The ability of some animals to regenerate body parts has been a source of fascination since at least the eighteenth century. In recent years, the power of molecular genetics has been brought to bear on the question of how this is actually achieved. Among the invertebrates, the planarian Schmidtea mediterranea is emerging as perhaps the most robust model for this purpose.

Planaria are flatworms that can rapidly regenerate any body part, which requires the function of a special type of radiation-sensitive stem cell called the neoblast. These cells are distributed throughout the body and are required for regeneration both after injury and during normal homeostasis. Peter Reddien and his colleagues at the Massachusetts Institute of Technology, in work published about a year ago, showed that a single neoblast can give rise, upon transplant, to the entire flatworm. The cells can thus stringently be defined as pluripotent.

In the course of these studies, graduate student Dan Wagner found that sublethal doses of irradiation would yield a few surviving neoblasts throughout the body of the flatworm. Subsequent expansion of these cells yields in vivo clonal colonies. When the researchers imaged these growing clones, they observed a linear relationship between the number of dividing, pluripotent cells and of postmitotic, differentiated cells. Furthermore, the relationship appeared to be autonomous to the neoblasts: it holds true irrespective of location within the body. “This observation presented the opportunity to use clones as a method to compare expansion and differentiation from colony to colony, animal to animal and in multiple conditions,” explains Reddien.

The radiation dose used to kill off most of the planarian neoblasts turned out to be key. Too low a dose gives colonies that are crowded together and are difficult to reliably image; too high a dose kills the flatworm or yields too few cells to analyze statistically. The researchers typically use irradiated flatworms with three or fewer starting neoblasts.

Defining three simple phenotypes—colony loss, failed proliferation and failed differentiation—they initially examined genes with already reported functions in regeneration in the flatworm. They irradiated the flatworms, allowed the few remaining neoblasts to form colonies either in the presence or absence of short interfering RNA for genes of interest, then imaged colony phenotypes 1 or 2 weeks later and quantified the numbers of proliferating and differentiating cells. They observed phenotypes consistent with the known functions of the tested genes (in neoblast self-renewal and differentiation).

Encouraged, they went on to use the in vivo colony assay to study 28 irradiation-sensitive genes they had previously identified by expression profiling, which are candidates for involvement in some aspect of neoblast function. Sixteen of these genes, including a transcription factor (a class of gene that has not been implicated in neoblast function before), showed perturbation of colony growth when knocked down. Several of these genes they also confirmed as functioning in normal homeostasis.

Although it is a relatively time-intensive method and therefore more suited for screening a list of already defined candidates, in vivo measurement of clonal neoblast outgrowth is likely to be a powerful way to study gene function in planarians. In particular, Reddien emphasizes the rapidity with which colonies grow as well as the synchronicity of the assayed cells. “Often when you study stem-cell phenotypes, things are unfolding in a complex way, it's easy to miss the primary defect by studying the whole system at some late time point after perturbing gene function. With the colonies, we've got the process stripped down to a short time window; every gene can be assessed in the same time course, and we know roughly what happened from start to finish.”