Silencing of β-catenin1 blocks tail regeneration, but does not induce head regeneration in the flatworm Macrostomum lignano

β-Catenin has two major functions conserved across metazoans. It enables the interaction of classical cadherin with actin and it is the main transcription factor activated by the canonical Wnt pathway. In these two functions, it acts in patterning of the major body axis in early embroys, in cell differentiation and proliferation. In flatworms, four β-catenin homologs have been identified in the planarian Schmidtea mediterranea. Functional studies revealed that only Smed-β-catenin1 can transduce the Wnt signal and plays a part in defining posterior identity. We made a phylogenetic reconstruction of β-catenin homologs retrieved from a wide range of flatworms and analysed the role of β-catenin during regeneration in Macrostomum lignano . We identified at least two copies of β-catenin in all major taxa except in Catenulida; this suggests that duplication of β-catenin occurred ancestrally in Rhabditophora. In M. lignano , we detected 3 β-catenin homologs. Upon knockdown of the 3 homologs, only Mlig-β-catenin1 blocked blastema formation and regeneration of a tail. After knockdown, posterior regenerates restored normal tails but with instances of imperfect or duplicated tails. Different to planarians, knockdown of Mlig-β-catenins individually or in combination failed to rescue a head in all amputation levels in anterior regenerates. Together, these findings suggest that β-catenin1 is responsible for posterior identity specification also in M. lignano . Our study demonstrates that the ability to rescue a head upon knockdown of β-catenin1 may be an apomorphy of planarians, but is not an ancestral character in flatworms.