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Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development
 
Kristin Tessmar-Raible1, Patrick R.H Steinmetz1, Heidi Snyman1, Monika Hassel2, Detlev Arendt1
1, European Molecular Biology Laboratory, Heidelberg
2, University of Marburg, Marburg, Germany
BioTechniques, Vol. 39, No. 4, October 2005, pp. 460–464
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Supplementary Material

Fast and reliable whole mount in situ hybridization (WMISH) techniques are essential for high-throughput characterization of expression patterns of single genes and for co-expression studies. Complementing time-consuming and expensive generation of antibodies, double WMISH allows the fast spatial correlation of gene expression, in many cases down to the single cell level. In addition, fluorescent WMISH as established here for Platynereis allows the use of laser confocal microscopy. Whereas robust double WMISH protocols exist for conventional model organisms (1,2), no protocol has been established for protostome marine organisms, despite their importance for comparative studies (3) and increasing availability of expressed sequence tag (EST) and genomic resources. Here we report two protocols for double WMISH in the marine model Platynereis dumerilii that allow co-expression determination starting from cDNA-containing plasmid preparation within 1 week. These new protocols are based on preexisting protocols for single WMISH in Platynereis (4) and for multiple color WMISH in mouse (5) and Drosophila (6).

Our protocols allow different combinations of RNA label and detection techniques, which should be chosen based on staining efficiency observed with single nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) staining (note that staining efficiency depends on the amount of digoxigenin incorporation, transcript abundance, probe binding efficiency, and activity of the enzyme coupled to the antibody). While strongly staining probes (staining reaction stopped within 6 h) are best compared by double fluorescent WMISH (protocol 1, see (Figure 1)), weakly staining probes (staining reaction at least overnight) or probes that detect transcripts in a few isolated cells can be compared with stronger probes by combining nonfluorescent NBT/BCIP with fluorescent tyramide-cyanine 3/tyramide-fluorescein precipitate (protocol 2, see (Figure 2)). The reason for the use of two different protocols is that the overall signal-to-noise ratio is lower for the fluorescent WMISH (for background quantification, see (Figure 1) legend) than for the NBT/BCIP staining (which in most cases is devoid of background). We see no significant difference in signal intensity (fluorescent or nonfluorescent) between the single or double WMISH protocols. Notably, the widely used fluorescent substrate FastRed (Roche Applied Science, Indianapolis, IN, USA) fails to work in Platynereis.

Figure 1.


Partial co-expression of Platynereis otx (Pdu-otx, red tyramide-cyanine 3 precipitate) and gbx (Pdu-gbx, green tyramide-fluorescein staining) in a 24 hours postfertilization (hpf) larva (ventral view). (A) otx-expressing cells [overlay of the tetramethyl rhodamine isothiocyanate (TRITC) and UV channels]. (B) gbx-expressing cells [overlay of the fluorescein isothiocyanate (FITC) and UV channels]. (C) Partial co-expression of otx and gbx (overlay of the TRITC, FITC, and UV channels). (D) Schematized 24 hpf Platynereis larva with cells expressing otx (red), gbx (green), or co-expressing both (light brown, note the yellow staining in panel C and compare with panels A and B). Dark gray, the developing brain. The blue nuclear staining in panels A, B, and C outlines the V-shaped boundary between the stomodaeum (light gray in panel D; spaced nuclei, large cells) and the neural plate (light blue in panel D; densely packed with nuclei, small cells). The otx probe has been labeled with digoxigenin, and the gbx probe has been labeled with fluorescein. Nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; blue). Anterior is to the top. TRITC excitation/detection wavelengths: 543/555–680 nm. FITC excitation/detection wavelengths: 488/500–550 nm. We determined the signal-to-noise ratio in the original pictures by measuring the mean grayscale values of a given channel in an area with signal versus a comparable area with background using ImageJ [National Institutes of Health (NIH)]. Signal-to-noise ratio (TRITC channel) = 91:2; signal-to-noise ratio (FITC channel) = 87:5. Pictures were taken on a Leica TCS SP2 confocal system (Leica, Heidelberg, Germany) using a 40× oil-immersion objective.

Figure 2.


Co-expression of Platynereis r-opsin (Pdu-r-opsin, nonfluorescent blue NBT/BCIP precipitate) and pax6 (Pdu-pax6, red fluorescent tyramide-cyanine 3 precipitate) in the larval eyes (arrows), but not in the adult eyes (arrowheads) at 72 hpf. (A and B) apical views and (C and D) lateral views of the same animals and focal planes, under Nomarski optics versus tetramethyl rhodamine isothiocyanate (TRITC) channel. Pictures were taken using the 40× objective and an additional 1.25× inside magnification of a Zeiss Axiophot microscope (Carl Zeiss, Oberkochen, Germany) connected to a Leica DC500 camera (Leica). NBT/BCIP, ni-troblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate; hpf, hours postfertilization.

The antisense RNA probes are differentially labeled with fluorescein-UTP or digoxigenin-UTP (the latter for weaker probes). Antisense RNA probes were synthesized by in vitro transcription similar to previous protocols (see Reference 7, and Supplementary Material available online at www.BioTechniques.com). After fixation (4), embryos were rehydrated in different methanol/1×PTW [phosphate-buffered saline (PBS) with 0.1% Tween® 20] dilutions (25% PTW, 50% PTW, 75% PTW, and 2× 100% PTW), proteinase K digested, fixed in 4% paraformal-dehyde (PFA)/PTW for 15 min, and prehybridized in hybridization mixture (see Supplementary Material) for 1 h at 65°C. Proteinase K (100 µg/mL) digestion times were significantly shortened compared with the previous protocol (4). This is essential because longer digestion produces blurry staining, probably due to enhanced diffusion of the RNA and staining precipitate. Sixteen to twenty-four hours postfertilization (hpf) specimens were digested for 30 s, 25–72 hpf specimens were digested for 45 s, 73 hpf to 6 days postfertilization (dpf) specimens were digested for 2 min, and 1- to 6-week-old worms were digested for 3 min. Hybridization was at 65°C overnight in a final probe dilution of 10–25 µL/200 µL hybridization mixture. Specimens were washed at 65°C, twice in 50% formamide/2× SSCT [standard saline citrate (SSC) containing 0.1% Tween 20] for 30 min, once in 2×SSCT for 15 min, and twice in 0.2× SSCT for 30 min.

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