# Canonical WNT signalling governs Echinococcus metacestode development

## TL;DR

This study shows that the WNT signaling pathway is essential for the development of the metacestode larva of Echinococcus multilocularis, offering new drug targets for treating alveolar echinococcosis.

## Contribution

The study identifies β-catenin and canonical WNT signaling as key regulators of posteriorization in Echinococcus metacestode development.

## Key findings

- RNAi-mediated knockdown of bcat-1 impaired vesicle formation and caused stem-cell hyperproliferation and muscle-fibre changes.
- bcat-1(RNAi) cultures showed anteriorization of gene expression, with upregulation of head-inducing genes like sfrp and downregulation of metacestode-specific genes like muc-1.
- Anterior markers like frizzled-10 and follistatin were overexpressed in bcat-1(RNAi) cultures, indicating a shift in body-axis patterning.

## Abstract

Alveolar echinococcosis (AE) is a lethal zoonosis caused by infiltrative growth of the metacestode larva of the tapeworm Echinococcus multilocularis in host organs. We previously showed that the Echinococcus metacestode is an evolutionarily unique, broadly posteriorized tissue, leading us to hypothesize that canonical WNT (cWNT) signalling, which patterns the body axis across metazoans, might be critical for metacestode formation. Here, we report effective RNAi-mediated knockdown of the E. multilocularis β-catenin gene (bcat-1), the central effector of cWNT signalling, in a primary parasite cell culture system that produces metacestode vesicles. bcat-1(RNAi) cultures were markedly impaired in vesicle formation, exhibited stem-cell hyperproliferation, and displayed changes in muscle-fibre organisation. Genome-wide transcriptomics revealed a general anteriorization of gene expression, and in situ hybridization showed an overproduction of cells expressing head-inducing factors such as sfrp upon bcat-1 knockdown. Conversely, metacestode-specific genes including the tegumental factors muc-1, TNFR, and antigen B as well as the posterior marker post2b were significantly downregulated, consistent with the observed vesicle-formation defects. In situ analyses further identified anterior markers frizzled-10, nou-darake, notum, and follistatin that were overexpressed in bcat-1(RNAi) cultures and localized to the future anterior pole at the earliest stages of protoscolex formation. Together, these findings establish a central role for cWNT signalling in directing Echinococcus body-axis formation and the posteriorization events driving metacestode growth within the host, providing insight into asexual parasite proliferation mediated by this biologically unique larval stage and pointing to potential targets for chemotherapy against AE.

Alveolar echinococcosis (AE) is a lethal disease caused by the cancer-like growth of the metacestode larva of the tapeworm Echinococcus multilocularis. From a developmental perspective, the Echinococcus metacestode is an unusual biological structure and even atypical among tapeworms. Previous work indicated that metacestode formation involves re-patterning of the body axis, eliminating head structures and producing broadly posteriorized tissue. How this is controlled at the molecular and cellular levels, however, was unknown. In this study, we perturbed expression of the β-catenin gene (bcat-1), a central regulator of canonical WNT signalling, using RNA interference (RNAi). bcat-1(RNAi) parasite cultures failed to generate metacestode vesicles and instead showed stem-cell hyperproliferation and muscle-cell alterations. Genes required for posteriorized metacestode tissue were downregulated, whereas genes directing head formation in adult worms (follistatin, sfrp, fz10, ndk) were upregulated, indicating a general anteriorization of the culture system. These findings identify β-catenin and the canonical WNT pathway as crucial regulators of the posteriorization that underlies metacestode formation. Given that WNT signalling is deregulated in many human cancers and that small-molecule inhibitors are available, our results suggest new avenues for anti-AE drug development.