible, additional fixation which occurs after ceil interiors are exposed by sectioning could increase the linkage of mRNA to tissue. Alternatively, additional fixation could decrease the degradation of mRNA by tissue RNAses. The decrease in nonspecific hybridization seen with pre-soaking is more difficult to explain. However, it is possible that positive charges on the tissue, which could attract and nonspecifically bind the riboprobes, were blocked by the EDTA. In any case, post-sectioning immersion of sections in EDTA/paraformaldehyde markedly increased signal-to-noise for the Brn-3 riboprobe. As has previously been noted with fetal tissue (He et al., 19891, proteinase K digestion is too harsh for use on cochlear sections. However, Triton X permeablization appears to provide sufficient permeablization to allow the penetration of riboprobes up to 1200 bases in length, with excellent preservation of cochlear cytoarchitecture.

Acknowledgements Supported by grants DC00139 from the NIH/NIDCD, by the Research Service of the Veterans Adminis-

tration, and by the Duaei Hearing Research Fund. The technical contributions of Duane Brumm and Graciela Sanchez-Watts are gratefully acknowledged.

References Cox, K.H., DeLeon, D.V., Angerer, L.M. and Angerer, R.C. (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Devel. Biol. 101. 485-502. Crenshaw, E.B., Ryan, A.F., Dillon, S.R., Kalla, K. and Rosenfeld, M.G. (1991) Wocko, a neurological mutant generated in a transgenie mouse pedigree. J. Neurosci. 1I, 1524-1530. Douglas, J., Civelli, 0. and Herbert, E. (lY84) Polyprotein gene expression: generation of diversity of neuroendocrine peptides. Annu. Rev. Biochem. 53, 665571.5. He, X., Treaty, M., Simmons. D., lngraham H., Swanson, L. and Rosenfeld, M.G. (1989) Expression of a large family of POU-domain regulatory genes in mammalian brain development. Nature 340. 35-42. Jones, P.S., Elias, J.M. and Schechter, N. (1986) An improved method for embedding retina for cryosectioning. J. Histotechnol. 0, 181-182. Kawasaki, ES. (1990) Amplification of RNA. In: M.A. Innes, D.H. Gelfand, J.J. Sninsky and T.J. White (Eds.). PCR Protocols. Academic Press, New York, 21-27. Lewin, B. (1990) Genes IV. Oxford Univ. Press, Oxford. Ryan, A.F., Brumm, D. and Kraft, M. (1991a) Glutamate receptor mRNA expression in the rat cochlea. Neurosci. Abstr. (in press). Ryan A.F., Simmons D.M. and Crenshaw E.B. (lY91b) Gene expression in normal and abnormal inner ears. Proc. N.Y. Acad. Sci. (in press). Ryan. A.F. and Watts, A.G. (1991) Expression of genes coding for cy and p isoforms of Na/K-ATPase in the cochlea of the rat. Molec. Cell. Neurosci. 2, 179-187. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Simmons, D.M., Arriza, J.L. and Swanson, L.W. (1989) A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J. Histotechnol. 12, 169-181. Wackym, P.A., Popper, P., Ward, P.H. and Micevych, P.E. (1990) In situ hybridization for the study of gene expression in neuro-otologic research. Otolaryngol. Head Neck Surg. 103, 519-526. Watts, A.G., Sanchez-Watts, G. Emanuel, J. and Levenson, R. tlY91) Complex cell -specific expression patterns of the mRNAs encoding Na,K-ATPase (Y- and p-subunit isoforms within the rat CNS. PNAS (in press). White, B.A. and Bancroft, F.C. (1982) Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J. Biol. Chem. 257, 8569-8576.

Preservation of mRNA during in situ hybridization in the cochlea.

Specific nucleic acid sequences can be identified within cells using in situ hybridization. Hybridization for mRNA can document the distribution and a...
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