Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos

Abstract

We describe a high-throughput protocol for RNA in situ hybridization (ISH) to Drosophila embryos in a 96-well format. cDNA or genomic DNA templates are amplified by PCR and then digoxigenin-labeled ribonucleotides are incorporated into antisense RNA probes by in vitro transcription. The quality of each probe is evaluated before ISH using a RNA probe quantification (dot blot) assay. RNA probes are hybridized to fixed, mixed-staged Drosophila embryos in 96-well plates. The resulting stained embryos can be examined and photographed immediately or stored at 4 degrees C for later analysis. Starting with fixed, staged embryos, the protocol takes 6 d from probe template production through hybridization. Preparation of fixed embryos requires a minimum of 2 weeks to collect embryos representing all stages. The method has been used to determine the expression patterns of over 6,000 genes throughout embryogenesis.

Figure 1

Flowchart for high-throughput RNA in situ hybridization to whole-mount Drosophila embryos. The major steps required for in situ hybridization to whole mount embryos are shown in boxes linked by arrows. Boxes on the left describe preparation of embryos and boxes on the right describe probe preparation. Approximate duration of each step is indicated in parentheses inside the box.

Figure 2. Size verification of PCR products for probe templates

A negative image of an agarose gel shows 5 ul from each of 96 PCR reactions. Size markers (Roche molecular weight marker X) are in the outside lanes. Reactions yielding a single visible band of the appropriate size were used to generate RNA probes.

Figure 3. Quantification of RNA probes

Dot blot of 96 probes, with 1 ul of a 1:300 dilution of each probe, shows acceptable variability of probe concentrations. Two reference dilutions series of 1:3.3, 1:10, 1:33, 1:100, and 1:330 are spotted above row A. On this blot the 1:300 reference dilution is not visible for either reference control probe and the 1:100 dilution is barely visible for the weaker reference probe (set on the right). Of the 96 probes, all but 5 spots are darker than the 1;100 dilution of the weaker reference control. The weakest probes, D3, G11, G12, were noted as weak and marked for rework, pending hybridization results.

Figure 4

Anticipated results. Stained embryos were mounted in 70%glycerol in PBS on a microscope slide without a coverslip to record production images (a, b) and screen for genes with patterned expression in the embryo. Production images were captured using a dissection microscope (Leica Wild M10) and a ProGres 3012 digital camera. Shown are images for embryos hybridized with probes for snail (a) and odd skipped (odd) (b). Higher resolution images for both embryos and imaginal discs were taken with a Spot RT digital camera mounted on a Zeiss Axiophot microscope with DIC optics, using a 20× objective. Embryos in 70% glycerol were mounted on slides under a 22×40 mm cover slip with 18mm coverslip spacers. Imaginal discs were mounted in 70% glycerol under a 22 mm coverslip without spacers. Shown are stage 5 (c) and stage 9 (d) embryos hybridized with a probe for odd and a leg disc (e) and an eye-antennal disc (f) stained with a probe for the forkhead domain gene fd96Cb. Embryos in (c) and (d) are oriented anterior to the left and dorsal up. The leg disc (e) is oriented anterior left and dorsal up. The eye-antennal disc (f) is oriented eye portion at the top and antennal portion on the bottom, lateral to the left and posterior up. In both the leg disc and antennal portion of the eye-antennal disc, expression is detected primarily in the ventral regions with patches on either side of the anterior/posterior boundary. Scale bars are 50 µm for c–f and 200 µm for a–b.