Following amputation, freshwater planarians properly regenerate a head or tail from the resulting anterior or posterior wound. The mechanisms that differentiate anterior from posterior and direct the replacement of the appropriate missing body parts are unknown. Here we report that RNA interference (RNAi) of β-catenin or dishevelled causes the inappropriate regeneration of a head instead of a tail at posterior amputations. Conversely, RNAi of the β-catenin antagonist adenomatous polyposis coli (APC) results in the regeneration of a tail at anterior wounds. In addition, the silencing of β-catenin is sufficient to transform the tail of uncut adult animals into a head. We suggest that β-catenin functions as a molecular switch to specify and maintain anteroposterior (A/P) identity during regeneration and homeostasis in planarians.

Regeneration is widespread throughout the animal kingdom, but our molecular understanding of this process in adult animals remains poorly understood. Wnt/β-catenin signaling plays crucial roles throughout animal life from early development to adulthood. In intact and regenerating planarians, the regulation of Wnt/β-catenin signaling functions to maintain and specify anterior/posterior (A/P) identity. Here, we explore the expression kinetics and RNAi phenotypes for secreted members of the Wnt signaling pathway in the planarian Schmidtea mediterranea. Smed-wnt and sFRP expression during regeneration is surprisingly dynamic and reveals fundamental aspects of planarian biology that have been previously unappreciated. We show that after amputation, a wounding response precedes rapid reorganization of the A/P axis. Furthermore, cells throughout the body plan can mount this response and reassess their new A/P location in the complete absence of stem cells. While initial stages of the amputation response are stem cell independent, tissue remodeling and the integration of new A/P address with anatomy are stem cell dependent. We also show that WNT5 functions in a reciprocal manner with SLIT to pattern the planarian mediolateral axis, while WNT11-2 patterns the posterior midline. Moreover, we perform an extensive phylogenetic analysis on the Smed-wnt genes using a method that combines and integrates both sequence and structural alignments, enabling us to place all nine genes into Wnt subfamilies for the first time.

The Hedgehog (Hh) signaling pathway plays multiple essential roles during metazoan development, homeostasis, and disease. Although core protein components are highly conserved, the variations in Hh signal transduction mechanisms exhibited by existing model systems (Drosophila, fish, and mammals) are difficult to understand. We characterize the Hh pathway in planarians. Hh signaling is essential for establishing the Anterior/Posterior axis during regeneration by modulating wnt expression. Moreover, RNAi methods to reduce signal transduction proteins Cos2/Kif27/Kif7, Fused, or Iguana do not result in detectable Hh signaling defects; however, these proteins are essential for planarian ciliogenesis. Our study expands the understanding of Hh signaling in the animal kingdom and suggests an ancestral mechanistic link between Hh signaling and the function of cilia.The Hh signaling pathway plays numerous evolutionarily conserved roles in the regulation of cell growth and patterning during the embryonic and postembryonic development of animals as diverse as frutiflies and humans. The misregulation of this pathway has equally profound consequences resulting in defects such as holoprosencephaly (cyclopia) and tumorigenesis. Secreted Hh protein alters gene transcription by binding the cell-surface receptor Patched (Ptc), preventing repression of the 7 membrane spanning receptor Smoothened (Smo) by Ptc. This activates Gli transcription factors and inactivates their inhibitor Suppressor of Fused (SuFu). Despite conservation of these core components and their mode of function (1,2), Hh signal transduction mechanisms appear to have diversified throughout evolution (3). Drosophila Hh signaling is cilia-independent and requires the kinesin Costal2 (4) (Kif7/27 in vertebrates) and the kinase Fused (5). The mouse Hh pathway requires primary cilia (6,7) and Kif7 (8-10), but not Fused (11,12). Zebrafish utilize cilia, Kif7, Fused, and Iguana/Dzip1 (Igu) (13)(14)(15)(16)(17)(18)(19). C. elegans has lost a functional Hh pathway altogether (20). Since planarians belong to a group of animals that evolved independently from flies, fish, and mammals (Sup. Fig. 1) an analysis of planarian Hh signaling could reveal how the mechanistic differences in a highly conserved signaling pathway arose.Systematic sequence homology searching of the S. mediterranea genome identified single homologs for planarian Hh (Smed-hh), Patched (Smed-ptc), Smoothened (Smed-smo) and Supressor of Fused (Smed-sufu), but three Gli homologs (37) (Sup. Fig. 2,3). Of the Gli homologs, only Smed-gli-1 exhibited an obvious role in Hh signaling (see below). We cloned (see SOM) and analyzed the expression of these planarian Hh components by in-situ hybridization (Fig. 1A-C,Sup. Fig. 4). ptc expression was reduced by RNAi of pathway activators (hh, smo, gli-1) and elevated by RNAi of pathway inhibitors (ptc, sufu) (Fig. 1B), suggesting that as in other animals (21-23),