Deficient autophagy in epithelial stem cells drives aging in the freshwater cnidarian Hydra.

Hydra possesses three distinct stem cell populations that continuously self-renew and prevent aging in Hydra vulgaris However, sexual animals from the H. oligactis cold-sensitive strain Ho_CS develop an aging phenotype upon gametogenesis induction, initiated by the loss of interstitial stem cells. Animals stop regenerating, lose their active behaviors and die within 3 months. This phenotype is not observed in the cold-resistant strain Ho_CR To dissect the mechanisms of Hydra aging, we compared the self-renewal of epithelial stem cells in these two strains and found it to be irreversibly reduced in aging Ho_CS but sustained in non-aging Ho_CR We also identified a deficient autophagy in Ho_CS epithelial cells, with a constitutive deficiency in autophagosome formation as detected with the mCherry-eGFP-LC3A/B autophagy sensor, an inefficient response to starvation as evidenced by the accumulation of the autophagosome cargo protein p62/SQSTM1, and a poorly inducible autophagy flux upon proteasome inhibition. In the non-aging H. vulgaris animals, the blockade of autophagy by knocking down WIPI2 suffices to induce aging. This study highlights the essential role of a dynamic autophagy flux to maintain epithelial stem cell renewal and prevent aging.

Model systems for regeneration: Hydra.

The freshwater polyp Hydra provides a potent model system for investigating the conditions that promote wound healing, reactivation of a developmental process and, ultimately, regeneration of an amputated body part. Hydra polyps can also be dissociated to the single cell level and can regenerate a complete body axis from aggregates, behaving as natural organoids. In recent years, the ability to exploit Hydra has been expanded with the advent of new live-imaging approaches, genetic manipulations that include stable transgenesis, gene silencing and genome editing, and the accumulation of high-throughput omics data. In this Primer, we provide an overview of Hydra as a model system for studying regeneration, highlighting recent results that question the classical self-enhancement and long-range inhibition model supposed to drive Hydra regeneration. We underscore the need for integrative explanations incorporating biochemical as well as mechanical signalling.

Trends in tissue repair and regeneration.

The 6th EMBO conference on the Molecular and Cellular Basis of Regeneration and Tissue Repair took place in Paestum (Italy) on the 17th-21st September, 2016. The 160 scientists who attended discussed the importance of cellular and tissue plasticity, biophysical aspects of regeneration, the diverse roles of injury-induced immune responses, strategies to reactivate regeneration in mammals, links between regeneration and ageing, and the impact of non-mammalian models on regenerative medicine.

Impact of cycling cells and cell cycle regulation on Hydra regeneration.

Hydra tissues are made from three distinct populations of stem cells that continuously cycle and pause in G2 instead of G1. To characterize the role of cell proliferation after mid-gastric bisection, we have (i) used flow cytometry and classical markers to monitor cell cycle modulations, (ii) quantified the transcriptomic regulations of 202 genes associated with cell proliferation during head and foot regeneration, and (iii) compared the impact of anti-proliferative treatments on regeneration efficiency. We confirm two previously reported events: an early mitotic wave in head-regenerating tips, when few cell cycle genes are up-regulated, and an early-late wave of proliferation on the second day, preceded by the up-regulation of 17 cell cycle genes. These regulations appear more intense after mid-gastric bisection than after decapitation, suggesting a position-dependent regulation of cell proliferation during head regeneration. Hydroxyurea, which blocks S-phase progression, delays head regeneration when applied before but not after bisection. This result is consistent with the fact that the Hydra central region is enriched in G2-paused adult stem cells, poised to divide upon injury, thus forming a necessary constitutive pro-blastema. However a prolonged exposure to hydroxyurea does not block regeneration as cells can differentiate apical structures without traversing S-phase, and also escape in few days the hydroxyurea-induced S-phase blockade. Thus Hydra head regeneration, which is a fast event, is highly plastic, relying on large stocks of adult stem cells paused in G2 at amputation time, which immediately divide to proliferate and/or differentiate apical structures even when S-phase is blocked.

Injury-induced immune responses in Hydra.

The impact of injury-induced immune responses on animal regenerative processes is highly variable, positive or negative depending on the context. This likely reflects the complexity of the innate immune system that behaves as a sentinel in the transition from injury to regeneration. Early-branching invertebrates with high regenerative potential as Hydra provide a unique framework to dissect how injury-induced immune responses impact regeneration. A series of early cellular events likely require an efficient immune response after amputation, as antimicrobial defence, epithelial cell stretching for wound closure, migration of interstitial progenitors toward the wound, cell death, phagocytosis of cell debris, or reconstruction of the extracellular matrix. The analysis of the injury-induced transcriptomic modulations of 2636 genes annotated as immune genes in Hydra identified 43 genes showing an immediate/early pulse regulation in all regenerative contexts examined. These regulations point to an enhanced cytoprotection via ROS signaling (Nrf, C/EBP, p62/SQSMT1-l2), TNFR and TLR signaling (TNFR16-like, TRAF2l, TRAF5l, jun, fos-related, SIK2, ATF1/CREB, LRRC28, LRRC40, LRRK2), proteasomal activity (p62/SQSMT1-l1, Ced6/Gulf, NEDD8-conjugating enzyme Ubc12), stress proteins (CRYAB1, CRYAB2, HSP16.2, DnaJB9, HSP90a1), all potentially regulating NF-κB activity. Other genes encoding immune-annotated proteins such as NPYR4, GTPases, Swap70, the antiproliferative BTG1, enzymes involved in lipid metabolism (5-lipoxygenase, ACSF4), secreted clotting factors, secreted peptidases are also pulse regulated upon bisection. By contrast, metalloproteinases and antimicrobial peptide genes largely follow a context-dependent regulation, whereas the protease inhibitor α2macroglobulin gene exhibits a sustained up-regulation. Hence a complex immune response to injury is linked to wound healing and regeneration in Hydra.

Robust G2 pausing of adult stem cells in Hydra.

Hydra is a freshwater hydrozoan polyp that constantly renews its two tissue layers thanks to three distinct stem cell populations that cannot replace each other, epithelial ectodermal, epithelial endodermal, and multipotent interstitial. These adult stem cells, located in the central body column, exhibit different cycling paces, slow for the epithelial, fast for the interstitial. To monitor the changes in cell cycling in Hydra, we established a fast and efficient flow cytometry procedure, which we validated by confirming previous findings, as the Nocodazole-induced reversible arrest of cell cycling in G2/M, and the mitogenic signal provided by feeding. Then to dissect the cycling and differentiation behaviors of the interstitial stem cells, we used the AEP_cnnos1 and AEP_Icy1 transgenic lines that constitutively express GFP in this lineage. For the epithelial lineages we used the sf-1 strain that rapidly eliminates the fast cycling cells upon heat-shock and progressively becomes epithelial. This study evidences similar cycling patterns for the interstitial and epithelial stem cells, which all alternate between the G2 and S-phases traversing a minimal G1-phase. We also found interstitial progenitors with a shorter G2 that pause in G1/G0. At the animal extremities, most cells no longer cycle, the epithelial cells terminally differentiate in G2 and the interstitial progenitors in G1/G0. At the apical pole ~80% cells are post-mitotic differentiated cells, reflecting the higher density of neurons and nematocytes in this region. We discuss how the robust G2 pausing of stem cells, maintained over weeks of starvation, may contribute to regeneration.

Hydra, a powerful model for aging studies.

Cnidarian Hydra polyps escape senescence, most likely due to the robust activity of their three stem cell populations. These stem cells continuously self-renew in the body column and differentiate at the extremities following a tightly coordinated spatial pattern. Paul Brien showed in 1953 that in one particular species, Hydra oligactis, cold-dependent sexual differentiation leads to rapid aging and death. Here, we review the features of this inducible aging phenotype. These cellular alterations, detected several weeks after aging was induced, are characterized by a decreasing density of somatic interstitial cell derivatives, a disorganization of the apical nervous system, and a disorganization of myofibers of the epithelial cells. Consequently, tissue replacement required to maintain homeostasis, feeding behavior, and contractility of the animal are dramatically affected. Interestingly, this aging phenotype is not observed in all H. oligactis strains, thus providing a powerful experimental model for investigations of the genetic control of aging. Given the presence in the cnidarian genome of a large number of human orthologs that have been lost in ecdysozoans, such approaches might help uncover novel regulators of aging in vertebrates.

The Wnt/ß-catenin/TCF/Sp5/Zic4 Gene Network That Regulates Head Organizer Activity in Hydra Is Differentially Regulated in Epidermis and Gastrodermis.

Hydra head formation depends on an organizing center in which Wnt/ß-catenin signaling, that plays an inductive role, positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/ß-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in either the epidermis or gastrodermis, we show that Sp5 promoter activity is differentially regulated in each epithelial layer. In intact animals, epidermal HySp5:GFP activity is strong apically and weak along the body column, while in the gastrodermis, it is maximal in the tentacle ring region and maintained at a high level along the upper body column. During apical regeneration, HySp5:GFP is activated early in the gastrodermis and later in the epidermis. Alsterpaullone treatment induces a shift in apical HySp5:GFP expression towards the body column where it forms transient circular figures in the epidermis. Upon ß-catenin(RNAi), HySp5:GFP activity is down-regulated in the epidermis while bud-like structures expressing HySp5:GFP in the gastrodermis develop. Sp5(RNAi) reveals a negative Sp5 autoregulation in the epidermis, but not in the gastrodermis. These differential regulations in the epidermis and gastrodermis highlight the distinct architectures of the Wnt/ß-catenin/TCF/Sp5/Zic4 network in the hypostome, tentacle base and body column of intact animals, as well as in the buds and apical and basal regenerating tips.

RNAseq versus genome-predicted transcriptomes: a large population of novel transcripts identified in an Illumina-454 Hydra transcriptome.

BACKGROUND: Evolutionary studies benefit from deep sequencing technologies that generate genomic and transcriptomic sequences from a variety of organisms. Genome sequencing and RNAseq have complementary strengths. In this study, we present the assembly of the most complete Hydra transcriptome to date along with a comparative analysis of the specific features of RNAseq and genome-predicted transcriptomes currently available in the freshwater hydrozoan Hydra vulgaris. RESULTS: To produce an accurate and extensive Hydra transcriptome, we combined Illumina and 454 Titanium reads, giving the primacy to Illumina over 454 reads to correct homopolymer errors. This strategy yielded an RNAseq transcriptome that contains 48'909 unique sequences including splice variants, representing approximately 24'450 distinct genes. Comparative analysis to the available genome-predicted transcriptomes identified 10'597 novel Hydra transcripts that encode 529 evolutionarily-conserved proteins. The annotation of 170 human orthologs points to critical functions in protein biosynthesis, FGF and TOR signaling, vesicle transport, immunity, cell cycle regulation, cell death, mitochondrial metabolism, transcription and chromatin regulation. However, a majority of these novel transcripts encodes short ORFs, at least 767 of them corresponding to pseudogenes. This RNAseq transcriptome also lacks 11'270 predicted transcripts that correspond either to silent genes or to genes expressed below the detection level of this study. CONCLUSIONS: We established a simple and powerful strategy to combine Illumina and 454 reads and we produced, with genome assistance, an extensive and accurate Hydra transcriptome. The comparative analysis of the RNAseq transcriptome with genome-predicted transcriptomes lead to the identification of large populations of novel as well as missing transcripts that might reflect Hydra-specific evolutionary events.

Injury-induced asymmetric cell death as a driving force for head regeneration in Hydra.

The freshwater Hydra polyp provides a unique model system to decipher the mechanisms underlying adult regeneration. Indeed, a single cut initiates two distinct regenerative processes, foot regeneration on one side and head regeneration on the other side, the latter relying on the rapid formation of a local head organizer. Two aspects are discussed here: the asymmetric cellular remodeling induced by mid-gastric bisection and the signaling events that trigger head organizer formation. In head-regenerating tips (but not in foot ones), a wave of cell death takes place immediately, leading the apoptotic cells to transiently release Wnt3 and activate the ß-catenin pathway in the neighboring cycling cells to push them through mitosis. This process, which mimics the apoptosis-induced compensatory proliferation process deciphered in Drosophila larvae regenerating their discs, likely corresponds to an evolutionarily conserved mechanism, also at work in Xenopus tadpoles regenerating their tail or mice regenerating their skin or liver. How is this process generated in Hydra? Several studies pointed to the necessary activation of the extracellular signal-regulated kinase (ERK) 1-2 and mitogen-activated protein kinase (MAPK) pathways during early head regeneration. Indeed inhibition of ERK 1-2 or knockdown of RSK, cAMP response element-binding protein (CREB), and CREB-binding protein (CBP) prevent injury-induced apoptosis and head regeneration. The current scenario involves an asymmetric activation of the MAPK/CREB pathway to trigger injury-induced apoptosis in the interstitial cells and in the epithelial cells a CREB/CBP-dependent transcriptional activation of early genes essential for head-organizing activity as wnt3, HyBra1, and prdl-a. The question now is how bisection in the rather uniform central region of the polyp can generate this immediately asymmetric signaling.

Combining RNAi-Mediated ß-Catenin Inhibition and Reaggregation to Study Hydra Whole-Body Regeneration.

In addition to its ability to regenerate any amputated body part, the Hydra freshwater polyp shows the amazing ability to regenerate as a full polyp after a complete dissociation of its tissues. The developmental processes at work in reaggregates undergoing whole-body regeneration can be investigated at the molecular level by RNA interference (RNAi). Here we provide a protocol that combines ß-catenin RNAi with reaggregation. This protocol serves as a basis to generate "RNAi-reaggregates," followed by the extraction of high-quality RNA for the precise quantification of gene expression by real-time PCR. This protocol is efficient, providing both a molecular signature, with the significant downregulation of ß-catenin and Wnt3, as well as a robust phenotype, the lack of axis formation, which is observed in all reaggregates.

Studying Stem Cell Biology in Intact and Whole-Body Regenerating Hydra by Flow Cytometry.

The freshwater Hydra polyp is a versatile model to study whole-body regeneration from a developmental as well as a cellular point of view. The outstanding regenerative capacities of Hydra are based on its three populations of adult stem cells located in the central body column of the animal. There, these three populations, gastrodermal epithelial, epidermal epithelial, and interstitial, continuously cycle in homeostatic conditions, and their activity is locally regulated after mid-gastric bisection. Moreover, they present an unusual cycling behavior with a short G1 phase and a pausing in G2. This particular cell cycle has been studied for a long time with classical microscopic methods. We describe here two flow cytometry methods that provide accurate and reproducible quantitative data to monitor cell cycle regulation in homeostatic and regenerative contexts. We also present a cell sorting procedure based on flow cytometry, whereby stem cells expressing a fluorescent reporter protein in transgenic lines can be enriched for use in applications such as transcriptomic, proteomic, or cell cycle analysis.

The transcription factor Zic4 promotes tentacle formation and prevents epithelial transdifferentiation in Hydra.

The molecular mechanisms that maintain cellular identities and prevent dedifferentiation or transdifferentiation remain mysterious. However, both processes are transiently used during animal regeneration. Therefore, organisms that regenerate their organs, appendages, or even their whole body offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4, whose expression is controlled by Wnt3/ß-catenin signaling and the Sp5 transcription factor, plays a key role in tentacle formation and tentacle maintenance. Reducing Zic4 expression suffices to induce transdifferentiation of tentacle epithelial cells into foot epithelial cells. This switch requires the reentry of tentacle battery cells into the cell cycle without cell division and is accompanied by degeneration of nematocytes embedded in these cells. These results indicate that maintenance of cell fate by a Wnt-controlled mechanism is a key process both during homeostasis and during regeneration.

A two-step process in the emergence of neurogenesis.

Cnidarians belong to the first phylum differentiating a nervous system, thus providing suitable model systems to trace the origins of neurogenesis. Indeed corals, sea anemones, jellyfish and hydra contract, swim and catch their food thanks to sophisticated nervous systems that share with bilaterians common neurophysiological mechanisms. However, cnidarian neuroanatomies are quite diverse, and reconstructing the urcnidarian nervous system is ambiguous. At least a series of characters recognized in all classes appear plesiomorphic: (1) the three cell types that build cnidarian nervous systems (sensory-motor cells, ganglionic neurons and mechanosensory cells called nematocytes or cnidocytes); (2) an organization of nerve nets and nerve rings [those working as annular central nervous system (CNS)]; (3) a neuronal conduction via neurotransmitters; (4) a larval anterior sensory organ required for metamorphosis; (5) a persisting neurogenesis in adulthood. By contrast, the origin of the larval and adult neural stem cells differs between hydrozoans and other cnidarians; the sensory organs (ocelli, lens-eyes, statocysts) are present in medusae but absent in anthozoans; the electrical neuroid conduction is restricted to hydrozoans. Evo-devo approaches might help reconstruct the neurogenic status of the last common cnidarian ancestor. In fact, recent genomic analyses show that if most components of the postsynaptic density predate metazoan origin, the bilaterian neurogenic gene families originated later, in basal metazoans or as eumetazoan novelties. Striking examples are the ParaHox Gsx, Pax, Six, COUP-TF and Twist-type regulators, which seemingly exert neurogenic functions in cnidarians, including eye differentiation, and support the view of a two-step process in the emergence of neurogenesis.

Injury-induced activation of the MAPK/CREB pathway triggers apoptosis-induced compensatory proliferation in hydra head regeneration.

After bisection, Hydra polyps regenerate their head from the lower half thanks to a head-organizer activity that is rapidly established at the tip. Head regeneration is also highly plastic as both the wild-type and the epithelial Hydra (that lack the interstitial cell lineage) can regenerate their head. In the wild-type context, we previously showed that after mid-gastric bisection, a large subset of the interstitial cells undergo apoptosis, inducing compensatory proliferation of the surrounding progenitors. This asymmetric process is necessary and sufficient to launch head regeneration. The apoptotic cells transiently release Wnt3, which promotes the formation of a proliferative zone by activating the beta-catenin pathway in the adjacent cycling cells. However the injury-induced signaling that triggers apoptosis is unknown. We previously reported an asymmetric immediate activation of the mitogen-activated protein kinase/ribosomal S6 kinase/cAMP response element binding protein (MAPK/RSK/CREB) pathway in head-regenerating tips after mid-gastric bisection. We show here that pharmacological inhibition of the MAPK/ERK pathway or RNAi knockdown of the RSK, CREB, CREB binding protein (CBP) genes prevents apoptosis, compensatory proliferation and blocks head regeneration. As the activation of the MAPK pathway upon injury plays an essential role in regenerating bilaterian species, these results suggest that the MAPK-dependent activation of apoptosis-induced compensatory proliferation represents an evolutionary-conserved mechanism to launch a regenerative process.

The ULK1 kinase, a necessary component of the pro-regenerative and anti-aging machinery in Hydra.

Hydra vulgaris (Hv) has a high regenerative potential and negligible senescence, as its stem cell populations divide continuously. In contrast, the cold-sensitive H. oligactis (Ho_CS) rapidly develop an aging phenotype under stress, with epithelial stem cells deficient for autophagy, unable to maintain their self-renewal. Here we tested in aging, non-aging and regenerating Hydra the activity and regulation of the ULK1 kinase involved in autophagosome formation. In vitro kinase assays show that human ULK1 activity is activated by Hv extracts but repressed by Ho_CS extracts, reflecting the ability or inability of their respective epithelial cells to initiate autophagosome formation. The factors that keep ULK1 inactive in Ho_CS remain uncharacterized. Hv_Basel1 animals exposed to the ULK1 inhibitor SBI-0206965 no longer regenerate their head, indicating that the sustained autophagy flux recorded in regenerating Hv_AEP2 transgenic animals expressing the DsRed-GFP-LC3A autophagy tandem sensor is necessary. The SBI-0206965 treatment also alters the contractility of intact Hv_Basel1 animals, and leads to a progressive reduction of animal size in Hv_AEP2, similarly to what is observed in ULK1(RNAi) animals. We conclude that the evolutionarily-conserved role of ULK1 in autophagy initiation is crucial to maintain a dynamic homeostasis in Hydra, which supports regeneration efficiency and prevents aging.

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in Hydra.

Here we discuss the developmental and homeostatic conditions necessary for Hydra regeneration. Hydra is characterized by populations of adult stem cells paused in the G2 phase of the cell cycle, ready to respond to injury signals. The body column can be compared to a blastema-like structure, populated with multifunctional epithelial stem cells that show low sensitivity to proapoptotic signals, and high inducibility of autophagy that promotes resistance to stress and starvation. Intact Hydra polyps also exhibit a dynamic patterning along the oral-aboral axis under the control of homeostatic organizers whose activity results from regulatory loops between activators and inhibitors. As in bilaterians, injury triggers the immediate production of reactive oxygen species (ROS) signals that promote wound healing and contribute to the reactivation of developmental programs via cell death and the de novo formation of new organizing centers from somatic tissues. In aging Hydra, regeneration is rapidly lost as homeostatic conditions are no longer pro-regenerative.

More constraint on ParaHox than Hox gene families in early metazoan evolution.

Hox and ParaHox (H/P) genes belong to evolutionary-sister clusters that arose through duplication of a ProtoHOX cluster early in animal evolution. In contrast to bilaterians, cnidarians express, beside PG1, PG2 and Gsx orthologs, numerous Hox-related genes with unclear origin. We characterized from marine hydrozoans three novel Hox-related genes expressed at medusa and polyp stages, which include a Pdx/Xlox ParaHox ortholog induced 1 day later than Gsx during embryonic development. To reconstruct H/P genes' early evolution, we performed multiple systematic comparative phylogenetic analyses, which identified derived sequences that blur the phylogenetic picture, recorded dramatically different evolutionary rates between ParaHox and Hox in cnidarians and showed the unexpected grouping of [Gsx-Pdx/Xlox-PG2-PG3] families in a single metagroup distinct from PG1. We propose a novel more parsimonious evolutionary scenario whereby H/P genes originated from a [Gsx-Pdx/Xlox-PG2-PG3]-related ProtoHox gene, the "posterior" and "anterior" H/P genes appearing secondarily. The ProtoHOX cluster would have contained the three Gsx/PG2, Pdx/PG3, Cdx/PG9 paralogs and produced through tandem duplication the primordial HOX and ParaHOX clusters in the Cnidaria-Bilateria ancestor. The stronger constraint on cnidarian ParaHox genes suggests that the primary function of pre-bilaterian H/P genes was to drive cellular evolutionary novelties such as neurogenesis rather than axis specification.

Origins of neurogenesis, a cnidarian view.

New perspectives on the origin of neurogenesis emerged with the identification of genes encoding post-synaptic proteins as well as many "neurogenic" regulators as the NK, Six, Pax, bHLH proteins in the Demosponge genome, a species that might differentiate sensory cells but no neurons. However, poriferans seem to miss some key regulators of the neurogenic circuitry as the Hox/paraHox and Otx-like gene families. Moreover as a general feature, many gene families encoding evolutionarily-conserved signaling proteins and transcription factors were submitted to a wave of gene duplication in the last common eumetazoan ancestor, after Porifera divergence. In contrast gene duplications in the last common bilaterian ancestor, Urbilateria, are limited, except for the bHLH Atonal-class. Hence Cnidaria share with Bilateria a large number of genetic tools. The expression and functional analyses currently available suggest a neurogenic function for numerous orthologs in developing or adult cnidarians where neurogenesis takes place continuously. As an example, in the Hydra polyp, the Clytia medusa and the Acropora coral, the Gsx/cnox2/Anthox-2 ParaHox gene likely supports neurogenesis. Also neurons and nematocytes (mechanosensory cells) share in hydrozoans a common stem cell and several regulatory genes indicating that they can be considered as sister cells. Performed in anthozoan and medusozoan species, these studies should tell us more about the way(s) evolution hazards achieved the transition from epithelial to neuronal cell fate, and about the robustness of the genetic circuitry that allowed neuromuscular transmission to arise and be maintained across evolution.

Autophagy in Hydra: a response to starvation and stress in early animal evolution.

The Hydra polyp provides a powerful model system to investigate the regulation of cell survival and cell death in homeostasis and regeneration as Hydra survive weeks without feeding and regenerates any missing part after bisection. Induction of autophagy during starvation is the main surviving strategy in Hydra as autophagic vacuoles form in most myoepithelial cells after several days. When the autophagic process is inhibited, animal survival is actually rapidly jeopardized. An appropriate regulation of autophagy is also essential during regeneration as Hydra RNAi knocked-down for the serine protease inhibitor Kazal-type (SPINK) gene Kazal1, exhibit a massive autophagy after amputation that rapidly compromises cell and animal survival. This excessive autophagy phenotype actually mimics that observed in the mammalian pancreas when SPINK genes are mutated, highlighting the paradigmatic value of the Hydra model system for deciphering pathological processes. Interestingly autophagy during starvation predominantly affects ectodermal epithelial cells and lead to cell survival whereas Kazal1(RNAi)-induced autophagy is restricted to endodermal digestive cells that rapidly undergo cell death. This indicates that distinct regulations that remain to be identified, are at work in these two contexts. Cnidarian express orthologs for most components of the autophagy and TOR pathways suggesting evolutionarily-conserved roles during starvation.