Molecular and functional characterization of Echinococcus multilocularis delta/notch signalling components

Pluripotent somatic stem cells are the drivers of unlimited growth of Echinococcus multilocularis metacestode tissue within organs of the intermediate host. For developing anti-infectives against the underlying disease, alveolar echinococcosis, it is vital to understand the basic mechanisms of Echinococcus stem cell maintenance, proliferation, and differentiation. We herein undertake first steps towards characterizing the role of an evolutionarily old system of metazoan cell differentiation, delta/notch signalling, in Echinococcus cell fate decisions. Bioinformatic analyses revealed that all central components of this pathway are encoded by the Echinococcus genome and are expressed in parasite larval stages. By in situ hybridisation we analysed the expression patterns of two delta-like ligands, delta1 and delta2 , as well as two notch receptors, notch1 and notch2 . We show that these components display ‘salt-and-pepper’-like expression patterns in the Echinococcus protoscolex, indicative of lateral inhibition mechanisms. Two of these genes, delta2 and notch2 , are posteriorly expressed in the protoscolex and are the major components of delta/notch signalling in the metacestode. EdU incorporation assays revealed that none of the delta/notch signalling factors is expressed in germinative cells nor in their immediate progeny, indicating that Echinococcus delta/notch dependent cell fate decisions are exclusively determined in post-mitotic cells. Finally, RNA interference against delta2 and notch2 led to significantly diminished production of metacestode vesicles from Echinococcus primary cell cultures, thus underlining the importance of this pathway for parasite development within the host. These analyses are relevant for understanding the interplay of fate determining signalling pathways in Echinococcus cell differentiation and may be exploited for the development of novel anti-infectives against echinococcosis.