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An optimised whole mount in situ hybridisation protocol for the mollusc Lymnaea stagnalis.

Hohagen J, Herlitze I, Jackson DJ - BMC Dev. Biol. (2015)

Bottom Line: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis.These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH.We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal.

View Article: PubMed Central - PubMed

Affiliation: Department of Geobiology, Geosciences Centre, Georg-August University of Göttingen, Goldschmidtstrasse 3, 37077, Göttingen, Germany. jhohage@uni-goettingen.de.

ABSTRACT

Background: The ability to visualise the expression of individual genes in situ is an invaluable tool for developmental and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and through inter-specific comparisons, the evolutionary history of unique morphological features. For well-established model organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automated for high-throughput analyses. While the overall concept of in situ hybridisation is simple (hybridise a single-stranded, labelled nucleic acid probe complementary to a target of interest, and then detect the label immunologically using colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the final result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an in situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogenetic stage within a species.

Results: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal.

Conclusions: This method that we present here will be of value to investigators employing L. stagnalis as a model for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), and brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.

No MeSH data available.


Related in: MedlinePlus

A pre-hybridisation treatment with different SDS concentrations significantly affects the WMISH signal.L. stagnalis larvae of different ages were subjected to pre-hybridisation treatments with varying amounts of SDS and then hybridised with anti-sense probes to beta tubulin(A-I), engrailed(J-O) and COE(P-U). For all genes and larval ages, treatment with 0.1% SDS did not generate consistent or strong WMISH signals (A, D, G, J, M, P and S). Treatments with both 0.5% and 1% SDS produced strong WMISH signals for beta tubulin and engrailed in larvae aged three to five days post first cleavage (dpfc), with high spatial resolution (inlet in K). For COE 0.5% SDS outperformed the 1% SDS treatment (T vs. U). Black stars indicate optimal treatments. Note that some treatments produced equally good results. The most consistent results (defined as constantly good signals among genes and ontogenetic stages with little variation between individuals within an experiment) were achieved with 0.5% SDS (examples shown in B’, E’, H’, K’, N’, Q’ and T’). Larvae in A-C and M-R are shown from an apical perspective, larvae in D-F are viewed ventrally, G-I laterally and J-L and S-U dorsally.
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Fig3: A pre-hybridisation treatment with different SDS concentrations significantly affects the WMISH signal.L. stagnalis larvae of different ages were subjected to pre-hybridisation treatments with varying amounts of SDS and then hybridised with anti-sense probes to beta tubulin(A-I), engrailed(J-O) and COE(P-U). For all genes and larval ages, treatment with 0.1% SDS did not generate consistent or strong WMISH signals (A, D, G, J, M, P and S). Treatments with both 0.5% and 1% SDS produced strong WMISH signals for beta tubulin and engrailed in larvae aged three to five days post first cleavage (dpfc), with high spatial resolution (inlet in K). For COE 0.5% SDS outperformed the 1% SDS treatment (T vs. U). Black stars indicate optimal treatments. Note that some treatments produced equally good results. The most consistent results (defined as constantly good signals among genes and ontogenetic stages with little variation between individuals within an experiment) were achieved with 0.5% SDS (examples shown in B’, E’, H’, K’, N’, Q’ and T’). Larvae in A-C and M-R are shown from an apical perspective, larvae in D-F are viewed ventrally, G-I laterally and J-L and S-U dorsally.

Mentions: Between one and five dpfc old embryos and larvae of L. stagnalis were treated with different amounts of the anionic detergent SDS prior to hybridisation (Figure 3). A permeabilising treatment with 0.1% SDS did not produce strong WMISH signals for all studied genes and larval ages (Figure 3A, D, G, J, M, P and S) whereas treatments with higher concentrations of SDS generated strong WMISH signals. For two of the genes we studied here, beta tubulin and engrailed, treating larvae between three to five dpfc with 0.5% or 1% SDS produced equally good results. In contrast, the staining was more intense after treatment with 0.5% SDS than with 1% SDS for COE (Figure 3Q vs. R and T vs. U) as well as for beta tubulin in two dpfc old larvae (Figure 3B vs. C), which may suggest a loss of the target transcripts due to excess permeabilisation of these younger stages. Additionally, embryos between one and two dpfc tend to adhere to plastic surfaces in 1% SDS. While in other animal systems SDS is commonly used at a concentration of 1% [18-20], in L. stagnalis strong WMISH signals were most consistently achieved with 0.5% SDS across different genes and ontogenetic stages.Figure 3


An optimised whole mount in situ hybridisation protocol for the mollusc Lymnaea stagnalis.

Hohagen J, Herlitze I, Jackson DJ - BMC Dev. Biol. (2015)

A pre-hybridisation treatment with different SDS concentrations significantly affects the WMISH signal.L. stagnalis larvae of different ages were subjected to pre-hybridisation treatments with varying amounts of SDS and then hybridised with anti-sense probes to beta tubulin(A-I), engrailed(J-O) and COE(P-U). For all genes and larval ages, treatment with 0.1% SDS did not generate consistent or strong WMISH signals (A, D, G, J, M, P and S). Treatments with both 0.5% and 1% SDS produced strong WMISH signals for beta tubulin and engrailed in larvae aged three to five days post first cleavage (dpfc), with high spatial resolution (inlet in K). For COE 0.5% SDS outperformed the 1% SDS treatment (T vs. U). Black stars indicate optimal treatments. Note that some treatments produced equally good results. The most consistent results (defined as constantly good signals among genes and ontogenetic stages with little variation between individuals within an experiment) were achieved with 0.5% SDS (examples shown in B’, E’, H’, K’, N’, Q’ and T’). Larvae in A-C and M-R are shown from an apical perspective, larvae in D-F are viewed ventrally, G-I laterally and J-L and S-U dorsally.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4379745&req=5

Fig3: A pre-hybridisation treatment with different SDS concentrations significantly affects the WMISH signal.L. stagnalis larvae of different ages were subjected to pre-hybridisation treatments with varying amounts of SDS and then hybridised with anti-sense probes to beta tubulin(A-I), engrailed(J-O) and COE(P-U). For all genes and larval ages, treatment with 0.1% SDS did not generate consistent or strong WMISH signals (A, D, G, J, M, P and S). Treatments with both 0.5% and 1% SDS produced strong WMISH signals for beta tubulin and engrailed in larvae aged three to five days post first cleavage (dpfc), with high spatial resolution (inlet in K). For COE 0.5% SDS outperformed the 1% SDS treatment (T vs. U). Black stars indicate optimal treatments. Note that some treatments produced equally good results. The most consistent results (defined as constantly good signals among genes and ontogenetic stages with little variation between individuals within an experiment) were achieved with 0.5% SDS (examples shown in B’, E’, H’, K’, N’, Q’ and T’). Larvae in A-C and M-R are shown from an apical perspective, larvae in D-F are viewed ventrally, G-I laterally and J-L and S-U dorsally.
Mentions: Between one and five dpfc old embryos and larvae of L. stagnalis were treated with different amounts of the anionic detergent SDS prior to hybridisation (Figure 3). A permeabilising treatment with 0.1% SDS did not produce strong WMISH signals for all studied genes and larval ages (Figure 3A, D, G, J, M, P and S) whereas treatments with higher concentrations of SDS generated strong WMISH signals. For two of the genes we studied here, beta tubulin and engrailed, treating larvae between three to five dpfc with 0.5% or 1% SDS produced equally good results. In contrast, the staining was more intense after treatment with 0.5% SDS than with 1% SDS for COE (Figure 3Q vs. R and T vs. U) as well as for beta tubulin in two dpfc old larvae (Figure 3B vs. C), which may suggest a loss of the target transcripts due to excess permeabilisation of these younger stages. Additionally, embryos between one and two dpfc tend to adhere to plastic surfaces in 1% SDS. While in other animal systems SDS is commonly used at a concentration of 1% [18-20], in L. stagnalis strong WMISH signals were most consistently achieved with 0.5% SDS across different genes and ontogenetic stages.Figure 3

Bottom Line: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis.These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH.We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal.

View Article: PubMed Central - PubMed

Affiliation: Department of Geobiology, Geosciences Centre, Georg-August University of Göttingen, Goldschmidtstrasse 3, 37077, Göttingen, Germany. jhohage@uni-goettingen.de.

ABSTRACT

Background: The ability to visualise the expression of individual genes in situ is an invaluable tool for developmental and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and through inter-specific comparisons, the evolutionary history of unique morphological features. For well-established model organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automated for high-throughput analyses. While the overall concept of in situ hybridisation is simple (hybridise a single-stranded, labelled nucleic acid probe complementary to a target of interest, and then detect the label immunologically using colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the final result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an in situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogenetic stage within a species.

Results: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal.

Conclusions: This method that we present here will be of value to investigators employing L. stagnalis as a model for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), and brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.

No MeSH data available.


Related in: MedlinePlus