In situ Protocol for Butterfly Pupal Wings Using Riboprobes

DAY 1

1. Prepare the following solutions:
   • 10x PBS
   • 1x PBS
   • 1x PBT
   • ddH₂O
     • Add DEPC for 0.1% total volume and shake solutions vigorously. 
     • Autoclave.
   • 4% Paraformaldehyde Fix - using PBS-DEPC
   • Proteinase K solution - if precipitate is present, vortex vigorously before removing aliquot
   • Digestion Stop Buffer
   • Prehybridization Buffer
   • 50:50 PBT:Prehybridization Buffer
   • Hybridization Buffer
     • Solutions should be kept RNase-free. Use either glass-ware that has been baked (4 hours at 250°C) or disposable plastic. Serological pipettes are very useful for making up solutions
2. Dissect wings from time-staged pupae in PBS
3. Move wings directly to Fix Buffer in wells of 24 well culture plate at room temperature
4. Fix discs for 2 hours
5. Wash 5 x 5 minutes in PBT
6. Incubate wings for 3 minutes in Proteinase K solution
7. Rinse 2 x in Digestion Stop Buffer
8. Wash 5 x 5 minutes in PBT
9. Wash 2 x 5 minutes in 50:50 PBT : PHB
10. Wash 1 x 10 minutes in PHB
11. Incubate in PHB at least 1 hour at 55°C.
12. Heat-denature RNA probe (20-50ng needed per well) (80°C for 5 minutes) and add to hybridization buffer (100 μl needed per well)
13. Add probe to wells and incubate 48 hours at 55°C 

DAY 3

1. Wash 4 x 5 minutes in 55°C PHB
2. Incubate overnight in PHB at 55°C

DAY 4

1. Prepare the following solutions:
   • Antibody Block Buffer
2. Wash 1 x 5 minutes in 50:50 PBT : PHB
3. Wash 4 x 5 minutes in PBT
4. Incubate wings for 1 hour in Block Buffer at 4°C
5. Incubate wings in a 1:2000 dilution of anti-Dig antibody overnight at 4°C

DAY 5

1. Prepare following solutions:
   • Detection Buffer
   • Developing Solution - light sensitive (wrap in foil)
2. Wash 3 x 5 minutes in PBT at room temperature
3. Wash 7 x 5 minutes in PBT
4. Rinse wings two times in Detection Buffer
5. Develop wings in 1 ml of Developing Solution - time of development must be monitored each time - developing times can change even when using the same probes and developing solution - generally check after 5-10 minutes and then increase intervals up to overnight. Plate should be wrapped in foil to prevent light exposure when not checking samples.
6. Rinse five times in Developing Stop Buffer
7. Mount wings.

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.