Peter W. Reddien
Regeneration is one of the great mysteries of biology. Planarians are bilaterally symmetric metazoans that possess almost unlimited regenerative capacities and that have been a classic regeneration model for over a century. Since planarian regeneration involves a population of adult pluripotent stem cells (the neoblasts), planarians are an excellent organism for studies of in vivo stem cell regulation. We use RNA interference (RNAi) for high-throughput studies of gene function in the planarian S. mediterranea. Our aim is to understand how planarian neoblasts control regeneration.
RNAi screening: Planarians have not historically been accessible to extensive genetic manipulation. We developed a highly effective RNAi strategy that introduces large-scale gene inhibition studies to planaria. 1065 genes have been inhibited during regeneration. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. 85% of these 240 genes have homology to genes in other organisms. We assessed the effects of inhibiting genes on tissue homeostasis in intact animals and on neoblast proliferation in amputated animals, identifying candidate stem cell, regeneration, and homeostasis regulators. Our RNAi screen establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.
Neoblast regulation: We identified a planarian gene, smedwi-2, that is needed for regeneration and homeostasis. We found that smedwi-2 and the related smedwi-1 gene are expressed in dividing neoblasts. The SMEDWI-2 protein is homologous to Drosophila PIWI, a germline stem cell regulator. Irradiation of planarians specifically eliminates neoblasts and causes defects that closely resemble those seen in smedwi-2(RNAi) animals. Therefore, smedwi-2(RNAi) neoblasts may be dysfunctional. We used FACS, antibody labeling, and BrdU to study the neoblasts of smedwi-2(RNAi) animals. Our data indicate a primary defect not in neoblast maintenance, but in the function of neoblast progeny to replace aged cells in homeostasis and to replace missing cells in regeneration. Other genes with similar RNAi-induced defects from the RNAi screen are being examined .
How do planarians specify what to regenerate? Planarians can regenerate entire heads, sides, and tails. Regeneration from irregularly shaped fragments involves a robust ability to generate bilateral symmetry: new tissue is produced in a regeneration blastema at the wound site and remaining body regions are rearranged to produce symmetrical and properly proportioned animals. We identified three genes that are needed for asymmetric regeneration. These genes are candidate components of a BMP signaling pathway that controls the dorsal-ventral patterning of many animal embryos: a BMP1/Tolloid-like gene ( smedolloid-1 ), a SMAD4-like gene ( smedsmad4-1 ), and a DPP-like gene ( smeddpp-1 ). Intact adult animals rely upon the action of this pathway to maintain their body plans: RNAi of smedsmad4-1 in normal adults causes a slow transformation in which animals develop two ventral sides. Animal fragments with perturbed DPP signaling and left-right symmetry regenerate blastemas with midline defects. smeddpp-1 is expressed on the dorsal midline indicating this pathway may normally regulate midline regeneration. Animal fragments with perturbed DPP signaling but that are asymmetric about the midline cannot produce blastemas. We suggest the patterning activity of the midline is necessary for the initiation of lateral blastema formation in asymmetric fragments. This work provides a starting point for investigations into how planarians "know" what part of their body is missing.
The invertebrate animals C. elegans and Drosophila have served as potent research subjects for the discovery of basic principles in developmental biology. We believe that the processes prominent in planarians--regeneration, extensive tissue turnover as part of homeostasis, and adult pluripotent stem cells--combined with our ability to systematically study planarian gene function for the first time provide a new and powerful venue for exploration of biology.