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

BMC Developmental Biology

Daniel John Jackson¹  Ines Herlitze¹  Jennifer Hohagen¹ 

[1]Department of Geobiology, Geosciences Centre, Georg-August University of Göttingen, Goldschmidtstrasse 3, Göttingen, 37077, Germany

关键词: Development;    Evolution;    Gene expression;    Lymnaea stagnalis;    Mollusc;    Whole mount in situ hybridisation;   

Others  :  1160974
DOI  :  10.1186/s12861-015-0068-7

 received in 2014-12-15, accepted in 2015-03-04,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Hohagen et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Christodoulou F, Raible F, Tomer R, Simakov O, Trachana K, Klaus S, et al.: Ancient animal microRNAs and the evolution of tissue identity. Nature 2010, 463:1084-8.
• [2]Arendt D: The evolution of cell types in animals: emerging principles from molecular studies. Nat Rev Gen 2008, 9:868-82.
• [3]Hashimoto N, Kurita Y, Wada H: Developmental role of dpp in the gastropod shell plate and co-option of the dpp signaling pathway in the evolution of the operculum. Dev Biol 2012, 366:367-73.
• [4]Raven CP: Morphogenesis in Limnaea stagnalis and its disturbance by lithium. J Exp Zool 1952, 121:1-77.
• [5]Timmermans LPM: Studies on shell formation in molluscs. Neth J Zool 1969, 19:63-78.
• [6]Kniprath E: Zur Ontogenese des Schalenfeldes von Lymnaea stagnalis. Dev Gen Evol 1977, 181:11-30.
• [7]Liu MM, Davey JW, Banerjee R, Han J, Yang F, Aboobaker A, et al.: Fine mapping of the pond snail left-right asymmetry (Chirality) locus using RAD-Seq and Fibre-FISH. PLoS One 2013, 8:e71067.
• [8]Hohagen J, Jackson D: An ancient process in a modern mollusc: early development of the shell in Lymnaea stagnalis. BMC Dev Biol 2013, 13:27. BioMed Central Full Text
• [9]Voronezhskaya E: Apical sensory neurones mediate developmental retardation induced by conspecific environmental stimuli in freshwater pulmonate snails. Development 2004, 131:3671-80.
• [10]Raven CP: The development of the egg of limnaea stagnalis L. From the first cleavage till the trochophore stage, with special reference to its “Chemical Embryology”. Arch Neer Zool 1946, 7:353-434.
• [11]Elbers PF, Bluemink JG: Pinocytosis in the developing egg of Limnaea stagnalis L. Exp Cell Res 1960, 21:619-22.
• [12]Morrill JB: Development of the Pulmonate Gastropod, Lymnaea. In Developmental biology of freshwater invertebrates. Edited by Harrison FW, Cowden RR. Alan R. Liss, Inc, New York; 1982:399-483.
• [13]Bayne CJ: Histochemical studies on the egg capsules of eight gastropod molluscs. J Moll Stud 1968, 38:199-212.
• [14]Iijima M, Takeuchi T, Sarashina I, Endo K: Expression patterns of engrailed and dpp in the gastropod Lymnaea stagnalis. Dev Genes Evol 2008, 218:237-51.
• [15]Shimizu K, Sarashina I, Kagi H, Endo K: Possible functions of Dpp in gastropod shell formation and shell coiling. Dev Genes Evol 2011, 221:59-68.
• [16]Shimizu K, Iijima M, Setiamarga D, Sarashina I, Kudoh T, Asami T, et al.: Left-right asymmetric expression of dpp in the mantle of gastropods correlates with asymmetric shell coiling. EvoDevo 2013, 4:15. BioMed Central Full Text
• [17]Pearson BJ, Eisenhoffer GT, Gurley KA, Rink JC, Miller DE, Sánchez Alvarado A: Formaldehyde-based whole-mount in situ hybridization method for planarians. Dev Dyn 2009, 238:443-50.
• [18]King RS, Newmark PA: In situ hybridization protocol for enhanced detection of gene expression in the planarian Schmidtea mediterranea. BMC Dev Biol 2013, 13:8. BioMed Central Full Text
• [19]Browne W, Schmid B, Wimmer E, Martindale MQ: Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis. Dev Gen Evol 2006, 216:581-95.
• [20]Price A, Patel N: Investigating divergent mechanisms of mesoderm development in arthropods: the expression ofPh-twist andPh-mef2 in Parhyale hawaiensis. J Exp Zool 2008, 310B:24-40.
• [21]Jackson D, McDougall C, Green K, Simpson F, Worheide G, Degnan B: A rapidly evolving secretome builds and patterns a sea shell. BMC Biol 2006, 4:40. BioMed Central Full Text
• [22]Jackson DJ, Wörheide G, Degnan BM: Dynamic expression of ancient and novel molluscan shell genes during ecological transitions. BMC Evol Biol 2007, 7:160. BioMed Central Full Text
• [23]Zeller R, Rogers M, Haramis AG, Carrasceo AE: In situ hybridization to cellular RNA. Current Protocols in, Molecular Biology; 2001.
• [24]Jowett T: Double in situ hybridization techniques in Zebrafish. Methods 2001, 23:345-58.
• [25]Hauptmann G, Gerster T: Multicolor whole-mount in situ hybridization. In Methods in molecular biology. Edited by Tuan RS, Lo CW. Humana Press, Totowa; 2000:139-48.
• [26]Shain DH, Zuber MX: Sodium dodecyl sulfate (SDS)-based whole- mount in situ hybridization of Xenopus laevis embryos. J Biochem Biophys Methods 1996, 31:185-8.
• [27]Jezzini SH, Bodnarova M, Moroz LL: Two-color in situ hybridization in the CNS of Aplysia californica. J Neurosci Methods 2005, 149:15-25.