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

doi:10.21203/rs.3.rs-5020183/v1

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

https://doi.org/10.21203/rs.3.rs-5020183/v1

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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.

echinococcosis

notch

RNAi

differentiation

stem cell

The metacestode larval stage of the tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, one of the most dangerous zoonoses of the Northern Hemisphere (Thompson 2017). Upon initial infection of the intermediate host (e.g. rodents, humans) by oral uptake of the oncosphere larva (contained in ‘infectious eggs’) and penetration of the intestinal barrier through the parasite, the oncosphere undergoes a true metamorphosis towards the metacestode, mostly within the liver of the host (Brehm and Koziol 2017). Subsequently, the metacestode tissue grows infiltratively, like a malignant tumour, within the host liver and even metastasizes to other host organs, eventually leading to organ failure and death. In natural intermediate hosts such as rodents, numerous protoscoleces are formed within the extended metacestode tissue that are passed on to the definitive host (foxes or dogs) when it takes the prey (Brehm and Koziol 2017; Thompson 2017).

The basic morphological unit of the metacestode is a cyst-like structure, surrounded by an acellular laminated layer that is produced by the parasite germinal layer, which consists of a syncytial tegument as well as few differentiated cell types such as calcareous corpuscles, muscle, nerve, and storage cells (Brehm and Koziol 2017). Around 25% of the germinal layer are made up of pluripotent stem cells (called ‘germinative cells’; GC) for which we previously demonstrated that they are the only mitotically active parasite cells, which give rise to all differentiated cell types (Koziol et al. 2014; Brehm and Koziol 2017). Since GC are highly regenerative, leading to complete metacestode regeneration in vitro within ~ 2 weeks of culture (Spiliotis et al. 2008, 2010), they are the key to anti-parasitic intervention strategies and must be eliminated if parasite killing is to be achieved (Brehm and Koziol 2014). Understanding the dynamics of stem cell self-renewal and differentiation towards muscle, nerve, tegumental, or storage cells in response to the host environment is therefore important in designing anti-parasitic intervention strategies.

Metazoan stem cell differentiation and cell fate decisions are governed by a complex interplay of several evolutionarily old signalling pathways such as wingless-related (wnt), hedgehog, fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor-β (TGF-β), or insulin signalling (Coutu and Galipeau 2011; Gupta et al. 2018; Katoh 2007; Oshimori and Fuchs 2018). Although several studies have so far been undertaken concerning the role of respective pathways in Echinococcus stem cell biology (Cheng et al 2017; Förster et al. 2019; Hemer et al. 2014; Kaethner et al. 2023; Koziol et al 2014), the basic mechanisms of how GC self-renewal and differentiation are regulated by parasite intrinsic or host-derived signals remain enigmatic.

One of the best studied metazoan pathways that determines cell fate decisions and differentiation is the delta/notch signalling pathway, which predominantly acts between neighbouring cells (Borggrefe and Oswald 2009; Bray 2006; Chiba 2006; Liao and Oates 2017; Wang et al. 2012). In this pathway, ligands of the delta or the related serrate/jagged families are expressed at the surface of signal transmitting cells (the ‘sender’) and directly bind to notch family receptors at the neighbouring cell (the ‘receiver’). Both the delta/serrate ligands and the notch receptor are single-pass transmembrane molecules that are composed of numerous extracellular EGF domains, alongside additional domains such as DSL (delta/serrate ligand) or cysteine-rich repeats, in the case of delta/serrate ligands, or LNR (Lin-notch repeat) and intracellular ankyrin (ANK) repeats, in the case of notch receptors (Borggrefe and Oswald 2009; Bray 2006). Ligand binding to heterodimeric notch receptors at the adjacent cell leads to metalloprotease-mediated cleavage of the extracellular receptor portion (called ‘S2-cleavage’), followed by an additional, γ-secretase-mediated processing step, called ‘S3-cleavage’ (Borggrefe and Oswald 2009; Bray 2006). The released intracellular notch receptor domain (NICD) is subsequently translocated to the nucleus of the signal receiving cell, where it associates with the constitutive DNA binding protein CSL (CBF1, Suppressor of hairless, LAG-1) and turns the CSL complex from a transcriptional repressor to an activator (Borggrefe and Oswald 2009; Bray 2006). The best-known targets for CSL are genes encoding basic helix-loop-helix (bHLH) transcription factors of the HES family, which govern terminal maturation of the signal receiving cell (Borggrefe and Oswald 2009; Bray 2006). Classical regulation mechanisms involving delta/notch signalling are lateral inhibition, in which differences between populations of equivalent cells are amplified, lineage decision, in which asymmetric inheritance of notch regulators leads to different fate of daughter cells, or boundary induction, in which organizer induction establishes developmental segregation of adjacent groups of cells (Borggrefe and Oswald 2009; Bray 2006; Liao and Oates 2017). While in most settings, delta/notch signalling determines terminal cell fate decisions among differentiating cells, this pathway is also involved in regulating stem cell self-renewal and asymmetric divisions in others (Abel et al 2014; Bray 2006; Xing et al. 2024).

Delta/notch signalling has so far little been studied in platyhelminths and even in the relatively well characterized systems of free-living planarians, only one report concerning the effects of γ-secretase inhibitors on regeneration (Dong et al. 2021) and few gene expression analyses in germ cells (Khan and Newmark 2022) are available. In schistosomes, Magalaes et al. (2016) bioinformatically identified core components of delta/notch signalling and demonstrated detrimental effects of the γ-secretase inhibitor DAPT on egg production. In E. granulosus, only one qRT-PCR expression study was performed, showing that one notch gene (orthologous to notch1 in the present study) was upregulated in the metacestode stage when compared to protoscolex or adult worms (Dezaki et al. 2016). We now present comprehensive bioinformatic analyses demonstrating that all core components of delta/notch signalling are present in Echinococcus genomes. We show that the expression patterns of selected signalling components point to lateral inhibition processes in differentiated parasite cells, and we demonstrate that RNA interference (RNAi) knockdown of delta/notch encoding genes negatively influences metacestode development. The implications of these data for cell fate decisions in Echinococcus are discussed.

Parasite material and in vitro cultivation

Experiments were carried out using the previously described parasite isolates Ingrid, GH09, and H95 (Herz et al. 2024). Metacestode tissue was continuously propagated by intraperitoneal passage in Mongolian jirds essentially as previously described (Spiliotis and Brehm 2009). Isolation and cultivation of parasite primary cell cultures was performed as previously described (Spiliotis et al. 2008, 2010; Spiliotis and Brehm 2009). Axenic cultivation was performed using conditioned medium from rat Reuber cells, nitrogen gas phase, and reducing conditions as described by Spiliotis and Brehm (2009) with medium changes every 3–4 days. Protoscoleces were isolated from in vivo cultivated parasite material as previously described (Koziol et al. 2013) and activated by low pH/pepsin/taurocholate treatment as described by Ritler et al. (2017).

Nucleic acid isolation, cloning, and sequencing

Total RNA was isolated from in vitro cultivated metacestode vesicles using the RNEasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and cDNA was generated using oligonucleotide CD3-RT essentially as previously described (Brehm et al. 2003). PCR products were cloned employing the TOPO TA cloning kit (Thermo Fisher Scientific) and sequenced by the Sanger method. Primer sequences for cloning of preparation of in situ hybridization probes were: notch1-fw (5’-CAG CAG GAT TCA TTC GTT A-3’) and notch1-rev (5’- ATA CTT GAC GGA GCC ATT AG-3’), notch2-fw (5’-CTG GCT TCG TTG GTC TAA-3’) and notch2-rev (5’-GAA CAC TGG CAG GTA AAG AG-3’), delta1-fw (5’-CCA CTT CCG TTA GCA CAG-3’) and delta1-rev (5’-CAT CGA TAA CTG CTG ATA AGG-3’), and delta2-fw (5’-TAC AAC CGC TCC TCA TCA-3’) and delta2-rev (5’-ACG ATA ACC AGA CAG GCA G-3’).

In situ hybridization and EdU incorporation labelling

Whole mount in situ hybridization (WISH) was performed on in vitro cultivated metacestode vesicles according to a previously established protocol (Koziol et al. 2014). Digoxygenin (DIG)-labeled probes were synthesized by in vitro transcription with T7 and SP6 polymerase (New England Biolabs) using the DIG RNA labelling kit (Roche) from cDNA fragment cloned into vector pJET1.2 (Thermo Fisher Scientific). After hybridization, all fluorescent specimens were processed and analysed essentially as described recently (Koike et al. 2022; Kaethner et al. 2023). Control experiments using labelled sense probes were always negative. In vitro staining of S-phase stem cells was carried out as previously described (Koziol et al. 2014) using 50 µM 5-ethynyl-2’-deoxyuridine (EdU) for a pulse of 5 h after vesicle isolation, followed by fluorescent detection with Alexa Fluor 555 azide as described previously (Koike et al. 2022; Kaethner et al. 2023). In the case of pulse-chase experiments, in vitro cultivated metacestode vesicles were first incubated for 5 h in EdU medium and then cultivated for another 72 h under axenic conditions, prior to in WISH as described above.

RNA interference (RNAi) experiments and RT-qPCR

RNAi was carried out on parasite primary cell cultures according to the method previously established by Spiliotis et al. (2010). Briefly, 1000 units of Echinococcus primary cell preparations, generated as described by Perez et al. (2022), were incubated in 5 ml culture medium (cDMEM-A6/B4) overnight at 37°C and nitrogen gas atmosphere. For electroporation, samples were transferred to a 1 mm gap electroporation cuvette (Plus BXTR) and pulsed once (200 V, 13 Ω, 50 µF, time constant 0.6 ms) in a GenePulser XCell (Biorad Laboratories) in the presence of desalted 23 nt duplex siRNAs (1.5 µg/µl; SIGMA-Genosys, Germany) specific for each gene or against GFP as a control (Spiliotis et al. 2010). siRNA sequences were 5’-GGU AAA UGU GAG GUU UCU AAA GA-3’ (notch2) and 5’-CAA CCU GGU UCU GUC UAC UGG AA-3’ (delta2). After electroporation, the cells were incubated for 12 min at 37°C, washed with fresh cDMEM-A6/B4 medium, transferred to culture wells, and further incubated for 3 weeks as described above. The generation of mature metacestode vesicles was assessed after 21 days essentially as described previously (Förster et al. 2019). To assess transcript knockdown, RT-qPCR analyses were performed after 4 days of cultivation essentially as previously described (Perez et al., 2019). Primer sequences for RT-qPCR were 5’-AAT GGC TGA TAT TGA AAT GGT TGA G-3’ and 5’-CTA CTC CTC CTC CCT GAT GCA-3’ for notch2 as well as 5’-CAT CAA CTT CCG TTG TGA ATG TCA G-3’ and 5’-TAC ATG GCG GAA AGT GCG G-3’ for delta2. As a reference gene, elp (EmuJ_000485800) was used, which is constitutively expressed in parasite larval stages and primary cells (Herz et al. 2024) and which is no affected by electroporation of primary cell cultures (Perez et al. 2022), using primers as described in Perez et al. (2019). Expression levels were calculated by the efficiency correction method using cycle threshold (Ct) values according to Ancarola et al. (2020). All experiments involving RNAi were performed in biological triplicates (independent primary cell culture setups) and technical triplicates.

Bioinformatic procedures and statistical analyses

Iterative BLASTP searches were carried out against the E. multilocularis genome (version 5, January 2016; Tsai et al. 2013) on WormBase ParaSite (Howe et al. 2016) as well as against the SwissProt database as available at GenomeNet (https://www.genome.jp/). Protein domain structure analysis was carried out using SMART 8.0 (http://smart.embl-heidelberg.de/). Multiple sequence alignments were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and MEGA 11 software. Statistical analyses were performed using GraphPad Prism (version 9) employing one-way t-test or one-way ANOVA (as indicated). Statistical analyses on positive signals in WISH were carried out essentially as previously described (Kaethner et al. 2023).

Identification of Echinococcus delta/notch components

Both delta/notch ligands and receptors are composed of extracellular arrays of multiple EGF domains (Bray 2006), making their bioinformatic identification challenging since EGF domains are also present in numerous other signalling molecules. To unequivocally identify Echinococcus delta/notch signalling factors, iterative BLASTP analyses were therefore performed on the E. multilocularis genome (Tsai et al. 2013) using as queries the respective amino acid sequences of human (available from SwissProt) and schistosome (Berriman et al. 2009; Magalhaes et al. 2016) delta/notch signalling components. Each of the identified Echinococcus gene models was then analysed by BLASTP against the SwissProt database to verify homologies to known delta/notch components. Furthermore, protein domain analyses were performed (SMART 8.0) to verify the presence of functionally conserved domains. By this strategy, we identified two genes that clearly encode notch receptor-like molecules, Notch1 and Notch2, with multiple extracellular EGF domains, NL and NOD domains, and intracellular ANK repeats (Fig. 1, Fig. 2). In the published genomes of E. multilolcularis (Tsai et al. 2013), E. granulosus (Korhonen et al. 2022; Tsai et al. 2013; Zheng et al., 2013), E. canadensis (Maldonado et al., 2017)d oligarthus (Maldonado et al., 2019), 4 additional loci (EmuJ_000343000, EmuJ_000111100, EmuJ_000851400, EmuJ_000252100 and respective orthologs) were annotated as encoding notch receptors, but the corresponding amino acid sequences did not contain intracellular ANK repeats nor NL or NOD domains, thus likely representing mis-annotations.

Ligands for notch receptors can be grouped into functionally distinct sub-families of the delta-like ligands and the serrate/jagged family based on the presence of a cysteine-rich sequence between the transmembrane domain and the first extracellular EGF domain (Fleming 1998). Furthermore, members of both sub-families often contain a so-called delta/serrate domain (DSL) that is essential to activate notch receptors (Bray 2006). Present in the E. multilocularis genome we found two genes encoding canonical members of the delta-like family, Delta1 and Delta2, with several extracellular EGF domains and a DSL region (Fig. 1, Fig. 2). Among the remaining positive hits, one locus (EmuJ_000744300) coded for a protein with DSL domain, 16 EGF domains, and a cysteine-rich region, thus indicating that it is a canonical member of the jagged/serrate family. Three additional loci lacked the cysteine-rich region, contained fewer EGF domains, and displayed highest overall homologies to mammalian members of the jagged family. Although we cannot clearly allocate those factors to one of the two sub-families, we assume that they are jagged family members (Fig. 1).

Several proteolytic cleavage steps are important in delta/notch signalling mechanisms, of which one (S1 cleavage,) which is essential for maturation of notch receptors, is mediated by the endo-protease furin (Bray 2006; Sachan et al. 2023). Two further cleavage steps that occur at the notch receptor are called S2-cleavage, mediated by metalloproteases, and S3-cleavage, which involves the γ-secretase complex. As shown in Fig. 1, we identified respective proteins encoded by the E. multilocularis genome such as one furin-convertase gene, one gene encoding an ADAM-like metalloprotease, and all four components (Presenilin, Nicastrin, PEN-2, and APH1) that form the γ-secretase complex.

Within the nucleus, delta/notch signalling converges on the transcription factor Su(H) (suppressor of hairless) which, together with co-activators and co-repressors, regulates target gene transcription (Borggrefe and Oswald 2009). In this context, we identified a clear Su(H) ortholog, two co-repressors of the GROUCHO family and one CtBP (C-terminal binding protein) co-repressor (Fig. 1), together with one SKIP-like co-activator, which we had already characterized in a previous study (Gelmedin et al. 2005). In mammals, binding of GROUCHO and CtBP to Su(H) requires the co-factor hairless, an ortholog for which we could not identify. However, previous analyses had revealed that, in Lophotrochozoa and other protostomes, the co-repressors can directly bind Su(H) (Miller et al. 2019) and we suggest that this is also the case in Echinococcus. Likewise, we could not detect an Echinococcus ortholog of the co-repressor mastermind, which is important for Su(H) function in vertebrates and Drosophila. However, as previously outlined by Gazave et al. (2009), due to little sequence conservation between vertebrate and invertebrate mastermind proteins, the respective genes are either very difficult to identify or, as an alternative, have indeed been lost in many invertebrate species.

Finally, we also identified several genes encoding bHLH transcription factors such as HES (hairy and enhancer of split) or SIM (single-minded) genes (Fig. 1), that are usual target genes for Su(H) (Davis and Turner 2001). It should be noted in this context that contrasting to all other factors mentioned above, which are well expressed in Echinococcus larvae, these genes only displayed very low expression levels in the metacestode (Fig. 1). On the one hand, it is possible that in most metacestode cells these genes are repressed by Su(H) under steady-state conditions. Alternatively, Echinococcus delta/notch signalling could mostly act on alternative targets such as cyclinD or hox genes, which are present in Echinococcus (Tsai et al. 2013), and which are Su(H) targets in other systems (Borggrefe and Oswald 2009).

Taken together, our bioinformatic analyses demonstrated that all core components of delta/notch signalling are encoded by Echinococcus genomes and are well expressed throughout development, although several aspects concerning Su(H) targets genes are still to be clarified. Interestingly, when we assessed the expression of identified delta/notch signalling components in recently generated transcriptomic profiles of metacestode vesicles with and without stem cells (Herz et al. 2024), we found no indication that these factors are among the stem cell expressed genes (Fig. 1). These data indicate that Echinococcus delta/notch signalling mostly acts in differentiating cells.

Delta/notch expression in the Echinococcus protoscolex

To further study Echinococcus delta/notch gene expression patterns, we then concentrated on the two identified notch receptor genes, notch1 and notch2, as well as on the two unequivocally identified delta-like ligand encoding genes, delta1 and delta2. We first carried out WISH analyses on the activated protoscolex, that mostly consists of terminally differentiated cells and shows stem cell proliferation almost exclusively in the neck region (Koziol et al. 2014). As shown in Fig. 3, we found a striking, alternating expression pattern for delta1 in the sucker region, reminiscent of lateral inhibition patterns that have been described in other biological systems (Liao and Oates 2017; Sjökvist and Andersson 2019). The delta1 expression domain is located at the edges of the sucker region that is rich in muscle and nerve cells (Koziol et al. 2013) and, although we cannot yet tell which specific cell type expresses delta1, we assume that its expression determines the differentiation of muscle and nerve cell fates.

delta2 displayed another distinct expression pattern with many positively staining cells in the interior region of the sucker, and a particularly striking domain close to the posterior pole (Fig. 3), adjacent to the region where we previously described the presence of two wnt1 expressing cells that are involved in anteroposterior axis formation (Koziol et al. 2016). Probably depending on the state of the activation process, delta2 was either expressed in two cells close to the posterior pole or, again, displayed a salt-and-pepper distribution at the posterior domain (Fig. 3). Hence, in contrast to delta1, which was exclusively anteriorly expressed in the sucker region, delta2 also showed prominent expression at the posterior pole.

As was the case for both delta-like ligands, notch1 displayed an expression pattern reminiscent of lateral inhibition processes in the neck region of the protoscolex, close to the surface tegument with alternating cells that are either notch1 + or notch1- (Fig. 3). Finally, notch2 was expressed in the central protoscolex neck region around the main and medial lateral nerve cords and the midline (Fig. 4). Furthermore, notch2 was also expressed in one single cell central to each sucker (Fig. 4), which was surrounded by delta2 + cells as shown in Fig. 3.

Taken together, in the terminally differentiated and activated protoscolex, all delta/notch signalling components which we have investigated displayed expression patterns clearly indicative of lateral inhibition, mostly in the protoscolex neck region and in the suckers.

Delta/notch expression in the Echinococcus metacestode

We next analysed expression patterns of delta-like ligands and notch receptors in the E. multilocularis metacestode under steady state conditions in which new cells are constantly formed through proliferative activity of parasite stem cells (Brehm and Koziol 2017). To investigate whether any of the components is expressed in the stem cell compartment, we combined WISH with EdU incorporation, thus staining stem cells in S-phase (Koziol et al. 2014).

For delta1, we did not obtain positive signals in the metacestode which agrees with transcriptome data (Herz et al. 2024; Tsai et al. 2013) showing that delta1 is exclusively expressed in the protoscolex (Fig. 1). delta2, on the other hand, was expressed in numerous cells distributed over the germinal layer without discernible pattern (Fig. 5). In total, around 4.7% (± 1.8%; n = 967) of all cells of the metacestode stained positive for delta2 and, interestingly, we could not detect any co-staining for delta2 and EdU, indicating that the gene is not expressed in GC. Like delta2, notch1 was expressed in the metacestode in cells distributed over the germinal layer (Fig. 5) with 4.4% (± 1.5%; n = 1056) of all cells staining positive for the receptor encoding gene. Again, no cells staining positive for notch1 and EdU were detected. Lastly, notch2 was intensely expressed over the germinal layer in 12.8% (± 2.5%; n = 1143) of all cells and, again, no notch2+/EdU + cells were detected (Fig. 5).

Taken together, these data indicated that delta2 and notch2 are the predominantly expressed delta/notch signalling components of the metacestode and that none of these genes is expressed in S-phase stem cells. It should also be noted that although notch2 and delta2 were well expressed in the germinal layer, we did not detect expression in developing brood capsules, except of one intense signal for delta2 at the anterior pole of the developing protoscolex (Fig. 5).

Since delta2 and notch2 were prominently expressed within the metacestode (Fig. 5) and showed expression in adjacent domains of the protoscolex (Fig. 3, Fig. 4), we assumed that both could represent a cognate ligand/receptor pair. We therefore carried out double WISH to analyse the relative expression of both genes in the metacestode. Again, we obtained clear signals for both genes and in few cases (around 5% of all notch2 + cells) we also obtained double positive delta2+/notch2 + cells as well as neighbouring cells expressing either of the genes (Fig. 6), indicating that both mechanisms of trans-activation and cis-inhibition (Sprinzak et al. 2010) could occur within metacestode tissue.

delta2 and notch2 are exclusively expressed in post-mitotic cells

Since delta/notch signalling is usually associated with cell differentiation, it was not necessarily expected that these genes are expressed in S-phase stem cells and as shown above, this was indeed the case in the Echinococcus metacestode. However, in several systems of asymmetric division of stem cells in response to the stem cell niche, delta/notch components are expressed directly after mitosis, thus regulating differentiation of one progeny cell and self-renewal of the other (Wang et al. 2012; Zamfirescu et al. 2022). To assess whether this occurs in the metacestode, we carried out pulse-chase experiments in which in vitro cultivated vesicles were first incubated for 5 h with EdU, followed by WISH 3 days later. Recent experiments of our group had shown that under these conditions the vast majority of metacestode stem cells had completed the cell cycle, yielding EdU + progeny cells (Herz et al. 2024). Accordingly, as shown in Fig. 7, we observed doubling of EdU + cells in metacestode tissue (from 8% ± 2% of all cells to 17% ± 3%, n = 1434) after 3 days of incubation, indicating that most stem cells had undergone mitosis. However, neither for delta2 nor for notch2 could we detect cells staining positive for EdU and WISH (Fig. 7). These data indicated that in the Echinococcus metacestode cell fate decisions involving delta/notch signalling are made very late after stem cell division, probably between differentiated and transiting cells that had left the cell cycle.

Figure 7 Pulse chase experiments indicate expression of delta2 and notch2 in post-mitotic cells. Shown are WISH EdU images (singel confocal slices) for notch2 and delta2 (as indicated). Channels are: blue (DAPI, nuclei), green (WISH), red (EdU). Shown are for notch2 (upper panel) 5 h EdU incorporation in red/green (left) and merge channels (right) as well as same channels for pulse chase after 3 d. Panel below shows respective images for delta2. Size bar represents 50 µm.

delta2 and notch2 are required for metacestode formation

In a final set of experiments, we utilized previously established protocols for targeted RNAi gene knockdown on Echinococcus primary cell cultures (Spiliotis et al. 2010) to investigate the role of delta2 and notch2 in metacestode formation. As shown in Fig. 8, we merely obtained gene knockdown rates to ~ 50% (delta2) or ~ 70% (notch2) of the original transcript levels. However, in both cases this was sufficient to yield statistically significantly reduced numbers of mature metacestode vesicles that were formed by these cell cultures. These data indicated that both delta/notch components are required for proper metacestode vesicle formation from parasite stem cells.

Unlike most cestode species, which undergo direct development from invading oncosphere larvae to head-like structures within the intermediate host, tapeworms of the genus Echinococcus display asexual multiplication by forming massive cysts (E. granulosus) or infiltratively growing and even metastasizing metacestode tissue (E. multilocularis) (Brehm and Koziol 2017; Koziol 2017). As we previously showed, this is most likely mediated by modulation of body-axis formation and delayed formation of the head organizing anterior pole, resulting in strongly posteriorized metacestode tissue (Koziol et al. 2016). We also demonstrated that metacestode growth is exclusively driven by a population of pluripotent stem cells (GC), which are the only parasite cells capable of undergoing mitosis, and which give rise to all differentiated cell types (Koziol et al. 2014). Since GC are highly regenerative and can replace metacestode tissue within 2–3 weeks (Spiliotis et al. 2008), they are of particular interest for the development of anti-infectives (Brehm and Koziol 2014). In this context, it is highly relevant to study mechanisms by which the germinative cell population undergoes stem cell renewal and maintenance, and which dynamics regulate the formation of differentiated cell types.

Since Echinococcus GC share several characteristics with stem cell systems of other flatworms (Herz et al. 2024; Koziol et al. 2014; Koziol 2017), including the well-studied planarian system, it is expected that at least some basic principles of stem cell dynamics are also shared between these groups. In planarians it is well established that the somatic stem cells (neoblasts), although morphologically indistinguishable, can be divided into functionally different sub-populations with differing gene expression profiles. According to the current model, one of these sub-popolations, the cNeoblasts, are truly pluripotent and give rise to more specialized subtypes (σ-, γ-, ζ-, ν-neoblasts), which eventually produce differentiated cells of distinct planarian tissues (Molina and Cebriá 2021). Evidence for the existence of different sub-populations of Echinococcus GC has already been obtained (Förster et al. 2019; Herz et al. 2024; Koziol et al. 2014; Stoll et al. 2021;) and several studies indicated that insulin (Hemer et al. 2014), FGF (Förster et al. 2019), and EGF pathways (Cheng et al. 2017) are likely involved in regulating Echinococcus stem cell dynamics. However, which pathways govern cell fate decisions between different Echinococcus stem cell sub-populations, or which components regulate terminal differentiation, are still elusive.

One of the evolutionarily very old systems of governing cell fate decisions in metazoans is the delta/notch signalling pathway (Bray 2006), and we herein carried out first steps towards a systematic characterization of this pathway in Echinococcus. Based on our bioinformatic analyses on Echinococcus genomes (Tsai et al. 2013) and transcriptomes (Herz et al. 2024; Tsai et al. 2013) it became clear that all essential components of this pathway are present in Echinococcus larval stages, although several aspects concerning delta/notch targeting genes still must be clarified in future studies. When compared to mammals, which express 4 members of the notch receptor family (Salazar and Yamamoto 2018), Echinococcus apparently expresses a reduced complement of only 2 receptors, which is, however, in agreement with other invertebrate species such as Caenorhabditis elegans (2 receptors) or Drosophila melanogaster (only one receptor) (Salazar and Yamamoto 2018). Likewise, the number of clearly identified delta- (two) and jagged- (one) like ligands is within the range of ligand molecules encoded by other invertebrate organisms (Salazar and Yamamoto 2018). Due to membrane bound expression of both delta/serrate ligands and notch receptors, functional assays concerning cognate ligand receptor pairs are difficult to perform. Nevertheless, based on our data, we suggest that at least the products of Echinococcus delta2 and notch2 are interacting partners since (i) the genes are expressed in adjacent domains within the protoscolex, and (ii) both genes show dominant expression within the metacestode stage. Based on our double WISH experiments, we also suggest that delta2 and notch2 are involved in trans-activation processes as well as in cis-inhibitory regulation.

Depending on the context, delta/notch signalling either regulates cell fate decisions in differentiating cells (Bray 2006; Sjökvist and Andersson 2019) or even self-renewal and specialization of stem cell systems (Abel et al. 2014; Gioftsidi et al. 2022; Wang et al. 2012). Our data indicate that in Echinococcus, delta/notch signalling exclusively governs fate decisions of differentiating cells. Neither in the activated protoscolex nor in the metacestode did we find EdU + cells that express delta/notch signalling components and even in pulse-chase experiments, no immediate progeny cell was positive for delta/notch gene expression. We therefore propose that Echinococcus delta/notch signalling is not involved in defining germinative cell sub-populations, but exclusively in determining fate of intermediate (differentiating) cells, probably depending on the presence of neighbouring cells that are terminally differentiated. This is also supported by recent transcriptomic analyses of our group (Herz et al. 2024), in which gene expression of mature metacestode vesicles was compared to vesicles in which stem cells were specifically depleted by treatment with hydroxyurea (HU) for 7 days. Although it is expected that this treatment also affects, at least to a certain extent, progeny cells that are immediately formed after stem cell division (see discussion in Herz et al. 2024), none of the delta/notch signalling components identified in the present study was affected by HU treatment (they were even higher expressed; Fig. 1).

We had previously shown that the metacestode represents posteriorized tissue that proliferates and differentiates in a wnt1 high environment (Koziol et al. 2016) and this is, interestingly, also reflected in the gene expression profiles of delta/notch components that we carried out herein. Only those factors that are posteriorly expressed in the protoscolex (delta2, notch2, and much less notch1) are also expressed in the metacestode. Of particular importance in this context could be delta2, for which we showed that it shows prominent expression at the protoscolex posterior pole, immediately adjacent to those cells that express posteriorizing wnt1. Since wnt1 is expressed in terminally differentiated muscle cells (Koziol et al. 2016), it is thus possible that delta2 expressing cells act as a signalling centre to direct the terminal differentiation of newly formed, intermediate cells, towards wnt1 + muscle cells. In this case we would expect that interference with delta2 expression would lead to reduced capacity of Echinococcus primary cell cultures to generate mature vesicles, which is what we had observed in our knockdown experiments. Even though we only achieved partial knockdown for delta2 and notch2, the primary cell cultures were significantly affected in their capacity to produce mature vesicles. Overall, this underlines the importance of delta/notch signalling in Echinococcus cell differentiation events, including the formation of the metacestode.

In the context of drug development, delta/notch signalling is in so far interesting as many of its components, particularly those for receptor processing such as furin convertase, ADAM-like metalloproteases, or the γ-secretase complex, are druggable enzymes (Majumder et al. 2021). To our knowledge, no investigations have so far been undertaken into one of these enzymes as possible drug targets for echinococcosis treatment. Having shown by RNAi that delta/notch signalling plays an important role in Echinococcus development, future endeavours into the role of these factors in Echinococcus signalling mechanisms and their exploitation as drug targets might be worthwhile.

Acknowledgements

The authors are indebted to Dirk Radloff for excellent technical assistance. We also wish to thank Uriel Koziol for fruitful discussions and valuable advice.

Ethical Approval

All animal experiments were carried out in accordance with European and German regulations on the protection of animals (Tierschutzgesetz, Sect. 6). Ethical approval of the study was obtained by the local ethics committee of the government of Lower Franconia (permit no. 55.2–2531.2-1824).

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Competing interests

The authors declare there are no competing interests.

Authors' contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Chris Speicher, Monika Bergmann and Klaus Brehm. The first draft of the manuscript was written by Klaus Brehm and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by a grant of the Bayerische Forschungsstiftung [AZ-1341-18] (to Klaus Brehm). Chris Speicher was supported by an individual grant of the Graduate School for Life Sciences, University of Würzburg.

Availability of data and materials

New sequences reported in this study were submitted GenBank and are available under the following accession numbers: PP657438 (notch1), PP657437 (notch2), PP657439 (delta1), and PP657440 (delta2).

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

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Nucleic acid isolation, cloning, and sequencing

RNA interference (RNAi) experiments and RT-qPCR

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