Investigation of the role of <i>HyBcl-2-l8</i> on cell death resistance in <i>Hydra</i> epithelial cells

Apoptosis, or programmed cell death, is vital for multicellular organisms, particularly during developmental processes but also throughout their entire lives, and must therefore be carefully balanced. Recently, it has been shown that Hydra epithelial cells show a graded sensitivity to apoptosis depending on their position along the body axis, significantly higher in the apical region than along the gastric column. Using RNA-seq analysis, one gene, HyBcl-2-l8, caught our attention as it is expressed only in epithelial cells and shows a graded expression with a higher level in the Hydra head than in the body column, thus mimicking the gradient of cell death sensitivity in epithelial cells. This gene is a member of the Bcl-2 family, involved in maintenance of the outer mitochondrial membrane integrity. These proteins prevent the leakage of certain components that are responsible for triggering molecular pathways that undoubtedly lead to programmed death of the cell. HyBcl-2-l8 is present in two isoforms in Hydra and is expressed in the gastrodermal epithelial lineage. Recent data have shown that when overexpressed in human cells and upon apoptosis induction, HyBcl-2-l8 has a pro-apoptotic effect. However, only a few studies have investigated the role of Bcl-2 family members in Hydra in vivo. The first method to interrogate the function of this gene was to silence it by delivering a hairpin carried by a vector plasmid. Under these conditions, HyBcl-2-l8 showed no pro-apoptotic effect. The second method used to study the function of HyBcl-2-l8 was by delivering siRNAs through electroporation. On Hv_Basel, the different assays showed a significant decrease in apoptosis after cell death induction and TUNEL assay. Furthermore, differences in the length of the animals were observed. Therefore, this suggests a pro-apoptotic effect of HyBcl-2-l8 in Hydra in vivo. The same experiments were repeated on Hv_AEP, this time no significant decrease in gene expression level was detected, nor any reduction in cell death after induction by wortmannin. Finally, the experiments were redone on Hv_AEP after treatment with hydroxyurea (HU), thereby suppressing most interstitial cells. Under these conditions, a small reduction in the gene expression level was detected and was correlated with a significant decrease in apoptosis in the body column after cell death induction. These results, allow us to assume a pro-apoptotic role of HyBcl-2-l8 in both distinct strains of Hydra, the intact Hv_Basel and in the Hv_AEP deprived of their interstitial cells but not in the intact Hv_AEP, probably due to the low gene silencing level obtained under the experimental conditions used in this study.L'apoptose, ou mort cellulaire programmée, est vitale pour les organismes multicellulaires, notamment au cours des processus de développement mais aussi tout au long de leur vie, elle doit donc être soigneusement contrôlée. Récemment, il a été démontré que les cellules épithéliales de l'Hydre présentent une sensibilité différentielle à l'apoptose en fonction de leur position le long de l'axe du corps. Cette sensibilité est nettement plus élevée dans la région apicale que le long de la colonne gastrique. Grâce à l'analyse RNA-Seq, un gène, HyBcl-2-l8, a attiré notre attention car il est exprimé uniquement dans les cellules épithéliales et montre une expression différentielle avec un niveau d'expression plus élevé dans la tête que dans le corps de l'Hydre, mimant ainsi le gradient de sensibilité à la mort cellulaire des cellules épithéliales. Ce gène est un membre de la famille Bcl-2, impliqué dans le maintien de l'intégrité de la membrane mitochondriale externe. Ces protéines empêchent la fuite de certains composants qui sont responsables du déclenchement de voies moléculaires qui conduisent indéniablement à la mort programmée de la cellule. HyBcl-2-l8 est présent sous deux isoformes chez l'Hydre et est exprimé dans la lignée épithéliale gastrodermique. Des données récentes ont montré que lorsqu'il est surexprimé dans des cellules humaines et que l'apoptose est induite, HyBcl-2-l8 présente un effet pro-apoptotique. Cependant, seules quelques études se sont intéressées au rôle des membres de la famille Bcl-2 chez l'Hydre in vivo. La première méthode pour interroger la fonction de ce gène a été de l'éteindre en délivrant un hairpin porté par un plasmide vecteur. Dans ces conditions, HyBcl-2-l8 n'a révélé aucun effet pro-apoptotique. La deuxième méthode utilisée pour étudier la fonction de HyBcl-2-l8 a été l'électroporation de siRNA. Sur Hv_Basel, les différentes analyses ont montré une diminution significative de l'apoptose après induction de la mort cellulaire et par le test TUNEL. De plus, des différences dans la longueur des animaux ont été observées. Par conséquent, cela suggère un effet pro-apoptotique de HyBcl-2-l8 chez l'Hydre in vivo. Les mêmes expériences ont été répétées sur Hv_AEP, cette fois, aucune diminution significative du niveau d'expression du gène n'a été détectée, ni aucune réduction de la mort cellulaire après induction par la wortmannine. Enfin, les expériences ont été réitérées sur Hv_AEP après un traitement à l'hydroxyurée (HU), supprimant ainsi la plupart de cellules interstitielles. Dans ces conditions, et bien que la réduction du niveau d'expression du gène soit faible, une diminution significative de l'apoptose a été détectée par le test de TUNEL dans la colonne gastrique des animaux après induction de l'apoptose</p

Loss of neurogenesis in <i>Hydra</i> 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

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

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

Non-developmental dimensions of adult regeneration in Hydra

An essential dimension of 3D regeneration in adult animals is developmental, with the formation of organizers from somatic tissues. These organizers produce signals that recruit surrounding cells and drive the restoration of the missing structures (organs, appendages, body parts). However, even in animals with a high regenerative potential, this developmental potential is not sufficient to achieve regeneration as homeostatic conditions at the time of injury need to be "pro-regenerative". In Hydra, we identified four distinct homeostatic properties that provide a pro-regenerative framework and we discuss here how these non-developmental properties impact regeneration. First, both the epithelial and the interstitial-derived cells are highly plastic along the animal body, a plasticity that offers several routes to achieve regeneration. Second, the abundant stocks of continuously self-renewing adult stem cells form a constitutive pro-blastema in the central body column, readily activated upon bisection. Third, the autophagy machinery in epithelial cells guarantees a high level of fitness and adaptation to detrimental environmental conditions, as evidenced by the loss of regeneration in animals where autophagy is dysfunctional. Fourth, the extracellular matrix, named mesoglea in Hydra, provides a dynamically-patterned environment where the molecular and mechanical signals induced by injury get translated into a regenerative process. We claim that these homeostatic pro-regenerative features contribute to define the high regenerative potential of adult Hydra

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 towards 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, metalloproteinase and antimicrobial peptide genes largely follow a context-dependent regulation, whereas the protease inhibitor α2macroglobulin exhibits a sustained up regulation. Hence a complex immune response to injury is linked to wound healing and regeneration in Hydra

Hydra, a versatile model to study the homeostatic and developmental functions of cell death

In the freshwater cnidarian polyp Hydra, cell death takes place in multiple contexts. Indeed apoptosis occurs during oogenesis and spermatogenesis, during starvation, and in early head regenerating tips, promoting local compensatory proliferation at the boundary between heterografts. Apoptosis can also be induced upon exposure to pro-apoptotic agents (colchicine, wortmannin), upon heat-shock in the thermosensitive sf-1 mutant, and upon wounding. In all these contexts, the cells that undergo cell death belong predominantly to the interstitial cell lineage, whereas the epithelial cells, which are rather resistant to pro-apoptotic signals, engulf the apoptotic bodies. Beside this clear difference between the interstitial and the epithelial cell lineages, the different interstitial cell derivatives also show noticeable variations in their respective apoptotic sensitivity, with the precursor cells appearing as the most sensitive to pro-apoptotic signals. The apoptotic machinery has been well conserved across evolution. However, its specific role and regulation in each context are not known yet. Tools that help characterize apoptotic activity in Hydra have recently been developed. Among them, the aposensor Apoliner initially designed in Drosophila reliably measures wortmannin-induced apoptotic activity in a biochemical assay. Also, flow cytometry and TUNEL analyses help identify distinctive features between wortmannin-induced and heat-shock induced apoptosis in the sf-1 strain. Thanks to the live imaging tools already available, Hydra now offers a model system with which the functions of the apoptotic machinery to maintain long-term homeostasis, stem cell renewal, germ cell production, active developmental processes and non-self response can be deciphered

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

Cellular, Metabolic, and Developmental Dimensions of Whole-Body Regeneration in <i>Hydra</i>

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

Generic and context-dependent gene modulations during <i>Hydra</i> whole body regeneration

The cnidarian Hydra is a classical model of whole-body regeneration. Historically, Hydra apical regeneration has received more attention than its basal counterpart, most studies considering these two regenerative processes independently. We present here a transcriptome-wide comparative analysis of apical and basal regeneration after decapitation and mid-gastric bisection, augmented with a characterization of positional and cell-type expression patterns in non-regenerating animals. The profiles of 25'637 Hydra transcripts are available on HydrATLAS (https://hydratlas.unige.ch), a web interface allowing a convenient access to each transcript profile. These data indicate that generic impulse-type modulations occur during the first four hours post-amputation, consistent with a similar integration of injury-related cues on both sides of the amputation plane. Initial divergences in gene regulations are observed in regenerating tips between four and eight hours post-amputation, followed by a dramatic transcriptomic reprogramming between eight and 16 hours when regulations become sustained. As expected, central components of apical patterning, Wnt3 and HyBra1, are among the earliest genes up-regulated during apical regeneration. During early basal regeneration, a BMP signaling ligand (BMP5-8c) and a potential BMP inhibitor (NBL1) are up-regulated, suggesting that BMP signaling is involved in the basal organizer, as supported by higher levels of phosphorylated Smad in the basal region and by the LiCl-induced extension of NBL1 expression. By contrast, upon ectopic activation of Wnt/b-catenin signaling, NBL1 is no longer expressed, basal differentiation is not maintained and basal regeneration is abolished. A tight cross-talk between Wnt/b-catenin apically and BMP signaling basally appears necessary for maintaining and regenerating Hydra anatomy