Sensitive two-color whole-mount in situ hybridizations using digoxygenin- and dinitrophenol-labeled RNA probes.

Two-color whole-mount in situ hybridization is a powerful technique for comparing the spatial expression patterns of two genes in developing embryos or within a given adult tissue. Many current procedures use two genespecific antisense RNA probes, each incorporating a different chemical moiety that is recognized by a specific antibody. Typically, the first gene-specific RNA probe is labeled with digoxygenin (DIG) and the second with fluorescein or biotin. After hybridization to the embryos, each bound antisense probe is then visualized using a specific enzyme-conjugated antibody and a distinctive color reaction. These procedures work well in situations when both of the target genes are expressed at high levels but are less reliable for cases in which one or both of the genes are expressed at moderate or low levels. Often, these difficulties can be attributed to the differing sensitivities of the two hapten/antibody/color detection systems that are used. Dinitrophenol (DNP) has been used as an alternative tag for labeling oligonucleotides for in situ hybridizations of tissue sections or for nonradioactive detection of DNA fragments (1,3,4,7,8). We have investigated the use of DNP for labeling antisense RNA probes for use in two-color wholemount in situ hybridizations. Specifically, we have replaced fluorescein-labeled RNA probes with their DNP-labeled counterparts and used an alkaline phosphatase (AP)-conjugated, anti-DNP Fab for immunostaining. In our hands, this hapten/antibody pair generally gives much less background staining, even after long development times, with signals comparable to or stronger than those produced with the corresponding fluorescein detection system. Consequently, using the combination of DIGand DNP-labeled antisense RNAs, we observe significantly improved reliability in two-color whole-mount in situ hybridizations, especially with probes to mRNAs of low or moderate abundance. DNP-labeled RNA probes were prepared by transcription in vitro with standard rNTPs and then tagged covalently with DNP by chemical modification using the Label IT® DNP Nucleic Acid Labeling System (Mirus, Madison, WI, USA). The DNP labeling procedure adds the DNP moiety to non-base-pairing sites on the guanine ring. According to the manufacturer’s specifications, only one DNP tag is added per 20–60 bp. Therefore, the “specific activity” of the DNP-labeled probe is theoretically less than that of a DIG probe, since a DIG group is reported to be present every 20–25 nucleotides in DIG-labeled transcripts (Roche Molecular Biochemicals, Indianapolis, IN, USA). To compare the DIG and DNP probes, we used equal concentrations of the corresponding probes for hybridization, identical dilutions of the AP-conjugated anti-DIG or anti-DNP Fab fragments, and the same staining times with BM Purple (Figure 1). Antisense probes to the zebrafish no tail (ntl) gene were compared by in situ hybridization with 4–7 somite-stage zebrafish embryos. The ntl gene was chosen because of its distinctive expression pattern in the notochord and tailbud (12). Surprisingly, the DNP and DIG ntl probes gave essentially the same staining intensity and specificity (Figure 1A). The DIG signal was slightly stronger, but the nonspecific background was also elevated slightly with the DIG probe. The lower specific activity of the DNP probe is presumably compensated by another factor, either increased hybridization efficiency or an antibody-dependent parameter. To test the suitability of DNP-labeled RNA probes in two-color wholemount in situ hybridizations, the DNPlabeled ntl probe was hybridized jointly with a DIG-labeled spadetail (spt) probe. The spt and ntl expression domains overlap in the tailbud of the zebrafish embryos but not in the notochord, where only ntl is expressed. (6,12). The DNP and DIG probes were visualized using p-iodonitrotetrazolium violet/X-gal 4-toluidine (INT RED/ BCIP; Sigma, St. Louis, MO, USA and Roche Molecular Biochemicals) and BM Purple as the AP substrates, respectively. The INT RED/BCIP staining was done using the method of Sagerström et al. (11), as modified by Liang et al. (9). The ntl and spt expression domains were easily detected with high specificity (Figure 1B). The punctate nature of the ntl staining (red) reflects the expression of ntl within specific notochord cells. This suggests that, using DNP probes, two-color whole-mount in situ hybridizations can provide single-cell resolution. This was confirmed separately by in situ hybridization using a DNP-labeled pitx2 probe and color development with INT RED/BCIP. We were able to see the staining of the individual cells (putative Rohon-Beard neurons; Figure 1C, arrow) where the pitx2 homeobox gene is expressed (5). Finally, we compared DNPand fluorescein-labeled antisense RNA probes. Higher, nonspecific background staining and/or lower signals were seen more often with the fluorescein probes. For example, Figure 1, D–G, illustrates the results from in situ hybridizations with either a fluoresceinor a DNP-labeled lefty2 probe; color development was with INT RED/BCIP. Lefty2 encodes a developmentally regulated growth factor (2). With the fluorescein-labeled probe, most embryos showed high (47%) (Figure 1G) or moderate (47%) background staining in addition to the specific staining of the heart tube (n = 34). Only a few embryos (6%) were stained with acceptably low levels of background (Figure 1F). In contrast, with the DNP-labeled lefty2 probe, 97% of the embryos (n = 32) showed little or no background (Figure 1D), and even the embryo with the highest background showed an acceptable signal-to-noise ratio (Figure 1E). Figure 1, H and I, shows a two-color/two-probe in situ hybridization experiment in which we tested a fluoresceinor DNP-labeled RNA probe in combination with a DIG-labeled RNA probe. We used a DIG-labeled cyclops probe with either the fluoresceinor DNP-labeled lefty2 probe. At the 19–23 somite stages, cyclops shows moderately strong expression in the brain and weak expression in the left lateral plate mesoderm, while lefty2 shows moderately strong expression on the left Benchmarks