Double-stranded RNA Specifically Disrupts Gene Expression During Planarian Regeneration
Sánchez Alvarado A, Newmark P
Metazoan regeneration is one of the least understood fundamental problems of biology. The lack of progress in understanding this phenomenon at the molecular level has been due to the poor regenerative abilities of the genetic organisms used for developmental studies, as well as the difficulties encountered with molecular and genetic manipulations of the commonly studied vertebrate models (the urodele amphibians). Here, we demonstrate that introduction of double-stranded RNA selectively abrogates gene function in planarians, a classic model of regeneration. The ability to eliminate gene function in a regenerating organism such as the planarian overcomes previous experimental limitations and opens the study of animal regeneration to unprecedented levels of molecular detail.
Among the metazoans, planarians have long been known to possess remarkable regenerative abilities. One example of these abilities was Morgan’s 1898 demonstration that a very small planarian fragment, corresponding to only 1/279th of the intact organism, was capable of regenerating a complete individual (1). Such striking regenerative powers have attracted generations of biologists to the study of this problem, and a vast literature, spanning more than 200 years, exists on the subject (2–6). In addition to their regenerative abilities, flatworms occupy a key position in the evolution of metazoans (7). They are widely acknowledged as being among the simplest organisms possessing three tissue layers (triploblasts), bilateral symmetry, cephalization, and complex organ systems (8). The most commonly studied planarians are the freshwater triclads of the class Turbellaria, phylum Platyhelminthes. Their regenerative properties, combined with their morphological simplicity and phylogenetic position, make planarians ideally suited for the study of regeneration mediated by the formation of a blastema (a bud with well defined epithelial and mesenchymal compartments). Despite their attractiveness as a model system, examination of planarians at the molecular level has just begun (9–11), and their use has been relegated mostly to descriptive, phenomenological studies.
Aiming to develop a model system in which to study the molecular basis of metazoan regeneration and to exploit the remarkable developmental plasticity of planarians, we have begun to screen for genes involved in planarian regeneration (12) and to develop methods with which to study gene function. In lieu of a classical genetic analysis to generate loss-of-function mutants in planarians, we have chosen to test genetic interference caused by double-stranded RNA (RNAi) as recently described in Caenorhabditis elegans (13, 14), Trypanosoma brucei (15), and Drosophila melanogaster (16), as well as in the plants Nicotiana tabaccum and Oryza sativa (17). In these organisms, the introduction of double-stranded (ds) RNA was found to inhibit specifically the expression of the gene from which that RNA was derived. Here, we extend the use of RNAi to eliminate gene expression in planarians, both in regeneration blastemas and in differentiated adult structures.