Hohagen J et al. (2015), An optimised whole mount in situ hybridisation ...

ECB-ART-43924

BMC Dev Biol 2015 Mar 28;15:19. doi: 10.1186/s12861-015-0068-7.

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

Hohagen J , Herlitze I , Jackson DJ .

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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.

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Species referenced: Echinodermata
Genes referenced: adcy10 ebf2 en1 LOC100887844 LOC583082 LOC588616 LOC594261 tubgcp2

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	Figure 1. Overview of the early larval development of L. stagnalis . During the first five days of development, embryos of L. stagnalis undergo drastic changes in size (A-E, images are to the same scale shown in E), tissue composition (F-J) and form all main larval structures (K-O). Indicated are the positions of the apical plate (ap), the eye (ey), the foot lobe (fl) or foot (f), the developing mantle margin (mm), the prototroch (pt), the shell (s) and the blastopore (bp) or stomodaeum (st). All ages are indicated in hours post first cleavage (h). F-H and K-M are ventral views with scale bars representing 50 Î¼m. I, J, N and O are lateral views with scale bars of 100 Î¼m. The scale bar in panel E is 500 Î¼m. Panels I, J, N and O are reflected about the vertical axis for consistency of presentation.
	Figure 2. Overview of WMISH signals generated after pre-hybridisation treatment with NAC and/or reduction. L. stagnalis larvae of different ages were subjected to a WMISH protocol similar to that described by Jackson et al. [22] (A, E, I, M and Q). This protocol was then modified by the addition of a reduction treatment (B, F, J, N and R), a NAC treatment (C, G, K, O and S) or a combination of both NAC and reduction treatment (D, H, L, P and T). Using this set of pre-hybridisation treatments, the optimal sample preparation regime for WMISH varies with respect to the target gene and the developmental stage. For engrailed and beta tubulin in younger larvae, the samples that underwent a reduction treatment display the best signal to noise ratio (B, J and N). Excess background that is revealed by reduced samples for COE and beta tubulin in older larvae (F and R) is diminished by a treatment with NAC (H and T). Black stars indicate optimal results after sample preparation involving NAC treatment and reduction. Panels A to H show larvae from a lateral perspective with the shell field oriented to the right. Larvae in I to P are viewed from dorsal and Q to T are viewed from apical.
	Figure 3. 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 Bâ€™, Eâ€™, Hâ€™, Kâ€™, Nâ€™, Qâ€™ and Tâ€™). 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.
	Figure 4. Non-specific probe binding to the shell field and radular is eliminated by treatment with TEAAA. We observe a well-defined and consistent WMISH stain for a variety of probes (represented here with a probe against the gene â€œcontig 380566â€) in the periphery of the shell field (arrows in B and C) and in the radular sac (arrow in D). Probes against other genes (for example beta tubulin) do not produce these patterns (A). The stain in the shell field periphery and the radular remains following a pre-hybridisation treatment with RNAse (F-H), while the specific signal against beta tubulin is abolished (E). This indicates that the signals in the shell field and the radular are the result of non-specific probe binding. Treatment with TEAAA abolishes this non-specific stain (J-L), while the specific signal against beta tubulin remains unaltered (I). B, F and J are dorsal views. A, C, E, G, I and K are lateral views of larvae with the shell gland oriented to the right. D, H and L are ventral views. Panels C, G and K are reflected about the vertical axis for consistency of presentation.
	Figure 5. Our optimised WMISH protocol is not improved by more antibody, PVA or hydrolysed riboprobes. Larvae three to four days post first cleavage (dpfc) were subjected to our optimised WMISH protocol (A, F, K and P). Using a beta tubulin probe, we investigated the effect of increasing the amount of anti-DIG antibody (B, G, L and Q), the addition of PVA to the colour detection solution (C, H, M and R) and the combination of more antibody and PVA (D and I). We also assessed the effect of hydrolysing the engrailed riboprobe individually (N and S) and in combination with a higher antibody concentration and the use of PVA (O and T). None of these modifications generated superior WMISH results to our baseline protocol. Samples incubated in more antibody and developed with PVA showed slightly more intense signals, but a lower signal to noise ratio (B, C, G, H, L, M, Q and R). PVA also appeared to compromise the morphological integrity of older larvae (H, I and R). Signals generated by the hydrolysed engrailed probe were much fainter and were partially masked by an increase in general background staining (N, O, S and T). The optimal treatment (A, F, K, and P) is indicated by a black star. Control WMISH experiments lacking a riboprobe and using the increased antibody concentration do not reveal any staining (E and J). All images of individual larvae are lateral views.
	Figure 6. Our WMISH protocol is suitable for fluorescent signal detection. The expression of COE (A), engrailed (B) and beta tubulin (C and D) in larvae treated with 0.5% SDS was detected using the fluorescent substrate Fast Red (A-D). Panel E is a scanning electron micrograph of a 62Â hours post first cleavage (hpfc) old larva with the approximate localisation of the COE expression indicated in red. Panel F is a lateral perspective of a 90 hpfc old larva with indicated engrailed expression. Panels G and H show the positions of beta tubulin expression in a 57 hpfc old larva (G, ventral perspective) and a 100 hpfc old larva (H, lateral perspective). Panels B-D are projections of confocal laser scanning micrographs. Panels B, D and H are reflected about the vertical axis for consistency of presentation. All scale bars are 50Â Î¼m.

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