Whole Mount In Situ Hybridization (WISH) is a laboratory technique used to visualize the location of a specific messenger RNA (mRNA) molecule within an entire tissue or organism. WISH is a variation of In Situ Hybridization (ISH), which localizes nucleic acid sequences directly inside cells. WISH is designed to work on entire, intact specimens, such as embryos from model organisms like zebrafish, fruit flies, or mice, without requiring prior sectioning. This capability helps researchers understand the spatial and temporal patterns of gene activity, particularly in developmental biology.
The Molecular Mechanism of Hybridization
The WISH technique relies on molecular hybridization, the selective binding between two complementary single strands of nucleic acid. The process begins with creating a specialized probe, a synthesized strand of RNA or DNA designed to exactly match the sequence of the target mRNA. To locate its target, the probe must be labeled with a detectable tag, often a hapten like digoxigenin.
The probe binds to the target mRNA inside the cell based on base-pairing complementarity (Adenine pairs with Uracil/Thymine, and Guanine pairs with Cytosine). This specific attraction ensures the labeled probe only sticks to the exact mRNA sequence of interest, forming a stable double-stranded hybrid.
The temperature and salt concentration of the reaction solution must be precisely controlled to maintain specificity, a condition known as stringency. High temperatures and low salt levels increase stringency, ensuring only perfectly matched sequences remain bound. Once hybridization is complete, the location of the target mRNA is marked by the tagged probe, which is then chemically visualized.
The Advantage of Three-Dimensional Visualization
The defining feature of WISH is its capacity to process an entire biological specimen without prior sectioning. This “whole mount” approach maintains the complete, three-dimensional architecture of the sample, such as a developing embryo. Traditional In Situ Hybridization requires slicing the tissue into thin sections before the hybridization step.
Keeping the specimen intact provides a comprehensive view of gene expression patterns across the entire organism in its native context. This is valuable in developmental studies, where spatial relationships between cells and organs are constantly changing. Researchers can observe precisely how a gene is turned on or off across different tissues, aiding the understanding of organ formation.
Visualizing expression in three dimensions eliminates the need to computationally reconstruct a complete image from multiple thin slices, which is necessary in section-based methods. This holistic view simplifies tracking gene expression boundaries and understanding how molecular activity dictates the physical structure of a developing organism.
Step-by-Step Procedure
The WISH technique involves several distinct steps to prepare the sample and visualize gene expression. The first step is Fixation, where the embryo or tissue is treated with a chemical like paraformaldehyde. This rapidly preserves the cellular structure and prevents the degradation of target mRNA molecules by cross-linking proteins and nucleic acids.
After fixation, the tissue undergoes Permeabilization to allow the large RNA probe access to the cell interior. This is commonly achieved by treating the tissue with a detergent, such as Tween 20, to create small pores in the cell membranes. For older or larger embryos, brief digestion with a protease enzyme, such as Proteinase K, may be necessary to improve probe penetration.
The next step is Hybridization, where the tissue is incubated with the labeled RNA probe, often overnight, in a high-temperature solution (typically 65°C to 70°C). This elevated temperature ensures the probe only binds to the perfectly complementary target mRNA sequence. Following hybridization, a series of Washing steps are performed using high-stringency buffers to remove non-specifically or weakly bound probes.
The final phase is Detection, which reveals the location of the bound probe and the target mRNA. This involves incubating the sample with an antibody that specifically recognizes the hapten tag. This antibody is linked to an enzyme, such as alkaline phosphatase. When a colorless substrate is added, the enzyme catalyzes a reaction that produces an insoluble, visible colored precipitate (typically blue or purple), marking the site of gene expression.
Decoding Gene Expression Patterns
The visible color precipitate generated by WISH provides a direct map of where the target gene is actively being expressed. The appearance of color in a specific region of the embryo, such as the developing brain or somites, indicates the presence of the corresponding mRNA molecules. This visual output is a qualitative measure, showing the spatial distribution of the gene transcript within the specimen.
Researchers use this spatial information to track the development of organs and tissues, observing how gene activity shifts over time. By performing WISH on embryos at different developmental stages, scientists build a temporal profile, revealing when a gene turns on, where it is most abundant, and when its expression fades. This process helps understand gene function, as mRNA expression location often dictates the function of the resulting protein.
For example, a gene might show strong staining in the anterior region of a young embryo, which later concentrates into the area that will form the heart. This observation allows developmental biologists to connect molecular events to the physical creation of an organism’s structure. By comparing expression patterns in normal and mutant organisms, WISH helps identify genes responsible for specific developmental disorders or malformations.