Open Biology

In this paper we describe the development of a new model system for Friedreichs Atax- ia (FA) using Dictyostelium discoideum. We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition multicellular development is affected as well as grow on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued The results of the study suggest that this new model system offers a wide range of pos- sibilities to easily explore diverse phenotypes in FA and develop drug or treatment screenings for designing and evaluating therapeutic strategies.

The Ras/cAMP/PKA pathway is one of the best studied signalling pathway in the budding yeast that regulates cellular responses to nutrients availability and stress. The cAMP levels and the PKA activity are also subjected to a strong negative feedback that operates either through the activity of the phosphodiesterase Pde1 and also on the amount of Ras2-GTP. We have previously made and simulated a dynamic model of the whole pathway and our results suggest the existence of stable oscillatory states that depend on the activity of the RasGEF (Cdc25) and RasGAP (Ira proteins) (Pescini et al. Biotechnol Adv 30, 99-107, 2012). Stochastic oscillations related to activity of the pathway were reported by looking at the nuclear localization of the trascription factors Msn2 and Msn4 (Gamedia-Torres et al. Curr Biol 17, 1044-9, 2007). In particular Medvedik et al. (PloS Biol 5, 2330-41, 2007) reported stable oscillations of the nuclear accumulation of Msn2 in condition of limited glucose availabiliy. We were able to reproduce the periodic accumulation of Msn2-GFP protein in yeast cells under condition of limiting glucose, and we tried to detect also in the same condition oscillations of cAMP levels in single yeast cells. We used a specific Fluorescence Resonance Energy Transfer (FRET) sensor based on a fusion protein between CFP-EPAC-YFP expressed in yeast cells. The FRET between CFP and YFP is controlled by cAMP concentration. This sensor allows us to monitor changes in cAMP concentrations in single yeast cell for a relative long time and a peak of cAMP was normally detected after addition of glucose to derepressed cells (Colombo et al. Biochem Biophys Res Commun 487, 594-99, 2017). Using this method we were able to detect noisy oscillations of cAMP levels in single yeast cells under condition of nutritional stress caused by limiting glucose availability (0.1%). We used Spectral analysis to discriminate between true oscillations and random noise. The oscillations were characterized by period of about 4-5 min, close to that observed for Msn2-GFP oscillations.

Mutations in the gene encoding the Adenomatous polyposis coli protein (APC) were discovered as driver mutations in colorectal cancers almost 30 years ago. Since then, the importance of APC in normal tissue homeostasis has been confirmed in a plethora of other (model) organisms spanning a large evolutionary space. APC is a multifunctional protein, with roles as a key scaffold protein in complexes involved in diverse signalling pathways, most prominently the Wnt signalling pathway. APC is also a cytoskeletal regulator with direct and indirect links to and impacts on all three major cytoskeletal networks. Here, we interrogate the enormous depth of sequencing data now available to reveal the conservation of APC across taxonomy and relationships between different APC protein families.

Progression through the cell cycle in eukaryotes is regulated on multiple levels. The main driver of the cell cycle progression is the periodic activity of cyclin-dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle is regulated by a transcriptional program that ensures the just-in-time gene expression. Many core cell cycle regulators are present in all eukaryotes, among them cyclins and CDKs; however, periodic transcriptional programs are divergent between distantly related species. In addition, many otherwise conserved cell cycle regulators have been lost and independently evolved in yeast, a widely used model organism for cell cycle research. To gain insight into the cell cycle regulation in a more representative opisthokont, we investigated the cell cycle regulation at the transcriptional level of Capsaspora owczarzaki, a species closely related to animals. We developed a protocol for cell cycle synchronization in Capsaspora cultures and assessed gene expression over time across the entire cell cycle. We identified a set of 801 periodic genes that grouped into five clusters of expression over time. Comparison with datasets from other eukaryotes revealed that the periodic transcriptional program of Capsaspora is most similar to that of animal cells. We found that orthologues of cyclin A, B and E are expressed at the same cell cycle stages as in human cells and in the same temporal order. However, in contrast to human cells where these cyclins interact with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, our results demonstrate that Capsaspora could be a useful unicellular model system for animal cell cycle regulation.\n\nAuthors summaryWhen cells reproduce, proper duplication and splitting of the genetic material is ensured by cell cycle control systems. Many of the regulators in these systems are present across all eukaryotes, such as cyclin and cyclin-dependent kinases (CDK), or the E2F-Rb transcriptional network. Opisthokonts, the group comprising animals, yeasts and their unicellular relatives, represent a puzzling scenario: in contrast to animals, where the cell cycle core machinery seems to be conserved, studies in yeasts have shown that some of these regulators have been lost and independently evolved. For a better understanding of the evolution of the cell cycle regulation in opisthokonts, and ultimately in the lineage leading to animals, we have studied cell cycle regulation in Capsaspora owczarzaki, a unicellular amoeba more closely related to animals than fungi that retains the ancestral cell cycle toolkit. Our findings suggest that, in the ancestor of Capsaspora and animals, cyclins oscillate in the same temporal order as in animals, and that expansion of CDKs occurred later in the lineage that led to animals.

Actin is one of the most highly conserved proteins--the actin cytoskeleton or actin-like structures are found in all kingdoms of life. In this work, Coronin-A, a central player involved in actin dynamics, is considered as an example to compare the effect of four mutation strategies on its function in Dictyostelium discoideum. Two distinct genetic knockout methods (selection-linked integration and classical gene disruption), RNAi knockdown, and sudden loss-of-function generated by chemically induced dislocation (CID) are compared and the cell lines are characterized according to actin dependent processes: appearance, development, and motility. These phenotypic features are affected to different degrees in the mutant cell lines, providing a basis to discuss the strengths and weaknesses of each approach. This comparative study highlights the advantages of the newly introduced selection-linked integration method to knock out genes over commonly used gene disruption, and for expressing a CID-based knock-sideways system under the native promotor of the protein of interest.

The vast majority of Chromosomal Instability (CIN) promoting mutations remain unknown. We assess the prevalence of Chromosomal Instability aiding Variants (CIVa) by collating Loss-of-Function (LoF) variants predicted in 135 chromosome segregation genes from over 150,000 humans, including consanguineous individuals. Surprisingly, we observe heterozygous and homozygous CIVa in Astrin and SKA3 genes that encode evolutionarily conserved microtubule-associated proteins essential for chromosome segregation. By combining high-resolution microscopy and controlled protein expression, we show the naturally occurring Astrin variant, p.Q1012*, as potentially harmful because it fails to localise normally, delays anaphase onset, induces chromosome misalignment and promotes chromosome missegregation. We show that N-terminal frameshift variants in Astrin and SKA3 are likely to generate shorter isoforms that do not compromise chromosome segregation revealing resilient mechanisms to cope with harmful variants. This study provides a framework to predict and stratify naturally occurring CIVa, an important step towards precision medicine for CIN syndromes.

In aneuploid cancer cells, the chromosome segregation apparatus is sensitive to increased chromosome number. The conserved protein kinase, Mps1, is a critical actor of this machinery, orienting the chromosomes properly on the spindle. Abnormally high levels of this kinase have been found in tumors with elevated chromosome number. However, it remains unclear, mechanistically, if and how cells with higher ploidy become dependent upon increased Mps1 levels. To answer these questions, we explored Mps1 dependence in yeast cells with increased sets of chromosomes. We discovered that having more chromosomes affects the ability of cells to orient chromosomes properly. The cells with increased numbers of chromosomes are particularly sensitive to the reduction of Mps1 activity. In mps1 loss of function mutants, cells display an extended prometaphase with a longer spindle and a delay in orienting properly the chromosomes. Altogether, our results suggest that increased numbers of chromosomes render cells more dependent on Mps1 for orienting chromosomes on the spindle. The phenomenon described here may be relevant in understanding why hyperdiploid cancer cells become excessively reliant on high Mps1 expression for successful chromosome segregation. Author summaryMost cells in solid tumors usually carry far more chromosomes than normal cells. Losing or gaining chromosomes during cell division can lead to aneuploidy (an abnormal number of chromosomes), cancer, and other diseases. Mps1 is a master regulator of cell division that is critical to keep the correct number chromosomes in each daughter cell. This master regulator has been shown to target and affect the function of various actors involved in cell division. Abnormally high levels of this master regulator are found in tumors with elevated chromosome numbers. The high levels of this regulator appear to be protecting these tumor cells. To answer if and how cells with higher ploidy become so dependent of Mps1, we generated yeast cells with increased set of chromosomes. Here, we report that cells with elevated chromosome number are particularly sensitive to the reduction of Mps1 level. In cells with higher ploidy and reduced level of Mps1, the progression during cell division is delayed. In the mutant cells, their ability to properly orient and segregate their chromosomes on the spindle is greatly reduced.

Human glutamate carboxypeptidase 2 (GCP2) from the M28B metalloprotease group is an important target for therapy in neurological disorders and an established tumor marker. However, its physiological functions remain unclear. To better understand general roles, we used the model organism Caenorhabditis elegans genetically manipulate its three existing orthologous genes and evaluate the impact on worm physiology. The results of gene knockout studies showed that C. elegans GCP2 orthologs affect the pharyngeal physiology, reproduction, and structural integrity of the organism. Promoter-driven GFP expression revealed distinct localization for each of the three gene paralogs, with gcp-2.1 being most abundant in muscles, intestine, and pharyngeal interneurons, gcp-2.2 restricted to the phasmid neurons, and gcp-2.3 located in the excretory cell. This study provides new insight into the unique phenotypic effects of GCP2 gene knockouts in C. elegans, and the specific tissue localizations. We believe that elucidation of particular roles in a non-mammalian organism can help to explain important questions linked to human GCP2 physiology and in extension to GCP2 involvement in pathophysiological processes.

Centrosome dysfunction is linked to developmental disorders affecting brain and body size, including microcephaly and primordial dwarfism. However, the cellular mechanisms underlying these rare conditions remain poorly understood. In this study, we investigate a rare variant of the centrosome-associated protein Pericentrin, which was discovered in a single family with Majewski/microcephalic osteodysplastic primordial dwarfism type II (MOPD II). Unlike the majority of pathogenic PCNT variants that cause severe protein truncation, the p.Lys3154del variant ({Delta}K3154) involves a single amino acid deletion in the proteins only conserved functional domain, providing a unique opportunity to explore PCNT function in MOPD II. To model PCNT{Delta}K3154, we examined the effects of Drosophila Pericentrin-like protein (PLP) carrying an orthologous deletion (Plp{Delta}R). Our results show that plp{Delta}R animals exhibit smaller tissues that recapitulate MOPD II phenotypes. Behavioral assays revealed defects in climbing and mechanosensation, suggesting impaired sensory cilia function. We also found that Plp{Delta}R cells exhibit accelerated mitosis, increased apoptosis, and reduced pericentriolar material recruitment. In silico structural modeling, yeast two-hybrid, and co-immunoprecipitation experiments show that Plp{Delta}R produces a protein that disrupts PLP dimerization and PLP interaction with Asterless, another centrosome protein. Overall, modeling the human MOPD II patient variant PCNT{Delta}K3154 in Drosophila reveals how a single amino acid deletion affects biological processes from the molecular level to the organismal level. Our work offers new insights into the defective cellular mechanisms underlying MOPD II in patients with the PCNT{Delta}K3154 variant, potentially linking the etiology of the disease in these individuals to the loss of a single protein-protein interaction.

Mitosis is a critical stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe may demonstrate catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from e.g. mutations in acetyl-CoA/biotin carboxylase (cut6), in fatty acid synthase (fas2/lsd1), or in the transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It was previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride. In this study we demonstrate that mitotic fidelity can be improved by multiple good nitrogen sources. Moreover, this rescue is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. We found that the TOR regulatory network plays a major role in mediating such rescue, highlighting a novel role for TOR in mitotic fidelity.

All cells of a multicellular organism usually share an identical genome, faithfully transmitted through successive divisions. Yet, a number of animal species deviate from this dogma, as parts of their DNA are systematically eliminated in all their somatic nuclei, in a process called Programmed DNA Elimination (PDE). PDE leads to the unexpected reorganisation of the genome at every generation in all somatic cells but its molecular mechanism, evolutionary origins, and functional significance remain unknown. This lack of understanding partially stems from limitations in genetically tractable model species. PDE can target an entire chromosome, or involve chromosome fragmentation followed by selective fragment retention and elimination, raising further questions on genome stability, genome integrity and mechanisms of DNA repair. PDE by chromosome fragmentation has been described in parasitic nematodes in the family Ascarididae, copepods in the genus Cyclops and unicellular ciliates. More recently, PDE has been discovered in three non-parasitic, lab-tractable nematode species from the Rhabditidae family, opening new perspectives. In this study, we used cytological approaches to screen 25 new Rhabditidae species for PDE. We found evidence of PDE in 17 species. Our work reveals that PDE is present in 12 out of 17 tested genera, demonstrating its widespread presence in Rhabditidae nematodes, with the notable exception of C. elegans. Genetic tools have already been established for some species. This work provides a collection of lab-tractable species that can be used to test many aspects of somatic Programmed DNA Elimination by chromosome fragmentation in animals.

Polyploidy is a common outcome of chemotherapies, but there is conflicting evidence as to whether this is a source of increased chemotherapy resistance and aggressive disease, or a benign or even favorable outcome. We have used Aurora B kinase (AURKB) inhibitors that efficiently promote polyploidy in many cell types to investigate the fate of polyploid cells. We demonstrate AURKB inhibitor treatment of cells that have loss of RB and p53 function causes them to become hyper-polyploid, undergoing continuous rounds of growth, replication and failed mitosis/cytokinesis (endomitosis), whereas RB and p53 functional cells will eventually exit the cell cycle. These hyper-polyploid cells (>4n DNA content) are viable and undergo continuous endomitotic cycles, but have lost the ability to form viable colonies in vitro or form tumours in vivo. Investigation of mitosis in these cells revealed that centrosome duplication remained coupled to DNA replication, with the hyper-polyploid cells containing high numbers of centrosome that were capable of supporting functional mitotic spindle poles, but these failed to progress to anaphase/telophase structures even when AURKB inhibitor was removed after 2-3 days. However, when AURKB inhibitor was removed after 1 day and cells had failed a single cytokinesis to become tetraploid, they retained long term colony forming ability. Collectively, these findings demonstrate that tetraploidy is well tolerated by tumour cells but higher ploidy states are incompatible with long term proliferative potential.

Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle assembly checkpoint (SAC) which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1{Delta} or mad2{Delta} strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die rapidly as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. Finally, we analyse the phenotype of mad and mps1 mutants during titan cell generation: quantitating viability of titan cells and their daughters generated during the ensuing reductive division. The mad1{Delta}, mad2{Delta} and mps{Delta} mutants show significantly reduced viability: many titans are dead and others produce slow growing colonies. We propose that these Cryptococcus neoformans checkpoint proteins have important roles in ensuring high fidelity chromosome segregation during stressful conditions, such that those heightened during its polyploid infection cycle.

Cell competition is a conserved phenomenon spanning from arthropods to humans. It involves the elimination of viable yet suboptimal "loser" cells when juxtaposed with their fitter "winner" counterparts. This process has received increased attention for its implications in cancer initiation and progression, neurodegeneration, and ageing. This study investigates the presence of the loser fitness fingerprint Flower LoseB (Fwe LB) and the fitness checkpoint Azot in the optic lobes over a period of 28 days. Notably, the absence of Azot is conventionally linked to the accumulation of loser cells over time. However, our investigation reveals that this accumulation is not perpetual and, intriguingly, Azot is not required for loser cell elimination in this context. Furthermore, we estimate that fewer than 50% of Fwe LB-expressing cells also express Azot and undergo apoptosis. Remarkably, our calculations also demonstrate that over 50% of cells undergoing apoptosis at any given time point are positive for the loser markers Fwe LB and Azot. This comprehensive analysis of fitness marker dynamics over a 28-day timeframe sheds new light on the intricate mechanisms governing Flower-dependent cell competition.

The study of Nuclear Matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogaster embryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol can be extended to larval tissues like salivary glands and imaginal discs with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant and transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture. SummaryNuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situ in the intact embryos and tissues of Drosophila melanogaster and its potential applications.

Tuberin is a major component of the protein regulatory complex known as the Tuberous Sclerosis Complex and plays a crucial role in cell cycle progression and protein synthesis. Mutations in the Tuberin gene, TSC2, lead to the formation of benign tumors in many organ systems and causes the Tuberous Sclerosis Complex disorder. Genotypes ranging from point mutations to large deletions in the TSC2 gene have been clinically characterized with a wide range of phenotypes from skin tumors to large brain tumors. Our current work investigates the molecular mechanisms behind Tuberin and its ability to regulate the cell cycle through its binding to the G2/M cyclin, Cyclin B1. After creating an early stop codon in a critical region of the Tuberin, our results show the in vitro phenotype that occurs from a truncated Tuberin protein. Herein we demonstrate that this clinically relevant truncated form of Tuberin promotes an increased nuclear accumulation of Cyclin B1 and a subsequent increase in cell proliferation supporting the phenotypic data seen in the clinic with Tuberous Sclerosis Complex patients showing deletions within the TSC2 gene. This data provides an insight into some of the functional and molecular consequences of truncated proteins that are seen in clinical patients.

Hypoxia and anoxia are known to suppress cell proliferation due to an increase in replication stress and activation of DNA damage checkpoints. Embryos of the annual killifish Austrofundulus limnaeus show a strong tolerance to extended anoxic exposure, indicating an improved genomic stability under oxygen starvation. Here we investigate the cell cycle regulation of the anoxia tolerant killifish embryonic cell line PSU-AL-WS40NE during anoxic exposure. Live cell imaging confirms continued cell proliferation of WS40NE cells for the first 24 hours of anoxic exposure with minimal cell death. Fluorescent imaging shows that cells begin to accumulate in G1 after the first day in anoxia with a pronounced and rapid entry into the S phase upon reoxygenation. Pharmacological inhibition tests show that this response appears to be reliant more on ATR signaling then ATM, suggesting that increased {gamma}H2AX levels are driven by increased replication stress instead of DNA damage. This conclusion is further supported by an apparent lack of induction of a G2 checkpoint in these cells suggesting that DNA damage during anoxic replication is minimal. Maintaining cellular proliferation during initial exposure to anoxia and accumulating cells in the G1 phase for extended anoxic exposure is likely one way that embryos of the annual killifish are able to survive prolonged anoxia and provides insight into mechanisms that enable cells to proliferate under metabolic stress.

Coleoid cephalopods, squids, cuttlefish, and octopuses, have emerged as powerful model organisms for studying neurobiology, development, and behavior, however, cellular tools for investigating their specialized tissues remain limited. In particular, their venom producing gland, the posterior salivary gland (PSG), has been extensively described anatomically and histologically, yet remains largely inaccessible to experimental investigation at the cellular level. Here, we report the first establishment of primary cell cultures derived from both the optical lobe and PSG tissues of Octopus bimaculoides. Building on recent advances in cephalopod brain cultures, we adapted and optimized dissociation and culture conditions to support short-term survival and attachment of cells in vitro. We show that passive cell release during tissue handling, rather than enzymatic treatment, yields viable cultures from both tissues, and poly-D-lysine markedly improves the adherence of PSG-derived cells. Morphological analyses and fluorescent staining confirm the presence and viability of distinct cell populations, while cell cycle analysis indicates that the majority of cells reside in G0/G1 phase. Notably, O. bimaculoides brain cultures exhibit features comparable to those previously described in squid, suggesting conserved aspects of coleoid cellular physiology. Together, our findings establish a foundational in vitro platform for studying octopus PSG and neural cell biology, providing a tractable system for probing venom biosynthesis, secretion, and neural regulation in coleoid cephalopods.

Cilia and flagella associated protein 410 (CFAP410) is a protein localized at the basal body of cilia/flagella and plays essential roles in ciliogenesis. Multiple single amino acid mutations in CFAP410 have been identified in patients. However, the molecular mechanism for how the mutations cause these disorders remains poorly understood due to a lack of high-resolution structures of the protein. Our studies demonstrate that CFAP410 adopts a bimodular architecture. We have previously reported our structural studies on the C-terminal domain (CTD) of CFAP410 from various organisms. Here we report a 1.0-[A] resolution crystal structure of the N-terminal domain (NTD) of Trypanosoma brucei CFAP410. We further examined how the disease-causing mutations in this domain may affect the folding and structural stability of CFAP410. Our results suggest that the single-residue mutations in the CFAP410-NTD cause human diseases by destabilizing the structure that subsequently disrupts its interaction with other partners.

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 {beta}-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 disrupted 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. Finally, pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior tissue in Echinococcus protoscoleces, killed metacestode vesicles, and reduced stem-cell proliferation at nanomolar concentrations. 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. Author SummaryAlveolar 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 {beta}-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 distortion. 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. Pharmacological inhibition of WNT signalling with pyrvinium pamoate caused complete loss of posterior structures in protoscoleces, reduced stem-cell proliferation, and killed metacestode tissue. These findings identify {beta}-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.