Multiplex Detection of RNA Expression in Drosophila Embryos

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Science  06 Aug 2004:
Vol. 305, Issue 5685, pp. 846
DOI: 10.1126/science.1099247


We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven different transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent transcript multiplex fluorescent in situ hybridization. These multiplex methods fill a current technological gap between high-resolution in situ hybridization with one or two fluorescently labeled probes and low-resolution but genome-wide microarray RNA profiling and should be of great utility in establishing gene networks.

Combinations of transcriptionally active genes control many aspects of cellular, organismal, and evolutionary diversity. Analysis of such gene networks is currently hampered by a gap between high-resolution in situ hybridization methods (13) and genome-wide coverage provided by microarray analysis (4). Here we report improved fluorescent in situ hybridization (FISH) methods, including a method for directly labeling RNA probes, that will help fill this gap. The key improvements are the use of bright Alexa Fluor dyes and a combination of directly labeled probes, haptenylated probes detected with secondary antibodies, and tyramide signal amplification. Figure 1A (supporting online text) shows a whole-mount Drosophila embryo in which five patterning genes were detected with these methods. Multiplex labeling also allows the researcher to genotype homozygous (Fig. 1, B and C) or heterozygous (Fig. 1J) transcript null embryos (or to measure levels of target RNA in embryos treated with RNA interference), while simultaneously testing transcript levels of downstream genes.

Fig. 1.

(A) Drosophila embryo, stained for sog (direct label, red), ind (green), msh (magenta), wg (yellow), and en (blue) transcripts. (B) Anterior of a wild-type embryo stained for vnd (blue), ind (green), msh (red), and dpp (yellow). (C) Anterior of a dpp-deletion mutant stained as in (B). (D) Cells stained for Dfd (red), Scr (blue), and ftz (green). Nuclear borders are indicated by dotted lines. (E) Diagram of two-color combinatorial coding scheme to detect transcripts from three genes. (F) Diagram of a nucleus from a male embryo, displaying nascent transcripts from these three genes at their chromosome positions. (G) Nuclei (blue) from an embryo stained with a two-color coding scheme possess two nascent transcript signals for rho (green) and one each for sog (yellow) and vnd (red). (H) An embryo stained for seven Hox transcripts: lab (light blue), Dfd (magenta), Scr (green), Antp (orange), Ubx (dark blue), abd-A (red), and Abd-B (yellow). (I) miR-10 transcripts (red) in a blastoderm embryo. (J) Close-up view of two nuclei from an embryo heterozygous for an miR-10 deletion, revealing two sites of Antp transcription (green) and one site of miR-10 transcription (red). White lines indicate nucleus borders.

It is possible to visualize the transcription patterns of numbers of genes by detecting nascent transcripts still associated with chromosomes at the site of RNA synthesis (2, 5). In Fig. 1D, we resolve the active transcription units of the Dfd, Scr, and ftz genes, spaced at ∼20-kb intervals in the primary DNA sequence. The spatial separation of nascent transcript signals, along with a coding system in which transcripts are labeled with different combinations of fluors, has allowed the simultaneous detection of 10 transcription units per nucleus in cultured cells (6). A similar coding scheme applied to an embryo is shown in Fig. 1, E to G, in which a two-color code identifies nascent transcripts from the X-linked sog gene. Nearly all (99.8%, n = 417) nuclei in sog expression domains have either one (males) or two (females) two-color signals. Such high coding accuracy is essential for nascent transcript multiplex FISH to have practical use in determining the precise extent and overlap of transcription patterns in embryos. With multicolor combinatorial codes, the simultaneous detection of large numbers of gene transcription patterns is feasible (6).

Another approach to multiplex detection is to reuse fluors for spatially separated transcripts. For example, as shown in Fig. 1H, seven Hox gene transcripts can be detected with low background staining with only four fluors. It is also possible to detect primary transcripts of microRNA genes. For example, the miR-10 gene, located between Dfd/Hox4 and Scr/Hox5 in animal Hox clusters, is transcribed in the thoracic and abdominal primordia of early embryos (Fig. 1, I and J).

Multiplex detection of RNA will facilitate the assignment of unique molecular signatures to individual cells in developing embryos. These data will assist both in the construction of gene expression maps for use in gene network modeling (7) and in the decoding of cis-regulatory sequence information in metazoan genomes (8).

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