Tracing back the origin of cell-cell communication: Hydra vulgaris releases extracellular vesicles delivering regulators of head and foot regeneration

Maria Moros Instituto deNanociencia y Materiales de Aragón(INMA) Eugenio Fergola Istituto di Scienze Applicate e Sistemi Intelligenti “E. Caianiello”, Consiglio Nazionale delle Ricerche Valentina Marchesano CNR-ISASI Margherita Mutarelli Istituto di Scienze Applicate e Sistemi Intelligenti “E. Caianiello”, Consiglio Nazionale delle Ricerche Giuseppina Tommasini Istituto di scienze Applicate e Sistemi Intelligenti "E.Caianiello", Consiglio Nazionale delle Ricerche Beata Miedziak Istituto di scienze Applicate e Sistemi Intelligenti "E.Caianiello", Consiglio Nazionale delle Ricerche Giuliana Palumbo Istituto di Scienze Applicate e Sistemi Intelligenti “E. Caianiello”, Consiglio Nazionale delle Ricerche Alfredo Ambrosone Istituto di scienze applicate e sistemi intelligenti https://orcid.org/0000-0002-1897-4028 Angela Tino Istituto di Scienze Applicate e Sistemi Intelligenti “E. Caianiello”, Consiglio Nazionale delle Ricerche Claudia Tortiglione (  claudia.tortiglione@cnr.it ) Istituto di scienze Applicate e Sistemi Intelligenti "E.Caianiello", Consiglio Nazionale delle Ricerche https://orcid.org/00000003-1447-7611

into the receiving cells nucleic acids, metabolites, lipid and proteins 12 . Of particular interest in cancer biology is their role in the 'education' of distant cells in order to create a pre-metastatic niche, thus promoting metastasis 18,19,20 . The intrinsic properties of exosomes in regulating complex intracellular pathways offer a new paradigm for their application as disease biomarkers, or as nanocarriers of therapeutic agents in cell-free therapies 21 .
The wide portfolio of possible applications makes even more appealing the evolutionary study of EVs in model organisms, to increase our understanding in how they work and how they exert their functions in different contexts and environments.
For instance, exosomes derived from Drosophila melanogaster have been identi ed as partly responsible for the establishment of Hedgehog (HH) and Wingless morphogenic gradients during the wing imaginal disc development 22,23 . In Caenorhabditis elegans HH-related proteins are released on apical exosomes in a MVB-dependent manner involving V0-ATPase and P-4 ATPase activities 24,25 . However, very little is known about the role of EVs in aquatic environment. By virtue of their ability to transport abundant biological signals, to protect them from the highly heterogeneous and possibly damaging environment, and to increase the local concentration of one or more co-acting biomolecules that reach the target cells, EVs can have a profound impact on aquatic community structure and trophic level-interactions 26 , providing important clues on interspecies and interkingdom relationships. Due to the conservation of key physiological mechanisms throughout the animal kingdom, evolutionary approaches to understanding EV biogenesis and bioactivity may profoundly impact on both fundamental and applied biology in vertebrates.
In this paper we report for the rst time the presence of EVs in the freshwater cnidarian polyp Hydra vulgaris and characterized main morphological, biochemical and functional features. Classically used as a model organism in developmental biology, the structural simplicity of Hydra body offers many advantages for a variety of investigations, from environmental ecotoxicology to nanobiotechnology, allowing to study the impact of exogenous nanostructured compounds 27,28,29,30 together with intracellular biotransformations and molecular responses 31,32,33 at animal, cell and molecular levels. The body wall is organized as a hollow tube, with an apical mouth opening surrounded by a ring of tentacles and a foot to anchor to a substrate. It is structured as a bilayer of epithelia, an ectoderm with epitheliomuscular cells facing the outer medium and an inner endoderm with all cells facing the body cavity, separated by an acellular mesoglea ( Figure 1A-B). This tissue-like organization, with no organs and body uids, is maintained by the continuous cell self-renewal and differentiation of three distinct stem cell lineages of endoderm, ectoderm, and interstitial stem cells, giving rise to a few differentiated cell type, i.e. neurons, gland cells, nematocytes and gametes 34,35 . The plasticity and remodelling capacity of the body reaches the maximum expression during the regeneration process 34,36,37,38 . Indeed, the ability to regrow the whole body from tissue pieces or even cell aggregates has made Hydra a one-of-a-kind platform to study regeneration processes and to shed some light on why some organisms have partially lost this ability 39 . The Wnt/b-catenin signalling pathway represents the major developmental pathway acting in adult Hydra to maintain the oro-aboral axis, and it is reactivated during injury and throughout the regeneration to rebuild missing structures from amputated polyps 40,41,42 . In addition, Wnt signalling is integrated by Notch-signalling, restricting the tentacle activation and re-establishing the head organizer during regeneration 43 . Functional and transcriptomic evidences have recently showed the early activation of the Wnt/b-catenin signalling pathway as part of a generic transcriptional response to injury, which initiates an oral patterning cascade independent from the tissue context 44,45 . The outcome of this activation appears regulated by long range signals generated by tissue organizers located at both animal poles 44,45,46,47,48 , whose existence was proposed 40 years ago, by means of classical tissue manipulation techniques 49,50 . To date, no vehicles have been isolated for lipid-modi ed and poorly soluble signalling molecules. The EV function in developmental signalling, promoting the release and spread of canonical ligands and/or mediating the transfer of a variety of biologically active molecules from one cell to another is well documented 23,51,52 . This prompted us to search for similar EV mediated signalling mechanisms also in Hydra, and to shed light on novel cell-cell communication strategies acting in homeostatic and in regenerative contexts, at the beginning of animal evolution. We developed a strategy for EV puri cation from the polyp culture medium, characterized their morphology and their molecular content at protein and RNA levels, and showed their active role in modulation of both head and foot regeneration. The shuttling of functional molecules via EV in target cells shows that this mechanism of cell-cell communication evolved early in animal evolution and may mediate not only physiological processes but also cross-species or cross-kingdom communication in aquatic ecosystems.

Results
Hydra vulgaris release round-shaped extracellular vesicles in the aquatic environment Groups of 250 polyps collected into Petri dishes were starved for 4 days into 10 ml of Hydra medium. EVs were isolated from the medium by differential centrifugation, following a well-established protocol 53 , slightly modi ed. Following removal of dead cells and cell debris the last centrifugations were performed at 70,000 x g rather than at 100,000 x g, as high speeds seem to promote the co-puri cation of contaminants such as protein complexes and aggregates 54 . Pelleted EVs were suspended in Phosphate buffered saline (PBS) and characterized by Transmission Electron Microscopy (TEM). Fig. 1C shows the round shape of isolated EV and the presence of double membrane around the electron dense vesicle (see also Fig.   S1). By micro uidic resistive pulse sensing (MRPS) a yield of 5x10 10 particles/mL was estimated from a typical preparation (250 polyps into 10 ml of medium), while the distribution of the particle size showed two peaks at 66 nm and 70.5 nm ( Fig   1D and Fig. S1). A good correlation with these measurements was obtained through other approaches, such as Dynamic Light Scattering (DLS) analysis and TEM (Fig S1), both con rming the average size of 66 nm, and suggesting an enrichment in exosome-like vesicles. The presence of EVs on Hydra ectodermal cell surface was previously supposed during an ultrastructural study describing several inward and outward mechanisms negotiating the entrance, tra cking, and clearance of gold nanoparticles in animal tissues 55 . To con rm this evidence, Hydra polyps were pulsed (24 h) with positively charged AuNPs, synthesised as previously described 55 and 48 h later processed for TEM (Fig. S2). Semi-thin slices showed the presence of round nanovesicles decorated with AuNPs on the external membrane of ectodermal cells, laying into the glycocalix ( Fig. 1E-F), con rming in vivo assembly of AuNP-EV structures as part of the secretory route 55 . The ability of EV to bound positively charged AuNP also in vitro was shown by incubating freshly isolated EVs with AuNPs, producing the same rosette-like nanostructures observed in vivo (Fig. S2). This self-assembly demonstrates the strong interaction between EV membranes and positively charged AuNPs, likely due to electrostatic interaction.
The ability of EVs to be internalized into Hydra tissues was investigated by labelling freshly isolated EVs with PKH67 and incubating living polyps up to 5 h. Distinct green uorescent signals were found on the body column and tentacles 2 h post incubation and in single cells obtained by maceration of treated polyps, indicating an e cient uptake of EV into Hydra cells (Fig 1 G-I; Fig. S3). These signals became diffuse after 5 h, suggesting the internalization and processing of the EV into the recipient cells Hydra EVs shuttle typical exosome-associated proteins and key components of axial patterning In order to characterize the molecular content of the EV, immunoblot analyses were initially performed to identify biochemical markers of EV subclasses, i.e. exosome speci c proteins such as the tetraspanin CD63 14 . By comparing immunoblot of protein extracts from whole animal lysates and EVs, a greater expression of the CD63 protein was found in EV extracts compared to lysates, while similar amount of proteins cross-reacted with antibody against the highly abundant membrane protein annexin B12. Actin expression was found greatly enhanced in whole lysates compared to EVs (Fig. 2). Overall these results demonstrate the presence of some exosome-speci c proteins on EV, and prompted us to perform a mass spectroscopy analysis for a more detailed protein characterization. Initially, a MALDI-TOF/TOF analysis performed on EV protein bands excised from an SDS-PAGE gel con rmed the identi cation of annexin B12, along with actin (Fig. S4). Then a large scale EV isolation was achieved from 8000 Hydra, and protein extracts were used for LC-ESI-MS/MS analysis and protein identi cation. The analysis from two biological replicates identi ed 52 proteins with a Mascot score >32 (Table S1). Table 1 shows selected unique Hydra proteins grouped for function. Remarkably, 29 proteins were present also into the Exocarta protein database (http://www.exocarta.org/), suggesting the exosome -like nature of the isolated EVs. The exosome-speci c proteins include two annexins, a synthenin1-like protein involved in the biogenesis of exosomes, a programmed cell death 6-interacting protein involved in the MVB formation and the CD151 antigen belonging to the Tetraspanin family. Among others, chaperones (heat shock proteins), cytoskeletal proteins (actin, Rab and tubulin), extracellular matrix and glycocalyx constituents ( brillin 1; Mucin 5AC), and important components of cell-cell adhesion (protocadherin fat 4) or signalling pathways (NOD2, Nav2.1, Notch) were found. Interestingly, two uncharacterized proteins containing thrombospondin type-1 domain were found, whose role as a feedback inhibitor of Wnt signalling during Hydra body axis patterning and maintenance has been recently identi ed 56 . Altogether the variety of putative proteins identi ed in the Hydra EVs, either structural or involved in cell-cell communication and signal transduction, suggests multiple functional roles played by EVs in adult Hydra, and their correlation with known exosome proteomes. A comprehensive transcriptomic analysis was performed on total RNA from puri ed EVs conducting pair-end next-generation sequencings. The sequenced reads were rst aligned against the NCBI Hydra vulgaris reference genome (assembly Hydra_RP_1.0), where a percentage of 32% and 22% could be mapped. For gene expression quanti cation, the reads were also directly aligned using the program kallisto on the Hydra_RP_1.0 transcript sequences, where the total mapped reads on transcripts were 50% and 45% of the reads mapped on the genomic sequence. The presence of a subset of genes was con rmed in EV-derived RNA by using reverse transcription polymerase chain reaction PCR (RT-PCR). The selection was based on manual screening of genes already identi ed in EVs and exosomes (hsp70 and actin) or belonging to the Wnt/bcatenin signalling pathway (b-catenin, and Wnt3). We then used the minimum expression level in TPM of the validated genes to de ne an empirical detection threshold (0.4 TPM in at least one library). Using this de nition, we selected 6100 detected genes, among which 5499 were protein-coding, 525 long non-coding and 31 tRNA genes. The detected genes were functionally annotated with the known associated Gene Ontology terms using OrthoDB to highlight the most important biological processes encoded by EV mRNA 57 . The full list of the GO categorized genes is provided in Supplementary le 1.
Interestingly, this analysis revealed that the most abundant transcripts within the Molecular Functions GO category are related to catalytic activity (alcohol dehydrogenase, sul te oxidase), proteases and proteinase inhibitors (cathepsin, several members of the astacin family, antistasin), hydrolase and transferase activity. Within the Cellular Component GO category the genes encoding for intracellular and cytoplasmic anatomical structures were highly abundant, such as actin, cophylin, αtubulin chain, β tubulin chain, talin-2, secreted glycoproteins (mucin 2-like mucin 5AC-like), and LAMP, a well-known exosome marker. The transcripts categorized into the Biological processes GO class included the classes of protein metabolism (tRNA synthase, eIF3, eIF4, eEF1a, tRNAs), signal transduction pathways (G-protein coupled receptors, Rho family, guanidine nuclear exchange factors, Wnt3, gremlin) and response to stress (Hsp70, Hsp90, Superoxide dismutase, Glutamate dehydrogenase, thioredoxin-like).
Beside transcripts identi cation, lncRNA species were detected from the RNA-seq analysis. In one case the lncRNA was mapped adjacent to the mucin5AC gene locus, detected both at mRNA and protein levels, suggesting the possibility that EVs may shuttle all molecular components for immediate and late availability of key information in target cells. The association of lncRNA with exosomes was rst demonstrated by the analysis of human plasma-derived exosomes by RNA-seq 58 , and has been recently functionally characterized and linked to disease, including a list of cancers, where they act via epigenetic regulation of key target genes. Their presence in Hydra EV might suggest a regulatory function in the receiving cells.
In order to map the EV cellular source in the animal body we used a recently-published single-cell RNA-seq (scRNA-seq) atlas 59 to match Hydra EV-associated genes with molecular signatures of speci c cell clusters, which were grouped according to cell lineage (ectodermal, endodermal and interstitial cell lineage), or anatomical location (head, body column and foot) (Fig. 3).
An alignment rate of 12% was obtained and 4997 transcripts were detected, of which 2975 were found in at least one cell type in the scRNA annotated on Swiss-prot (TPM 0.2 in at least one library, Supplementary le 2). A large percentage of transcripts (48%) was clustered as speci c of the interstitial cell lineage, while those derived from the ectodermal (12,7%) and endodermal cell lineage (9,45%) were less abundant. By subclustering the EV transcripts according to unique cell type expression, using the same cell categories and differentiation trajectories identi ed in the scRNA-seq 59 , a remarkable contribute of basal disk cells, battery cells, neurons and nematoblasts emerged ( Fig. 3B-C). A detailed analysis subclustering EV transcripts into cell types of each cell lineage is shown in Fig. S5. Overall this indicates the mixture of precise molecular information from multiple cell types in Hydra EVs. The list of the 5 top genes in each cell cluster is provided in Table S4 and the list of the 10 top genes identi ed matching the scRNAseq is shown in Table S5. Interestingly, many genes belonging to the Wnt/b catenin signalling pathway were identi ed (  56 , was present both at mRNA and protein levels, indicating a pivotal role played by EVs in the regulation of the head organizer formation. By comparing the EV transcriptome to the proteome the majority of annotated proteins (found also on exosome proteomes) was identi ed also at transcript level (actin, tubulin, annexinB12, mucin 5AC, hsp70, astacin, thrombospondin, Notch …) indicating the concomitant transfer of molecular information for immediate and programmed response in the target cells.
Genes involved in Wnt/β-catenin signaling pathway  Table 2 List of EV transcripts involved in the Wnt/β-catenin signalling pathway matching the scRNA-seq database

EVs modulate head and foot regeneration in Hydra
The nding in the EV transcriptome of both positive and negative regulators of the Wnt/b catenin pathway (Fig. 4A) prompted us to evaluate the EV bioactivity in receiving polyps. Wnt3 gene transcript levels were shown upregulated in polyps treated with EV, both at 24 h and 48 h post treatment (Fig. 4B), suggesting for the rst time the involvement of Hydra EV in the exogenous activation of Wnt signalling.
In addition to Wnt modulators, Notch was identi ed in the EV proteome and transcriptome, strongly supporting a role of EVs as contributing to the head organiser activity, carrying signals for local self-activation and long ranging inhibition. To verify this hypothesis, the role of Hydra EVs in the context of regeneration was tested. Freshly prepared EV were added to polyps amputated at midgastric level (50% of body column length), and the head regeneration e ciency was evaluated by morphometric analysis. Stumps of progressive developmental stage (stages 0, 1, 2 and 3) were monitored at regular intervals, starting at 24 h post amputation (p.a.) (Fig. 5A). At each time point signi cant differences were observed in the distribution of developmental stages, with the EV-treated stumps always presenting more advanced stages compared to the controls (Fig. 5B). These results indicate a clear enhancement effect played by EV in the regeneration of the head. Similar results were also obtained when the amputation was sub-hypostomal (80% of upper body length) (Fig. S6), suggesting that EV cargo, when received by upper body cells behaves as an exogenous source of head activators. The impact of EVs on the ability of the lower body cells to regenerate a new foot was evaluated by bisecting whole polyps at 20% of the body length (Fig. 5C). The absence of distinctive morphological changes that characterize this process led us to use biochemical assays to monitor the differentiation of foot speci c cells, based on the detection of peroxidase activity. This enzymatic activity is used as speci c marker of basal disk cell differentiation, producing a strong signal at 36-48 h p.a 64  in Hydra head organizer has been supposed, acting in adults polyps to maintain the oral-aboral polarity and preventing the formation of ectopic organizers elsewhere in the body. Amputated organizers may transiently increase the ability of wounded tissue throughout the body column to form secondary axes, while during foot regeneration these inhibitory signals would prevent ectopic head formation at aboral-facing amputations 44 . To date, these signals have not been identi ed and in the present work we provide evidence that they could rely on EV released in the extracellular medium.
We puri ed and characterized EVs from Hydra culture medium by differential centrifugation, characterized their roundshaped and double-layered morphology via TEM and estimated the size in the range consistent with exosome EV subtype 73 . Proteomic analysis revealed numerous exosome markers, often related to their biogenesis route and cargo sorting mechanisms 74 15 . This evidence, together with the morphology, size and mRNA loading strongly suggests that the EV isolated are exosomes or are highly enriched in this vesicle subtype. Most of the identi ed proteins correlate with the transcripts, including proteinases, catalytic proteins and transcription factors, suggesting the delivery of key molecular information for early and late responses in the receiving cells. Moreover, a multitude of signalling components belonging to BMP, Notch and Wnt/bcatenin pathways among others were identi ed. The alignment of EV transcriptome with the scRNAseq atlas recently produced 59 allowed to extract important information on the cell sources of EV transcripts. Under our condition of EV puri cation multiple cell types appeared involved in the loading and release of EVs. In addition to ectodermal cells expected to release EV, as facing the external medium, a large contribute to the EV transcriptome matched transcripts speci c of the interstitial stem cell lineage, dispersed throughout the body column, especially neurons. This suggests that EVs may mediate the information ow within the Hydra non overlapping neural networks 75 . Finally, a minor fraction of transcripts was speci c of endodermal cells, especially gland cells, which is consistent with their location, facing the gastric cavity, and their molecular repertoire of enzymatic activities and signalling factors, such as those restricting the Wnt signal in the hypostome 56,60,62 . A comparative analysis between EVs produced by transgenic lines tagged in each cell lineage would help in the future to con rm this in silico evidence.
Concerning the possible role in vivo, the EV information tool-box may be used for cell-cell communication to maintain homeostatic condition, i.e. axis polarity, regulate animal size and tissue growth. In other conditions, i.e. during budding, regeneration or stress response, they may act to orchestrate coordinated cell responses between distant tissue regions, or among different polyps. In this work we puri ed EV from the medium of healthy polyps mirroring the condition used to enhance EV production from cell culture, i.e. starvation and crowding 76 and our omics and functional analyses are the outcomes of this particular state. EV may vary their molecular cargo under certain conditions, such as stress, reproduction, temperature changes, adapting the molecular information to deliver diverse messages to the cells, from hydrophobic signals to lipids, second messengers, or nucleic acids. We believe that by comparing the EV content produced under different physiological states different outputs might be produced, leading to decipher the "code" used by cells to communicate to distant cells or organisms about the environmental challenges.
We have demonstrated that treating amputated polyps with EVs, at a dosage far higher compared to the physiological condition, induces an enhancement of the head regeneration and a decrease in the foot regeneration e ciency. We explain these nal outcomes as resulting from the release of key messengers acting as both positive and negative regulators of pivotal signalling pathways. Our focus on Wnt/bcatenin messengers identi ed several EV transcripts belonging to this pathway, especially Wnt inhibitors, leading to the hypothesis that the EV may represent the long distance signalling tools proposed since a long time ago for the determination of head and foot organizer tissue 44,49 . EVs were visualized using TEM following a slightly modi ed protocol to that reported by Thery et al 76 . Brie y, EVs were xed with an equal volume of 4% paraformaldehyde and deposited onto a Formvar-carbon coated electron microscopy grid. Once dried, the grids were washed with PBS, xed with 1% glutaraldehyde for 5 min and washed with distilled water 8 times. The samples were stained with 1% ammonium molybdate for 2 min, dried and observed using a FEI Tecnai 12 120 kV or JEOL USA, Inc., USA) at 80kV Hydra polyps were dissected into appropriate pieces to t into cup-shaped HPF specimen carriers. Tissue pieces were pipetted into a 0.  EV staining and uptake EVs obtained as described before were labelled with PKH67 Green Fluorescent Cell Linker (Sigma Aldrich) following manufacturer's protocol, with minor modi cations. Brie y, EVs obtained from 500 polyps in Hydra medium were added to 1 mL of diluent C. As a negative control, 1 mL of diluent C was mixed with the same volume of Hydra medium. Afterwards, 6 μL of PKH67 dye was added and mixed for 30 sec by gently pipetting. After 5 min incubation at room temperature, 2 mL of 10% BSA was added to quench the unbound dye. EVs were washed and isolated by ultracentrifugation as described before. Hydra were incubated with PKH67-labelled EVs and the negative control up to 5 h, washed with Hydra medium and imaged under a uorescence microscope (Zeiss).

Regeneration Experiments
All the experiments were carried out using adult polyps starved for 24 h. For the head regeneration experiments, batches of 25 budless polyps were bisected at 80 % body length (sub-hypostomal cut) or 50 % body length (mid-gastric cut) and left regenerating for up to 72 h in presence of EVs isolated from 250 polyps and resuspended in 300 µL of Hydra solution. The regeneration process was monitored at different time points using an optical microscope and polyps were grouped into four stages according to their tentacle morphogenetic features. and high (19000-28000 mm 2 ).
EV pre-treatment with proteinase and RNAse Prior to total RNA extraction, EVs isolated by ultracentrifugation were enzymatically treated to eliminate the free RNA derived from the ribonucleoprotein complexes that could be present in the conditioned medium. Isolated vesicles were treated with 1 mg/mL of proteinase K for 30 min at 37°C. Afterward, the samples were incubated 10 min at 65°C to inactivate the proteinase K and then treated with 10 µg/mL of RNAse A for 15 min at 37°C. In order to validate RNA-seq analysis, a presence/absence experiment of selected targets was performed using an EV sample isolated from 2000 polyps overall. Speci c primers of Hydra genes Wnt3a, b-catenin, actin and Hsp70 were designed using the Primer3 software (http://frodo.wi.mit.edu/primer3/) and are listed in Table S3, together with the corresponding GenBank accession numbers. Due to the qualitative nature of the presence/absence analysis, no internal control was used for the ampli cation reaction. The presence of such transcripts in the EV sample was con rmed by their cDNA ampli cation compared to negative controls, where no ampli cation occurred. Additional analysis by generation of melting curves (from 60 °C to 95 °C, 0.5 °C increment) con rmed sequence speci c ampli cation in EVs and the absence of ampli cation in No Template Control (NTC). Mean C t values for each gene and its NTC are shown in Table S2 RNA seq EVs were collected at different times and stored at -80 °C. Total RNA extraction was performed on EV samples collected from 10,000 polyps overall and in two replicates using "Total RNA Puri cation Kit" (Norgen Biotek Corp.) and following the manufacturer's supplementary protocol for exosomal RNA puri cation, with one modi cation: the optional DNA removal step was carried out using RQ1 RNase-Free DNAse (Promega) at 0.1 U/mL. The two samples were sent to the Sequentia Biotech company for quality check, cDNA library preparation using "Ovation® SoLo RNA-Seq Systems" (NuGEN Technologies, Inc), and sequencing via a paired-end chemistry on an Illumina platform. After quality inspection of the produced reads with fastqc (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore), reads were cleaned to remove the rst 5 bases of read1, the Illumina adapter sequence 'AGATCGGAAGAGC' and the bases with quality below 20 from the 3' end using the program TrimGalore! (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore); the cleaned reads shorter than 35bp were removed from further analysis (9.3% of the produced read pairs). Trimmed reads were then aligned using the program STAR (version 2.7.1a) 78 against the NCBI Hydra vulgaris reference genome (assembly Hydra_RP_1.0), while for gene expression quanti cation, trimmed reads were also aligned on the corresponding transcript sequences GCF_000004095.1_Hydra_RP_1.0_rna.fna.gz using the program kallisto 78, 79 . Kallisto was also used to quantify genes using the reference transcriptome used in Siebert et al. 59 in order to directly map the genes with the cell types where they were identi ed.

Protein Identi cation by MALDI-TOF/TOF
To perform the MALDI-TOF EV samples were rst run into a SDS-Page gel. EV total proteins were quanti ed using a Bradford assay, 4x Laemmli Sample Buffer with 2-mercaptoethanol (Biorad) was added, samples were heated to 95 °C for 5 min and they were run in a mini protean TGX precast 7.5% gel (Biorad). The gel was silver stained following standard procedures.
In-gel digestion-Protein bands were manually excised with a cutter, and in-gel digested with an automatic digestor (Intavis,   Protein identi cation in Hydra EV Western blot analysis of typical exosome biomarkers in whole-body protein extracts (Total) and EV fractions (EV) shows speci c cross reactions to Hydra anti Annexin B12, mouse anti actin and rabbit anti CD63 antibodies. An enrichment in the exosome speci c marker CD63 was observed in EV protein extracts, compared to whole body lysates.  Wnt signaling activation via Hydra EV A) Schematic view of the canonical Wnt/β-catenin pathway at cellular level, in the activated state, showing extracellular and intracellular regulators identi ed in the Hydra EV transcriptome. B) Relative mRNA expression levels of Wnt3/Ef1α. Whole polyps were treated for the indicated period with EVs freshly isolated from Hydra medium, then processed for RNA extraction and qRT-PCR. Data represent the average of three biological replicates, each performed in triplicate. Statistical evaluation was performed using the One sample t test, **P<0.005