In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea
© King and Newmark; licensee BioMed Central Ltd. 2013
Received: 15 January 2013
Accepted: 27 February 2013
Published: 12 March 2013
The freshwater planarian Schmidtea mediterranea has emerged as a powerful model for studies of regenerative, stem cell, and germ cell biology. Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical methods for determining gene expression patterns in planarians. While expression patterns for a number of genes have been elucidated using established protocols, determining the expression patterns for particularly low-abundance transcripts remains a challenge.
We show here that a short bleaching step in formamide dramatically enhances signal intensity of WISH and FISH. To further improve signal sensitivity we optimized blocking conditions for multiple anti-hapten antibodies, developed a copper sulfate quenching step that virtually eliminates autofluorescence, and enhanced signal intensity through iterative rounds of tyramide signal amplification. For FISH on regenerating planarians, we employed a heat-induced antigen retrieval step that provides a better balance between permeabilization of mature tissues and preservation of regenerating tissues. We also show that azide most effectively quenches peroxidase activity between rounds of development for multicolor FISH experiments. Finally, we apply these modifications to elucidate the expression patterns of a few low-abundance transcripts.
The modifications we present here provide significant improvements in signal intensity and signal sensitivity for WISH and FISH in planarians. Additionally, these modifications might be of widespread utility for whole-mount FISH in other model organisms.
KeywordsPlanarian Whole-mount in situ hybridization (WISH) Fluorescent in situ hybridization (FISH) Tyramide signal amplification (TSA) Autofluorescence Multicolor FISH Peroxidase quenching Regeneration Heat-induced antigen retrieval (HIAR)
Planarians are re-emerging as a choice animal model for studying regeneration, with the recent development of genomic resources and molecular tools in a few species, including Schmidtea mediterranea and Dugesia japonica. After captivating scientists with their remarkable regenerative capacity for over a century , significant progress is being made in understanding how planarians reestablish axial polarity following injury [3–5], how their stem cells regulate choices between self-renewal and differentiation [6, 7], and how their organ systems, including the central nervous system [8–11], intestine [12, 13], excretory [14, 15], and reproductive system [16–19], regenerate following injury.
Whole-mount in situ hybridization (WISH) and whole-mount fluorescent in situ hybridization (FISH) are critical techniques for determining gene expression patterns. Planarians present several challenges for (F)ISH: first, planarians secrete a layer of mucous that needs to be removed prior to fixation; second, some planarian tissues are “sticky”, resulting in non-specific binding or trapping of antibodies used for detection; third, planarian tissue autofluoresces across a broad range of wavelengths leading to a poor signal-to-noise ratio for low-abundance genes by FISH; and fourth, regenerating tissue is fragile during early stages of regeneration, necessitating a fine balance during permeabilization to allow even probe penetration of mature tissues while preserving morphology of regenerating tissue.
Early WISH protocols in planarians utilized treatment with hydrochloric acid and alcohol-based fixation to overcome issues with planarian mucous . More recently a formaldehyde-based WISH protocol, which utilizes N-acetyl-cysteine for mucous removal, was developed, providing vastly better sensitivity and maintenance of morphology for WISH of planarians . This protocol has been widely utilized and has been a critical advancement in the field. However, as with other model organisms, elucidation of the expression patterns for low-abundance transcripts remains challenging. In some cases the expression patterns for genes with known functions remain unclear or elusive [15, 22, 23].
Fluorescent detection of transcripts provides superior spatial resolution and enables visualization of overlapping expression patterns compared to development with chromogenic substrates. While the recently developed formaldehyde-based WISH protocol does provide improved signal sensitivity for FISH, we further improved signal sensitivity by systematically optimizing several key steps, including the bleaching, blocking, and washing steps. Furthermore, multicolor FISH using tyramide signal amplification (TSA) requires sequential rounds of amplification using peroxidase-conjugated reagents. Therefore, to prevent residual peroxidase activity from generating false signal during subsequent rounds of detection it is critical to efficiently quench peroxidase activity between TSA reactions. We directly compared several methods for quenching peroxidase activity and find that incubation with azide is the most effective at quenching peroxidase activity and the least detrimental toward detection of gene expression in subsequent rounds of TSA. These modifications represent a significant improvement for FISH in planarians, and we have utilized these advancements to clarify ambiguous or elusive gene expression patterns. Additionally, many of the modifications we present here can be applied directly to FISH protocols for other model organisms.
Results and discussion
Formamide bleaching increases signal intensity
Achieving maximal signal intensity in WISH requires balancing preservation of target mRNA with permeabilization of tissue to allow probe hybridization. Using the planarian WISH protocol established in  as a starting point, we began systematically testing modifications to improve signal sensitivity with the goal of improving detection of problematic transcripts by FISH. Because the TSA reaction used for fluorescent detection of transcripts rapidly proceeds to completion, we began by using alkaline phosphatase-based detection to directly compare the rate of development of various probes while varying conditions including fixation, bleaching, permeabilization, hybridization buffer, and hybridization temperature. We first tested the effects of these variations using readily detected transcripts, including the neoblast marker smedwi-1; moderately detected transcripts, including a vacuolar ATPase B subunit we have identified as being upregulated in the intestine (Smed-vatpaseB), and the midline marker Smed-slit-1; and with weakly detected transcripts including the hunchback-like transcription factor, Smed-hb, reported to be broadly expressed .
Modified blocking and wash buffers dramatically improve signal specificity
One of the challenges in achieving high signal sensitivity for FISH is that the TSA reaction proceeds rapidly to completion and cannot be monitored and stopped when an optimal signal-to-noise ratio has been reached. Therefore, eliminating weak background staining is vital for optimal signal sensitivity when using the TSA reaction for FISH.
Quenching endogenous autofluorescence with copper sulfate
An additional approach for improving signal sensitivity is to reduce or eliminate autofluorescence. While there are a variety of causes for autofluorescence, the broad range of autofluorescence in planarians, its increase following incubation at high temperatures, and its resistance to photobleaching (not shown) is similar to lipofuscin-based fluorescence observed in tissues of other animals [29, 30]. Incubation in copper sulfate solution has been reported to quench lipofuscin-based autofluorescence [29, 30]. To test the ability of copper sulfate to reduce background signal in planarians, we incubated heat-treated animals for 1 hour in copper sulfate solution (10 mM CuSO4, 50 mM ammonium acetate pH 5.0) and imaged using identical settings to the unheated and heat-treated samples. The copper sulfate treatment dramatically reduced autofluorescence at all wavelength ranges examined (Figure 4C, F, I, and L).
The nearly complete elimination of autofluorescence we observed was very encouraging. However, treatment with copper sulfate has been reported to quench some fluorophores . To test whether the benefits of copper sulfate outweigh its potential harm to signal, we analyzed expression of Smed-EGFR-5 (EGFR-5), which is detected at moderate levels in protonephridia , using TAMRA-conjugated tyramide before and after copper sulfate treatment. Prior to quenching, detection of EGFR-5 in protonephridia was discernible, but autofluorescence in the secretory cells, which have a similar tubular pattern, complicated visualization of signal (Figure 4M). When we imaged the same animal after treatment with copper sulfate we had to increase the gain to achieve a similar level of brightness. However, the signal-to-noise ratio was dramatically improved, greatly facilitating visualization of EGFR-5 (Figure 4N). The significantly enhanced signal-to-noise ratio we observed for EGFR-5 highlights the utility of copper sulfate treatment for analyzing the expression pattern of transcripts with weak-to-moderate signals. Copper sulfate treatment should also be useful for multicolor fluorescence experiments, as we have noticed only minor quenching of DyLight 405-, FAM-, Cy3-, and DyLight 633-tyramides as well as Alexa488-conjugated secondary antibodies following copper sulfate treatment (not shown).
Balancing signal sensitivity while preserving tissue morphology in regenerates
FISH analysis of planarians within the first few days following amputation presents a challenge, as the blastema tissue is particularly fragile and can be easily damaged by the aggressive treatments required to permeabilize mature tissues sufficiently. Therefore, it is important to achieve a balance during the permeabilization steps that allows for relatively even penetration of probe into mature tissues without excessively damaging blastema tissue. One strategy for accomplishing this is to adjust Proteinase K concentration and incubation time until a satisfactory result is obtained. Additionally, while experimenting with alternative methods for permeabilizing planarians, we noticed that heat-induced antigen retrieval (HIAR) resulted in slightly weaker signal in intact planarians, but allowed for consistent and even labeling throughout the animal while causing less damage to superficial layers compared to Proteinase K treatment (not shown).
Enhancing signal intensity through iterative TSA
For particularly low-abundance transcripts, gene expression patterns can be especially difficult to determine due to low signal intensity. While new and more sensitive imaging systems are vastly improving the ability to image weak signals, it can still be difficult to rapidly screen expression patterns in multiple samples by epifluorescence to identify animals or regions on which to focus imaging efforts. For example, Smed-nog1 (nog1) is weakly to moderately detected in portions of the central nervous system, around the body margins, at the base of the pharynx, and at the mouth [23, 33]. nog-1 FISH signal following a conventional single TSA is challenging to discern when viewed by eye under epifluorescence, but yet is capable of being detected by confocal microscopy (Figure 5C). In an attempt to boost signal intensity for nog1 we performed TSA first with DNP-conjugated tyramide, then incubated with peroxidase-conjugated anti-DNP antibody followed by a second, iterative, TSA with fluorophore-conjugated tyramide. In the first reaction signal is amplified by covalently depositing multiple DNP-tyramide molecules near the site of antibody binding. The signal is then further amplified by localizing additional peroxidase-conjugated antibody to the sites of DNP deposition and then performing an additional amplification with fluorophore-conjugated tyramide, which can then be visualized. When we performed iterative TSA for nog1 we noticed a dramatic increase in signal intensity that greatly facilitated observation (Figure 5D). At higher magnification it is easy to identify nog1-positive cells near the cephalic ganglia in animals processed with iterative TSA (Figure 5D’), whereas with single TSA, signal is detected just above background (Figure 5C’). nog1 detection is stronger in the ventral nerve cords, and easily observed following either single or iterative TSA (Figure 5C” and D”). The weak expression in cells around the body margin is almost undetectable following single TSA (Figure 5C”’), but after iterative TSA, nog1-expressing cells are easily identified (Figure 5D”’). The improved specificity from the optimized blocking and wash buffers has been particularly beneficial to iterative TSA, as minor background from non-specific antibody binding is greatly amplified with this method. While the extensive washing following TSA with DNP-conjugated tyramide appears critical, we have had success deploying this technique in multicolor FISH experiments without greatly extending the length of the experiment (see Additional file 1 for details).
Azide effectively quenches peroxidase activity without inhibiting subsequent gene detection in multicolor FISH
Application of the in situhybridization modifications to detect difficult transcripts
With the improved signal sensitivity we were able to achieve with our modifications to the planarian FISH protocol, we sought to resolve the expression patterns of a few genes with unclear expression. The transcription factor Smed-hb (hb) is required for normal maintenance and regeneration of protonephridia [15, 38]. However, based on the published expression pattern it is unclear whether hb is expressed in protonephridia. Therefore, whether it acts autonomously in protonephridia or non-autonomously remains unresolved. We used hb as a representative problematic transcript for optimization of our FISH protocol, and found dramatically improved signal sensitivity particularly following formamide bleaching (Figure 1O). While we did observe broad expression of hb following chromogenic detection, we noticed staining of tubular structures consistent with protonephridial expression as well as stronger, punctate expression throughout the animal. To determine whether the tubular expression coincides with protonephridia, we performed FISH for hb and immunostained with anti-acetylated α-Tubulin antibody, which labels cilia in the lumen of protonephridial tubules [39–41]. We observed clear signal for hb surrounding ciliated protonephridia, consistent with hb function being required autonomously in protonephridial cells (Figure 7A). Scimone et al. (2011) have described a population of protonephridial progenitor cells defined by overlapping expression of protonephridial transcription factors . To examine whether the punctate staining we observed for hb might represent protonephridial progenitor cells, we performed double FISH experiments with hb and several protonephridial transcription factors, including Smed-POU2/3, and found that a few of the hb-positive cells also expressed POU2/3 (Figure 7A). While we did observe expression of hb outside of the protonephridia, these results support the possibility that hb function may be required in protonephridial progenitor cells and mature cell types.
The planarian gene Smed-CHD4 (CHD4), is a homolog of the chromatin remodeling gene CHD4/Mi-2, and is required for the normal differentiation of neoblasts . CHD4 has been reported to be broadly expressed and enriched in the central nervous system . Consistent with CHD4 expression in neoblasts, mesenchymal expression is reduced following lethal irradiation, and CHD4 in situ hybridization of sorted cells results in labeling of a significant fraction of neoblast subtypes . While these data provide compelling evidence that neoblasts express CHD4, we wanted to see if we could verify coexpression of CHD4 with other neoblast markers in intact animals. For this we performed multicolor FISH for CHD4 and smedwi-1. While we noticed broad expression of CHD4 throughout the animal, there was clear punctate expression of CHD4 in the cytoplasm of neoblasts (Figure 7B). The punctate, cytoplasmic localization of CHD4 RNA in neoblasts is reminiscent of transcripts present in ribonucleoprotein complexes called chromatoid bodies that are believed to be important sites of post-transcriptional regulation . To examine the possibility that CHD4 mRNA localizes to chromatoid bodies we immunostained with the monoclonal antibody Y12 , which recognizes symmetrical dimethylarginine in proteins associated with chromatoid bodies . While we occasionally observed CHD4 puncta (magenta) near chromatoid bodies (yellow) (Figure 7B open arrowheads), we rarely observed overlap between Y12 immunostaining (Figure 7B arrows) and CHD4 FISH signals. This observation suggests that the punctate signals observed represent subcellular localization of transcripts to cytoplasmic regions other than chromatoid bodies.
The expression patterns for several members of the noggin gene family in planarians have been described . Smed-nog2 (nog2) is one of a few noggin gene family members whose mRNA distribution remained elusive despite confirmation of expression by Reverse Transcription-quantitative PCR. While knockdown of either nog1 or nog2 alone yields no phenotype, nog1; nog2 double knockdown leads to a dorsalization phenotype , further bolstering the likelihood that nog2 is expressed. In single FISH experiments we were able to detect nog2 expression (Additional file 2), and observed a pattern similar to that of nog1. We were curious to determine whether nog1 and nog2 are coexpressed, or whether different cells contribute either nog1or nog2 to regulate dorsoventral polarity. To examine this we performed double FISH for nog1 and nog2. Signal for nog2 was clear but significantly weaker than for nog1. We found only a small percentage of nog1-positive cells that also expressed nog2 in the body margin (Figure 7C). The more limited expression pattern for nog2 compared to nog1 could be real, or may indicate that expression of nog2 in some cells is below the limit of detection. Despite the latter possibility, our ability to at least partially detect gene expression for a gene that has been refractory to analysis highlights the utility of the modifications we have established for this protocol.
The FISH protocol we present here represents a significant improvement in signal sensitivity for the rapidly growing planarian field. Additionally, the modifications we have developed may be beneficial for FISH in other model systems. The short formamide bleaching step seems to improve tissue permeabilization properties, and therefore may be a useful addition to WISH protocols in other organisms where removal of pigment may or may not be a necessary step. Tissue autofluorescence is not unique to planarians, and while there are multiple causes of autofluorescence, treatment with copper sulfate may provide similar benefits for FISH in other organisms. The peroxidase-conjugated antibodies used here are commonly employed for FISH in numerous model systems, therefore the modified blocking and wash buffers described here will no doubt improve FISH sensitivity in other organisms, and the azide treatment will be directly applicable in other multicolor FISH experiments.
For large/complex organisms it can be difficult to rapidly identify cells or tissues expressing low-abundance transcripts. To improve initial screening we have developed methods for iterative TSA, which significantly improve signal intensity, facilitating identification of expression domains. Finally, while HIAR methods have been extensively used for tissue permeabilization in immunofluorescence protocols [46, 47], we show that this method can also be applied for FISH, enabling sufficient permeabilization with improved morphology of fragile tissues. Additionally, this method may be widely useful in allowing for the use of some antibodies that are sensitive to Proteinase K treatment following FISH. Together, the enhanced signal specificity resulting from the modifications presented here will no doubt be useful for shedding light on how planarians achieve their remarkable regenerative capacity.
Asexual Schmidtea mediterranea clonal line CIW4  was maintained in the dark at 20°C in deionized water containing 0.5 g/L Instant Ocean Sea Salts. Animals were fed pureed organic calf liver 1–2 times per week and starved for 1 week before use.
Hapten-labeled anti-sense RNA probes were generated from in vitro transcription reactions containing either DIG-12-UTP (Roche), DNP-11-UTP (PerkinElmer), or FAM-12-UTP (Roche) according to the manufacturer’s suggested protocol (Roche). DNA template for the in vitro transcription reaction was generated by PCR amplifying sequences from gene clones obtained from either the S. mediterranea EST Database  or from cDNA clones generated using standard methods. Primers and unincorporated nucleotides were removed from PCR products using a DNA clean and concentrator kit (Zymo Research) prior to use as template in transcription reactions. Probes were precipitated using LiCl/ethanol according to the manufacturer’s suggested protocol (Roche) and resuspended in 50 μl RNAse-free water. Probe quality and concentration were assessed on a 1% agarose gel and using a NanoDrop ND-1000 spectrophotometer. Probe concentration was adjusted to 50 ng/μl by adding hybridization buffer (see Additional file 1) and probes were stored at −20°C. In some cases, FAM-labeled probes were further purified using a sephadex G-50 quick spin column (Roche) according to the manufacturer’s recommendations.
Animal pretreatment and hybridization
Unless otherwise noted, asexual planarians 1–5 mm in length were processed for WISH essentially as described  with the following significant modifications: the reduction step prior to dehydration was omitted. Bleaching was performed for 2 hours in formamide bleaching solution (1.2% H2O2, 5% formamide, and 0.5xSSC ). For regenerating planarians, the Proteinase K/post fixation steps were replaced with a 10 minute boiling step in 10 mM sodium citrate pH 6.0 with 0.05% Tween20, followed by a 20 minute room temperature incubation in PBSTx (Phosphate Buffered Saline , 0.3% Triton X-100) with 1% SDS. Blocking and antibody incubation for peroxidase-conjugated anti-digoxigenin (1:2,000 [Roche]), anti-fluorescein (1:2,000 [Roche]), and anti-dinitrophenol (1:300 [PerkinElmer]) were performed with 5% horse serum and 0.5% RWBR in TNTx (100 mM Tris pH 7.5, 150 mM NaCl, 0.3% Triton X-100). For chromogenic detection using alkaline phosphatase-conjugated anti-digoxigenin antibody (1:2,000 [Roche]), antibody incubation and blocking were performed with 5% horse serum in TNTx, and post-antibody washes were with TNTx prior to development as described in .
Ciliated protonephridia were labeled with anti-acetylated α-Tubulin antibody (6-11B-1, Santa Cruz Biotech) diluted 1:1,000 with 5% horse serum and 0.5% RWBR in TNTx. Chromatoid bodies were labeled with the monoclonal antibody, Y12 (NeoMarkers) diluted 1:250 with 0.6% IgG-free BSA and 0.45% fish gelatin in PBSTx as described in .
Tyramide conjugates were synthesized as described  from N-hydroxy-succinimidyl-esters of 5/6-carboxyfluorescein (Pierce), 5-(and-6)-carboxytetramethylrhodamine (Molecular Probes), DyLight 633 (Pierce), and 6-(2,4-dinitrophenyl) amino hexanoic acid (Molecular probes). Tyramide signal amplification was performed by incubating planarians for 10 min in fluorophore-conjugated tyramide diluted 1:250–1:500 in 100 mM borate buffer pH 8.5, 2 M NaCl, 0.003% H2O2, and 20 μg/ml 4-iodophenylboronic acid. For double FISH experiments, residual peroxidase activity was quenched by incubating for 45 minutes in 100 mM glycine pH 2.0 or in PBSTx containing either 2% H2O2, 4% formaldehyde, or 100 mM sodium azide.
Animals were cleared in 80% (v/v) glycerol and mounted on slides. Planarians developed using the chromogenic alkaline phosphatase substrate NBT/BCIP were imaged on a Leica M205A microscope equipped with a Leica DFC420 camera and a Leica TL 4000 RC base that was adjusted for Rottermann contrast. Fluorescent images were collected on a Carl Zeiss LSM710 confocal microscope running ZEN 2011. Images were processed using ImageJ 1.47f .
In cases where animal autofluorescence was significant, animals were gently removed from slides, washed in PBSTx, washed two times in deionized water, and incubated for 1 hour in freshly prepared 10 mM copper sulfate; 50 mM ammonium acetate pH 5.0. Following the copper sulfate quench, animals were washed two times in deionized water and then PBSTx before being cleared in 80% glycerol and remounted on slides.
Whole-mount in situ hybridization
Whole-mount fluorescent in situ hybridization
5- and 6- carboxytetramethylrhodamine
Heat-induced antigen retrieval
Tyramide signal amplification
PerkinElmer Blocking Reagent
Roche Western Blocking Reagent.
We thank members of the Newmark laboratory for useful feedback, particularly Dave Forsthoefel and Bo Wang for insightful discussion and collaboration on aspects of the FISH optimization. We thank Tracy Chong, Melanie Issigonis, Rachel Roberts-Galbraith and Labib Rouhana for helpful comments on the manuscript. Some cDNA clones used in this study were generously provided by Rachel Roberts-Galbraith. P.A.N. is an investigator of the Howard Hughes Medical Institute.
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