Freshwater planarians are well known for their ability to regenerate from injuries considered catastrophic in other animals. When a planarian is decapitated, for example, the resulting trunk regenerates a new head with all the attendant organs (e.g.
, brain, photoreceptors, sensory neurons) within a week. Even a fragment accounting for roughly 1/279th
of the former body size can regenerate a complete animal (Morgan, 1898
). The fact that a large population of adult somatic stem cells is required for planarian regeneration has fuelled the recent surge in interest in understanding the molecular mechanisms behind this process (Newmark and Sánchez Alvarado, 2002
). A key molecular technique commonly used in planarian and other developmental biology studies is whole-mount in situ hybridization (WISH), which allows for the visualization of gene transcripts within cells and tissues. The central goal of a WISH method is to provide investigators with the technical ability to: a) define specific cell types by their gene expression pattern; b) analyze the distribution of cell types within the tissue architecture; and c) study the functional interactions between multiple cell types. However, as the need for more detailed analysis of planarian stem cells and regeneration has proceeded, so has the need for more robust and sensitive WISH methods.
A critical component of successful WISH involves balancing the often contrasting demands between sensitivity and preservation of morphology. While the retention of transcripts and preservation of overall tissue morphology necessitates use of a fixative, permeabilization of tissues and cells is a prerequisite for optimal access of antisense riboprobes to their targets. Any given organism presents its own set of challenges to achieve this balance. For example, in Drosophila embryos, the chorion and vitelline membrane must be removed in order to permeabilize, fix, and visualize expression patterns. Zebrafish embryos can be fixed in the chorion, but extensive permeabilization steps must be performed prior to WISH due to the relatively higher tissue density when compared to many invertebrates. While WISH is rarely performed in C. elegans, WISH protocols commonly use a histological fixative called Bouin's to permeabilize the tough cuticle.
Even though it has been empirically determined in the vast majority of systems that formaldehyde (FA) fixations are optimal for WISH, planarians have not been amenable to this approach. This is mainly due to two major distinguishing characteristics. First, these organisms secrete a dense mucus layer around their entire bodies thought to facilitate osmotic balance, locomotion, and predator avoidance (Hyman, 1951
). The mucus layer also presents a considerable barrier to the exchange of fluids and large molecules needed during WISH. Second, experimental animals are often several millimeters in length, posing special challenges with respect to detection efficiencies in deep tissues. Previously, it was found that an alcohol/acid based fixative called Carnoy's could penetrate the mucus barrier and yield WISH signals in planarians (Umesono et al., 1997
). This method quickly became the field standard and is now widely used. However, we find that the degree of staining in a batch of Carnoy's fixed animals can be highly variable. Moreover, Carnoy's is not a cross-linking fixative, and thus the balance between permeability and transcript retention is not optimal (Urieli-Shoval et al., 1992
). In contrast, the cross-linking capabilities of FA-based fixations offer higher cellular resolution and thus have emerged over the last 2 decades as the preferred WISH fixative across a wide variety of model systems. The need for cellular resolution and simultaneous labeling with multiple riboprobes has promoted our attempts to develop a more sensitive and robust WISH method for planarians.
Here we report an FA-based, sensitive, and reproducible whole-mount WISH methodology for planarians. Different techniques for fixation, permeabilization, hybridization and post-processing were tested for their general utility and were systematically optimized for maximal synergy. The effects of key innovations in the protocol are demonstrated with a set of markers labeling a range of planarian cell and tissue types. We find that the new methodology is superior to Carnoy's fixation for all tested markers, allowing the visualization of fine anatomical details at cellular resolution, yet at a sensitivity level sufficient even for the detection of microRNA (miR-124a) expression. Finally, we show that double fluorescent WISH along with antibody-staining of protein-epitopes provides robust access to multi-cell-type analysis using this optimized method. Hence the method described here represents a valuable asset for the planarian community and our optimizations should be widely applicable to WISH methods in other systems.