In order to examine gene expression in the pupal wing tissue of Bicyclus anynana, we present an optimized protocol for in situ hybridizations using riboprobes. We also provide guidelines for the further optimization of this protocol for use in pupal wings of other Lepidopteran species.
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Optimization of existing protocols
In situ hybridizations on larval wing discs have been successfully performed using discs from Precis coenia butterflies (Carroll et al. 1994; Keys et al. 1999; Weatherbee et al. 1999). The current protocol has been adapted from a detailed written protocol available upon request from the Carroll Lab for larval wing stainings. The changes we made serve to accommodate differences between larval and pupal wing tissues. During pupal hindwing dissections, the peripodial membrane should be removed and not included in the wells. The initial fix time is much longer in our protocol but the post-fix step has been removed. We found that a 10-fold dilution of the Proteinase K solution was necessary for the fragile pupal wing tissue while still allowing for successful probe penetration. We also found that blocking the wings before antibody staining greatly reduced background, whereas increasing the antibody incubation to overnight at 4°C increased signal strength.
Applications
The applications of this protocol are manifold. Being able to localize the expression of a candidate gene on a developing pupal wing will be the first step in implicating this gene in some functional role during wing or wing pattern development (Marcus et al. 2004; Ramos et al. 2006). If several genes that are known to interact in other systems are co-expressed together on the wings this may implicate the co-option of more elaborate gene networks in specifying novel wing patterns (Monteiro et al. 2006). Butterfly wing pattern evo-devo provides a rich system where pertinent evo-devo questions involving the processes of gene and gene network co-option, the evolution of novelties, the evolution of convergent and parallel traits, the evolution of serial homology, and of gene duplication and sub-functionalization can all be studied in an integrated fashion. Moreover, butterflies display a bewildering variety of wing patterns that play a role in species recognition, sexual selection, mimicry, thermoregulation, and predator avoidance. Understanding both the genetic and developmental basis behind the generation of these patterns, as well the ecological factors that favor certain patterns can bring us to a holistic understanding of the evolutionary process and of the biases and constraints imposed upon this process by developmental systems.
Optimization guidelines for adaptation to different species
When adapting this protocol to wings of different species, the main concern will be making the tissue permeable to the probe while maintaining the integrity of the tissue. Therefore, the fixation and permeablization steps will have to be optimized. For Bicyclus pupal wings, we have found that a 2 hour fix at room temperature in fresh PFA buffer and a gentle Proteinase K digestion is sufficient. Tissues may be fixed up to 12 hours at 4°C if necessary but it is possible to over-fix tissue which will reduce signal, so shorter fixation times are preferred. Both the enzyme concentration and the length and temperature of digestion should be determined empirically for each tissue. The age of the wings may also be a significant factor in the digestions as older wings will have more elaborate cuticular structures and nay require a longer digestion. It is important to remember that Proteinase K preparations that are available commercially are not standardized for a specific activity and, therefore, digestion conditions may also need to be adjusted when changing lots. The permeability of tissues can also be increased by incubation in detergent solutions, for instance a 1% Triton-X solution. This could be added prior to the prehybridization step.
Another concern will be to optimize probe size, general rule of thumb being bigger is better. Bob Reed, who has performed late pupal wing in situs in Heliconius, suggested 300 bp as a target size (personal communication). Larger probes, which may increase the specificity of the signal, may be hydrolyzed to aid their entry into the cells. In other systems, however, researchers routinely use 1 kb probes without hydrolyzing them. Here we used probes around 300bp as well which worked fine.
Working with riboprobes can be intimidating for labs not used to handling RNA. We have found this technique to be fairly robust and to contain several steps that minimize loss of probe due to RNase activity. The Proteinase K digestion will remove RNase contamination and the 50% formamide of the prehybridization/hybridization buffers will inhibit RNase activity (Chomczynski 1992). Therefore, we encourage people to use riboprobes which increase the specificity of the signal and are not as daunting as some may think.
We thank Jayne Selegue, Margaret Hollingsworth, Jin Berry, Ryo Futahashi, Najmus Sahar Mahfooz, Aleksandar Popadic, Bob Reed, Roche Technical Support for help in troubleshooting this protocol. We also thank William Piel for advice on editing the movie.
Material Name | Type | Company | Catalogue Number | Comment |
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Fix Buffer | 4% Paraformaldehyde in PBS | |||
PBT | 0.1% Tween 20 in PBS | |||
Proteinase K solution | 2.5g/ml Proteinase K in PBT | |||
Digestion Stop Buffer | 2 mg/ml glycine in PBT | |||
Pre-Hybridization Buffer | For 50 ml PHB: 12 ml DEPC treated water + 25 ml Formamide + 12.5 ml 20 x SSC + 50 ul Tween 20 + 500 ul 10 mg/ml salmon sperm (Rnase free, heat denatured prior to addition to solution). | |||
Hybridization Buffer | Add 1 mg/ml glycogen to prehybridization buffer | |||
Block Buffer | 50 mM Tris pH 6.8, 150 mM NaCl, 0.5% IGEPAL (NP40), 5 mg/ml BSA | |||
Anti-DIG | Ab | Roche Applied Science | 11 093 274 910 | Alkaline Phosphatase conjugated |
DIG Wash and Block Buffer Set | Roche Applied Science | 11 585 762 001 | ||
Crystal Mount Aqueous Mounting Medium | Sigma | C0612 | Mounting Medium |