Wanda Christa Buzgariu

Apoptotic Cells Provide an Unexpected Source of Wnt3 Signaling to Drive Hydra Head Regeneration

Decapitated Hydra regenerate their heads via morphallaxis, i.e., without significant contributions made by cell proliferation or interstitial stem cells. Indeed, Hydra depleted of interstitial stem cells regenerate robustly, and Wnt3 from epithelial cells triggers head regeneration. However, we find a different mechanism controlling regeneration after midgastric bisection in animals equipped with both epithelial and interstitial cell lineages. In this context, we see rapid induction of apoptosis and Wnt3 secretion among interstitial cells at the head- (but not foot-) regenerating site. Apoptosis is both necessary and sufficient to induce Wnt3 production and head regeneration, even at ectopic sites. Further, we identify a zone of proliferation beneath the apoptotic zone, reminiscent of proliferative blastemas in regenerating limbs and of compensatory proliferation induced by dying cells in Drosophila imaginal discs. We propose that different types of injuries induce distinct cellular modes of Hydra head regeneration, which nonetheless converge on a central effector, Wnt3.

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.

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.

Methods to investigate autophagy during starvation and regeneration in hydra

In hydra, the regulation of the balance between cell death and cell survival is essential to maintain homeostasis across the animal and promote animal survival during starvation. Moreover, this balance also appears to play a key role during regeneration of the apical head region. The recent finding that autophagy is a crucial component of this balance strengthens the value of the Hydra model system to analyze the implications of autophagy in starvation, stress response and regeneration. We describe here how we adapted to Hydra some established tools to monitor steady-state autophagy. The ATG8/LC3 marker used in biochemical and immunohistochemical analyses showed a significant increase in autophagosome formation in digestive cells after 11 days of starvation. Moreover, the maceration procedure that keeps intact the morphology of the various cell types allows the quantification of the autophagosomes and autolysosomes in any cell type, thanks to the detection of the MitoFluor or LysoTracker dyes combined with the anti-LC3, anti-LBPA, and/or anti-RSK (ribosomal S6 kinase) immunostaining. The classical activator (rapamycin) and inhibitors (wortmannin, bafilomycin A(1)) of autophagy also appear to be valuable tools to modulate autophagy in hydra, as daily-fed and starved hydra display slightly different responses. Finally, we show that the genetic circuitry underlying autophagy can be qualitatively and quantitatively tested through RNA interference in hydra repeatedly exposed to double-stranded RNAs.

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.

Loss of neurogenesis in Hydra leads to compensatory regulation of neurogenic and neurotransmission genes in epithelial cells

Hydra continuously differentiates a sophisticated nervous system made of mechanosensory cells (nematocytes) and sensory–motor and ganglionic neurons from interstitial stem cells. However, this dynamic adult neurogenesis is dispensable for morphogenesis. Indeed animals depleted of their interstitial stem cells and interstitial progenitors lose their active behaviours but maintain their developmental fitness, and regenerate and bud when force-fed. To characterize the impact of the loss of neurogenesis in Hydra, we first performed transcriptomic profiling at five positions along the body axis. We found neurogenic genes predominantly expressed along the central body column, which contains stem cells and progenitors, and neurotransmission genes predominantly expressed at the extremities, where the nervous system is dense. Next, we performed transcriptomics on animals depleted of their interstitial cells by hydroxyurea, colchicine or heat-shock treatment. By crossing these results with cell-type-specific transcriptomics, we identified epithelial genes up-regulated upon loss of neurogenesis: transcription factors (Dlx, Dlx1, DMBX1/Manacle, Ets1, Gli3, KLF11, LMX1A, ZNF436, Shox1), epitheliopeptides (Arminins, PW peptide), neurosignalling components (CAMK1D, DDCl2, Inx1), ligand-ion channel receptors (CHRNA1, NaC7), G-Protein Coupled Receptors and FMRFRL. Hence epitheliomuscular cells seemingly enhance their sensing ability when neurogenesis is compromised. This unsuspected plasticity might reflect the extended multifunctionality of epithelial-like cells in early eumetazoan evolution.

Multi-functionality and plasticity characterize epithelial cells in Hydra.

Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra.

Hydra, a model system for deciphering the mechanisms of aging and resistance to aging

The freshwater cnidarian polyp named Hydra, which can be mass-cultured in the laboratory, is characterized by a highly dynamic homeostasis with a continuous self-renewal of its three adult stem cell populations, the epithelial stem cells from the epidermis, the epithelial stem cells from the gastrodermis, and the multipotent interstitial stem cells, which provide cells of the nervous system, gland cells and germ cells. Two unusual features characterize these stem cells that cannot replace each other, they all avoid G1 to pause in G2, and the two epithelial populations are concomitantly multifunctional and stem cells. H. vulgaris that does not show any signs of aging over the years, resists to weeks of starvation and adapts to the loss of neurogenesis, providing a unique model system to study the resistance to aging. By contrast some strains of a distinct species named H. oligactis undergo a rapid aging process when undergoing gametogenesis or when placed in stress conditions. The aging phenotype is characterized by the rapid loss of somatic interstitial stem cells and the progressive reduction in epithelial stem cell self-renewal, the loss of regeneration, the disorganization of the neuro-muscular system, the loss of the feeding behavior, and the death of all animals within about three months. We review here the possible mechanisms that help H. vulgaris to sustain stem cell self-renewal and thus bypass aging processes. For this, FoxO seems to act as a pleiotropic actor, regulating stem cell proliferation, stress response and apoptosis. In H. oligactis, the regulation of the autophagy flux differs between aging-sensitive and aging-resistant animals, pointing to a key role for proteostasis in the maintenance a large pool of active and plastic epithelial stem cells.

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.

Deficient autophagy drives aging in Hydra

Hydra exhibits a negligible senescence as its epithelial and interstitial stem cell populations continuously divide. Here we identified two H. oligactis strains that respond differently to interstitial stem cell loss. Cold-resistant (Ho_CR) animals adapt and remain healthy while cold-sensitive (Ho_CS) ones die within three months, after their epithelial stem cells lose their selfrenewal potential. In Ho_CS but not in Ho_CR animals, the autophagy flux is deficient, characterized by a low induction upon starvation, proteasome inhibition or Rapamycin treatment, and a constitutively repressed Ulk activity. In the non-aging Hydra vulgaris, WIPI2 silencing suffices to induce aging. Rapamycin can delay aging by sustaining epithelial self-renewal and regeneration, although without enhancing the autophagy flux. Instead Rapamycin promotes engulfment in epithelial cells where p62/SQSTM1-positive phagocytic vacuoles accumulate. This study uncovers the importance of autophagy in the longevity of early-branched eumetazoans by maintaining stem cell renewal, and a novel anti-aging effect of Rapamycin via phagocytosis.