Whole mount in situ hybridization (WISH) is a scientific method used to create a three-dimensional map of gene expression within an intact organism or tissue. It shows the precise locations of messenger RNA (mRNA), which carries a gene’s instructions for making a protein. By revealing where a gene is turned on, WISH gives researchers clues about that gene’s function during development or disease.
The technique uses a labeled probe that seeks out and binds to a specific mRNA sequence. This process creates a visible signal, effectively “lighting up” the cells where a particular gene is working. This provides a blueprint, not of the organism’s structure, but of the genetic activity happening within it.
The WISH Process Explained
The first step is preparing the sample, often an embryo or small organ, through fixation. Chemicals like formaldehyde are used to preserve the tissue, freezing cellular components in place. This ensures the delicate mRNA molecules do not degrade and remain in their original locations for an accurate snapshot of gene activity.
Next, the sample is made permeable so the molecular probe can enter the cells. Detergents or enzymes, such as proteinase K, create microscopic pores in the cell membranes. This step requires a balance between allowing probe entry and maintaining the tissue’s structural integrity.
Hybridization is the main event, where the sample is incubated with the molecular probe. This probe is a complementary, mirror-image sequence to the target mRNA. The sample is heated, causing the probe to bind specifically to its target mRNA strand, forming a stable hybrid molecule.
After hybridization, a series of washes removes any unbound or loosely attached probes. These steps use solutions with varying salt concentrations at specific temperatures to ensure only the correctly bound probes remain. This cleanup is necessary for a clear signal without confusing background noise.
The final step is detection. The bound probe, which is tagged, is made visible using a multi-step chemical reaction. This reaction produces a colored precipitate, often deep purple or blue, that deposits at the site of the probe, revealing exactly where the gene is active.
The Molecular Probe
The molecular probe is a custom-designed tool at the heart of the WISH technique. It is a small, single-stranded piece of nucleic acid, usually RNA (a riboprobe), synthesized to be perfectly complementary to the target mRNA. This design allows it to bind with high precision to a specific gene’s transcript among thousands of others in the cell.
The probe’s specificity is its most important feature. Its sequence is designed using genetic databases to match a unique portion of the target gene. This prevents the probe from accidentally binding to similar mRNAs, ensuring the final pattern represents the activity of a single gene.
To make the probe detectable, it is labeled with a hapten, a small molecule recognized by an antibody. A common hapten is digoxigenin (DIG), which is not naturally present in the animals studied. During synthesis, DIG-labeled nucleotides are incorporated into the probe’s structure.
After the probe binds to the mRNA, an antibody engineered to recognize the DIG molecule is introduced. This antibody is linked to an enzyme, and when the correct chemical substrate is added, the enzyme starts a reaction that produces an insoluble colored product, marking the gene’s location.
Revealing Developmental Blueprints
WISH is a valuable technique in developmental biology for understanding how a fertilized egg becomes a complex organism. By visualizing when and where genes are active in an embryo, scientists can map the genetic instructions that guide the formation of organs and body parts.
The patterns revealed by WISH often correlate directly with the formation of anatomical structures. For example, a gene involved in segmentation may appear as stripes along the body axis, each corresponding to a future vertebra. A gene for eye formation, like Pax6, appears as a patch of cells in the head where the eye will develop, providing direct evidence of a gene’s role.
Model organisms have been studied extensively using WISH, including:
- The fruit fly (Drosophila)
- Zebrafish
- The frog (Xenopus)
- The mouse
In transparent zebrafish embryos, scientists can watch as a gene lights up in areas undergoing significant organization. In mouse embryos, researchers have used WISH to trace the migration of embryonic cells that form parts of the skull, nerves, and skin.
By comparing gene expression patterns, scientists can build regulatory networks and understand which genes act as master switches. The technique is also used to study genetic mutations, showing how an altered gene can disrupt the expression patterns of other genes and lead to developmental abnormalities.
Advantages Over Sliced Tissue Analysis
The main advantage of WISH is its ability to preserve the complete three-dimensional context of the organism. Traditional methods require cutting tissue into thin slices for analysis. While sectioning provides high cellular detail, it loses the overarching spatial relationships between different parts of the tissue.
Keeping the sample intact allows researchers to see the full scope of a gene’s expression pattern across complex structures. A gene active in a winding network of blood vessels or scattered migratory cells is immediately visible as a continuous pattern. In sliced tissue, this same pattern would appear as disconnected fragments across many slides, making the complete picture difficult to reconstruct.
This 3D perspective helps in understanding processes that occur over large distances, such as tracing the path of developing neurons. In a whole embryo, the continuous line of gene expression can be followed directly, whereas in sections, this path would be broken into pieces that need to be reassembled.
Sectioning can be performed after a WISH experiment to combine the benefits of both approaches. However, the initial whole-mount analysis provides a necessary overview. It ensures that complex or unexpected gene expression patterns are not missed, making WISH an effective method for initial explorations of gene function and large-scale patterning.