Hydra, a model for studying the role of injury-induced ROS signalling during regeneration and monitoring the autophagy flux in live animals

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Thesis

Reference

Hydra, a model for studying the role of injury-induced ROS signalling during regeneration and monitoring the autophagy flux in live animals

SUKNOVIC, Nenad Slavko

Abstract

Hydra is a freshwater cnidarian polyp that regenerates any missing part after amputation. This study focuses on the potential role of injury-induced ROS signaling in triggering head regeneration. We detected immediately after mid-gastric bisection symmetrical levels of mitochondrial superoxide and asymmetrical levels of hydrogen peroxide (H2O2), higher in head-regenerating tips than in basal-regenerating ones. This asymmetry likely results from a higher superoxide dismutase (SOD) activity in head-regenerating tips while catalase is lower.

Pharmacological treatments (Tiron, DPI) and transient gene silencing approaches (sod-1, catalase) indicate that signaling via mitochondrial ROS plays a role in wound healing while high levels of H2O2 are necessary for apical regeneration. Through paracrine signalling, H2O2 triggers CREB phosphorylation as well as death of interstitial cells, while H2O2 levels are amplified by apoptotic cells via a feedback loop. Thus, asymmetric ROS signaling immediately after bisection is critical to induce cell death and apical regeneration.

SUKNOVIC, Nenad Slavko. Hydra, a model for studying the role of injury-induced ROS signalling during regeneration and monitoring the autophagy flux in live animals. Thèse de doctorat : Univ. Genève, 2019, no. Sc. Vie 25

DOI : 10.13097/archive-ouverte/unige:125593 URN : urn:nbn:ch:unige-1255930

Available at:

http://archive-ouverte.unige.ch/unige:125593

Disclaimer: layout of this document may differ from the published version.

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Hydra, a Model for Studying the Role of Injury-induced ROS

Signalling during Regeneration and Monitoring the Autophagy Flux in Live Animals

THÈSE

présentée aux Facultés de médecine et des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences en sciences de la vie,

mention Biosciences moléculaires

par

Nenad Slavko SUKNOVIC

de

Novi Sad (Serbie)

Thèse N^o25

GENÈVE

Atelier d’impression REPROMAIL Juillet 2019

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES

Section de biologie

Département de génétique et évolution Professeure Brigitte Galliot

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The history of science shows that theories are perishable. With every new truth that is revealed we get a better understanding of Nature and our conceptions and views are modified.

Nikola Tesla

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Acknowledgements

I would like to thank first my thesis supervisor Prof. Brigitte Galliot. Even though she had other candidates available with existing, stronger background in biology, she decided to go for a guy educated mainly in chemistry. She understood how difficult it is to change fields, and with her patience and vast knowledge contributed to the quality of this doctoral thesis. Thank you for all the scientific discussions that we had, each of them improving the way I think, dissect and analyze scientific data.

Next, I would like to thank the members of my PhD thesis committee: Prof. Jean-‐

Claude Martinou, prof. Florenci Serras and Dr. Denis Martinvalet for taking interest in my work and accepting to be the part of my thesis’ jury.

Many thanks to all the current and past members of the lab of Prof. Brigitte Galliot:

Szymon Tomczyk, Chrystelle Perruchoud, Laura Iglesias, Wanda Buzgariu, Quentin Schenkelaars, Yvan Wenger, Salima Boukerch, Marie-‐Laure Curchoud, Delphine Colevret and Denis Benoni, as well as our secretaries Valérie Mino and Corrine Matthey.

I would like to additionally thank to Dr. Matthias Vogg, who helped me with numerous discussions to tackle new points of view on ROS signaling and for his general interest in my studies.

Special thanks to Dr. Silke Reiter, who started the ROS signaling project in the lab.

I would like to mention my previous mentors that along my studies each contributed to my professional development: Anđelka Carević and Mirjana Rašković, my high school biology and chemistry teachers who sparked my interest in these amazing fields of science and Dr. Dejan Orčić, a remarkably bright young principal investigator that I had the privilege to work with during my Bachelor and Master thesis.

Special thanks to my wonderful girlfriend Sarah Al Haddad, who was a constant emotional and professional support during my whole PhD and especially while writing my doctoral thesis. Could not have done it without you!

I also met some extraordinary people during my stay in Geneva, Szymon Tomczyk, Kamila Kowa, Adrien Valino, Chrystelle Perruchoud, Eleni Tavridou, Ambra Sartori, Alberto Ferrari and Aleksandra Ležaja. Thank you guys for many nice memories!

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Hvala

Prvenstveno mojim životnim učiteljima: majki Dragici i ocu Slavku. Moj tata, inače učitelj geografije po struci, je i moj veliki mentor. Jedno od mojih prvih jasnih i dragih sećanja je kako me on upoznaje sa brojevima i slovima. U više navrata su me kolege pitale odakle mi toliko visok nivo organizacije naučnih podataka. Ovo je nešto što sam bez sumnje nasledio od mame. Ona je sposobna da pronadje svaki detalj bilo iz privatnog ili poslovnog života koji je prethodno zaveden u ‘’specijalan tefter’’. Hvala Vam puno na svemu, ovaj doktorat je Vaših ruku delo.

Dalje bih želeo da se zahvalim mojoj bližoj porodici: babi Olgi, ‘mojoj drugoj majki’’ -‐

tetki Branki, teči Vladislavu i njihovim ćerkama, mojim sestrama Dragani i Svetlani sa svojim, sada višečlanim porodicama.

Mom najboljem drugu Nikoli, koga poznajem dugo koliko i sebe, na beskrajnoj podršci i razumevanju i skoro tri decenije neprestanog prijateljstva.

Mojim drugarima iz detinjstva Petru, Dušanu, Vladimiru, kao i onima koji su se priključili tokom srednje skole: Ratku, Sonji, Gagiju, Kobri, Milošu i Grbi.

Specijalno hvala mom kumu Borisu i kumi Ajumi.

Mojoj ekipi iz fakultetskih dana: Mileni, Ivanu, Vajku, Marini, Slavku i Zlatku.

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In a large number of species, including mammals, signaling via the Reactive Oxygen Species (ROS) plays a major role in wound healing and regeneration. In mammals, the regeneration potential has significantly dropped compared to non-‐mammalian species such as amphibians, zebrafish, planaria or Hydra. Hydra polyps show a remarkable ability to renew any missing body part after bisection, which, together with their early-‐branched position in the animal kingdom, make them a valuable model for studying regeneration. The Hydra model can be used to understand the fundamental mechanisms of regeneration, but also to identify how this property may have evolved. The objective of this doctoral project was to extend our knowledge on injury-‐induced signaling in the immediate phase of Hydra regeneration. We focused on ROS signaling and its potential role in amputation-‐induced cell death as observed in head-‐regenerating tips.

First, we characterized and quantified the different ROS signals from membrane or mitochondrial sources. We detected an immediate and symmetrical production of mitochondrial superoxide after mid-‐gastric bisection, mainly by gastrodermal epithelial cells. On the other hand, we have identified an immediate production of asymmetric hydrogen peroxide (H2O2), higher in head-‐regenerating tips than in basal-‐regenerating ones, probably due to asymmetric enzymatic activities, i.e. a higher superoxide dismutase (SOD) activity in head-‐regenerating tips while catalase is lower. High levels of H2O2 trigger CREB phosphorylation as well as death of interstitial cells by paracrine signaling, and H2O2 levels are probably amplified by apoptotic cells via a feedback loop. Pharmacological treatments (Tiron, DPI) and transient gene silencing approaches (sod-‐1, catalase) indicate that signaling via mitochondrial ROS plays a role in wound healing while high levels of H2O2 are necessary for apical regeneration. Thus, asymmetric ROS signaling immediately after bisection is critical to induce cell death and apical regeneration.

In the next chapter, our objective was to characterize the components of the ROS signaling pathway involved in Hydra regeneration. Among 31 Hydra proteins presumed to be redox sensitive, whose genes are immediately up-‐regulated after amputation, we found the autophagy cargo protein p62/SQSTM1 (sequestosome 1),

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the ribosylation factor 1 ADP, the transcription factors CCAAT/EBP, CREB3-‐L1, Jun-‐D, Fra-‐l1 and NFE2Fl/Nrfl, whose structure is similar to that of the mammalian factor Nrf3. We expressed the Hydra gene NFE2Fl/Nrfl in mammalian cells and analyzed the activity of NFE2Fl/Nrfl when the cells are exposed to drugs modulating the oxidative stress. When transactivation is measured on a reporter construction driven by antioxidant response elements (ARE), the NFE2Fl/Nrfl factor exerts a negative competition on human Nrf2, indirectly demonstrating its ability to bind AREs. In HEK293T cells where human Nrf2 expression is silenced, the transactivation exerted by NFE2Fl/Nrfl is constitutively low but increased upon exposure to sulforaphane, a known NFE2F/Nrf2 inducer, but at a level lower than Nrf2. The role of NFE2Fl/Nrfl remains to be tested in vivo, as well as that of other redox-‐sensitive transcription factors that could be key actors.

Autophagy is tightly regulated by ROS signaling and as a side project, I was involved in the analysis of autophagy deficiency in aging Hydra. I optimized a biosensor designed to monitor the autophagy flux in live animals. This autophagy reporter construct contributed to produce reliable results on the formation of autophagosomes in intact H. oligactis submitted to starvation or proteasome inhibition as well as in H. vulgaris knocked-‐down for WIPI2, a gene encoding a protein involved in the early phase of autophagosome formation.

In conclusion, this work provides us with new perspectives on (i) the role of ROS signaling, immediately asymmetric between the apical and basal sides of the amputation plane, relying on hydrogen peroxide to orchestrate cell death and head regeneration; (ii) the transcription factor NFE2Fl/Nrfl as a possible actor in the response to oxidative stress, although weakly sensitive; (iii) the new imaging technique obtained with genetically encoded biosensors as a powerful and quantitative tool for monitoring biological processes at the sub-‐cellular level.

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RESUME

Chez un grand nombre d'espèces, y compris les mammifères, la voie de signalisation ROS (Reactive Oxygen Species) joue un rôle majeur dans la cicatrisation des plaies et la régénération. Chez les mammifères, le potentiel de régénération a significativement chuté par rapport aux non mammifères tels que les amphibiens, les poissons zèbres, les planaires ou les Hydres. Les polypes d'Hydre régénérent toute partie manquante de leur corps après amputation, ce qui, avec leur branchement précoce au sein du règne animal, en fait un modèle précieux pour étudier la régénération. Le modèle de l’Hydre permet d’analyser les mécanismes fondamentaux de la régénération, mais aussi de comprendre comment cette propriété a pu évoluer. L'objectif de ce projet de doctorat était d'étendre nos connaissances sur la signalisation induite par l’amputation dans la phase immédiate de la régénération d'Hydre. Nous nous sommes concentrés sur la signalisation ROS et son rôle potentiel sur la mort cellulaire induite par l’amputation observée dans les bourgeons régénérant la partie apicale.

Dans un premier temps, nous avons caractérisé et quantifié les différents signaux ROS provenant de source membranaire ou mitochondriale. Nous avons détecté une production immédiate et symétrique de superoxyde mitochondrial après bissection à niveau mi-‐gastrique, principalement par les cellules épithéliales gastrodermiques.

Nous avons aussi identifié une production immédiate de peroxyde d'hydrogène (H2O2) asymétrique, plus élevée dans les bourgeons régénérant la partie apicale que dans ceux régénérant la partie basale, probablement en raison d'activités enzymatiques asymétriques, c'est-‐à-‐dire une activité superoxyde dismutase (SOD) plus élevée dans les bourgeons régénérant la tête tandis que la catalase est moins élevée. Des niveaux élevés d'H2O2 déclenchent la phosphorylation de CREB et la mort des cellules interstitielles, probablement par signalisation paracrine. Les niveaux d'H2O2 sont probablement amplifiés par les cellules apoptotiques via une boucle de rétroaction. Les traitements pharmacologiques (Tiron, DPI) et les approches d'éteignage transitoire des gènes (Sod-‐1, catalase) indiquent que la signalisation via les ROS mitochondriaux joue un rôle dans la cicatrisation de la plaie alors que des niveaux élevés de H2O2 sont nécessaires à la régénération apicale. Ainsi, une signalisation ROS asymétrique immédiatement après bissection est critique pour induire la mort cellulaire et la régénération apicale.

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Dans le chapitre suivant, notre objectif était de caractériser les composants de la voie de signalisation ROS impliqués dans la régénération de l'Hydre. Parmi 31 protéines d'Hydre présumées sensibles à l'oxydoréduction dont les gènes sont immédiatement régulés à la hausse après amputation, nous avons trouvé la protéine cargo d’autophagie p62/SQSTM1 (séquestosome 1), le facteur 1 de ribosylation ADP, les facteurs de transcription CCAAT/EBP, CREB3-‐L1, Jun-‐D, Fra-‐l1 et NFE2Fl/Nrfl, dont la structure est proche de celle du facteur humain Nrf3. Nous avons exprimé le gène d’Hydre NFE2Fl/Nrfl dans des cellules de mammifères et analysé l’activité de NFE2Fl/Nrfl sur une construction rapportrice pilotée par des éléments de réponse antioxydants (ARE). Le facteur NFE2Fl/Nrfl réprime l’activité du Nrf2 humain endogène, prouvant indirectement sa capacité à lier les ARE. Dans les cellules HEK293T où l’expression de Nrf2 est réduite, la transactivation exercée par NFE2Fl/Nrfl est constitutivement faible cependant augmentée lors de l'exposition au sulforaphane, un inducteur de NFE2F/Nrf2, mais à un niveau inférieur obtenu avec le Nrf2 humain. Le rôle de NFE2Fl/Nrfl reste à tester in vivo, ainsi que celui d'autres facteurs de transcription sensibles au redox qui pourraient jouer un rôle clé.

L'autophagie est étroitement régulée par la signalisation ROS et comme projet parallèle, j'ai participé à l'analyse de la déficience de l'autophagie dans le vieillissement de l’Hydre. J'ai optimisé un biocapteur génétique conçu pour monitorer le flux d'autophagie chez les animaux vivants. Ce biocapteur d'autophagie a permis d’obtenir des résultats fiables concernant la formation d'autophagosomes dans H.

oligactis intact, soumis à la famine ou à l'inhibition du protéasome ainsi que dans H.

vulgaris lorsque l’expression du gène WIPI2, codant pour une protéine impliquée dans la phase précoce de formation d'autophagosomes, est bloquée.

En conclusion, ce travail nous apporte de nouvelles perspectives sur (i) le rôle de la signalisation ROS, immédiatement asymmétrique entre le côté apical et basal du plan d’amputation, s'appuyant sur le peroxyde d'hydrogène pour orchestrer la mort cellulaire et la régénération de la tête; (ii) le facteur de transcription NFE2Fl/Nrfl en tant qu'acteur possible dans la réponse au stress oxydatif quoique faiblement sensible; (iii) la nouvelle technique d'imagerie utilisant des biocapteurs à codage génétique comme outil puissant et quantitatif pour suivre des processus biologiques

au niveau sub-‐cellulaire.

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List of Figures

Introduction

Figure 1: Anatomy of a Hydra polyp

Figure 2: Sources of new cells in regeneration

Figure 3: Phases of cellular remodeling during Hydra apical regeneration Figure 4: Molecular events taking place during apical regeneration Figure 5: The Drosophila Apoptotic pathway

Figure 6: Early injury signals in wound healing Figure 7: Cellular ROS metabolism

Figure 8: Summary of ROS – apoptosis crosstalk research from 2015-‐2019 done by Serras’ lab

Results Chapter 1

Figure 1: Mathematical modeling of the immediate injury-‐induced signaling that triggers cellular remodeling in head regenerating tips.

Figure 2: Characterization of different types of ROS production in bisected and injured Hydra

Figure 3: Injury-‐induced ROS production in animals exposed to Tiron or DPI Figure 4: ROS plays a critical role in wound healing and regeneration

Figure 5: Asymmetric H2O2 production leads to injury-‐induced cell death and cell death-‐ROS crosstalk

Figure 6: Impact of SOD expression and SOD activity on H2O2 production, wound healing and regeneration

Figure 7: Current working model

Figure S1: Fitting the model to previous experimental data.

Figure S2: The Hydra Sod1, Sod2, Sod3 sequences and deduced proteins Figure S3: The Hydra Catalase sequence and deduced protein

Figure S4: Expression profiles of the Hydra Sod1, Sod2, Sod3 and Catalase genes Figure S5: Toxicity tests of pharmacological inhibitors used in the study

Figure S6: Wound closure of Apical-‐Regenerating halves exposed to Tiron or DPI Figure S7: Wound closure of Basal-‐Regenerating halves exposed to Tiron or DPI

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Figure S8: Anatomies of regenerating polyps continuously exposed to Tiron (15 mM) or DPI (10 µM) for 76 hours from mid-‐gastric bisection

Figure S9: MitoSOX detection of mitO2 in AR and BR halves from untreated and HU-‐

treated animals

Figure S10: Kinetics of apical regeneration in animals knocked-‐down for Sod1 (left) or catalase (right)

Figure S11: Wound closure in animals knocked-‐down for Sod1

Chapter 2

Figure 1: Injury-‐induced modulations of gene expression in Tiron-‐treated animals Figure 2: Spatial, cell-‐type and regeneration expression profiles of three putative

redox-‐sensitive genes

Figure 3: Structure, phylogeny and expression of the Hydra NFE2l/Nrfl transcription factor

Figure 4: Human NRF2 transactivation activity measured in human HEK293T cells Figure 5: Transactivation activity of Hydra NFE2Fl/Nrfl in human HEK293T cells

Figure S1: Injury-‐induced modulations of gene expression in regenerating Hydra and during the homeostasis as detected by RNA-‐seq

Chapter 3

Preface figure: hyLC3a tandem-‐sensor is a powerful tool to follow the autophagy flux in vivo in adult Hydra

Figure 1: Inducible aging phenotype in cold sensitive Hydra oligactis (Ho_CS).

Figure 2: Somatic interstitial loss upon aging and pharmacological induction of aging in Ho_CS animals maintained at 18°C

Figure 3: Deficiency in the inducibility of the autophagy flux in Ho_CS animals Figure 4: Modulation of p62/SQSTM expression levels in Ho_CS animals

Figure 5: Rapamycin treatment delays aging in Ho_CS without enhancing the autophagy flux

Figure 6: Rapamycin promotes epithelial phagocytosis and lipid droplet formation in Hydra

Figure 7: Impact of WIPI2 silencing on autophagic flux, survival, and regeneration in H. vulgaris

Figure 8: Schematic view of the inducibility of the autophagic flux in epithelial cells of aging and non-‐aging Hydra

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Figure S1: Features and reversibility of the cold-‐induced aging phenotype in Ho_CS animals

Figure S2: RNA-‐seq profiles of 20 genes expressed in interstitial cell lineages in Ho_CS and Ho_CR animals maintained at 18°C or transferred to 10°C.

Figure S3: Starvation-‐induced phenotypes in Ho_CR, Ho_CS and Hv_sf1 animals Figure S4: Phylogenetic analysis of the metazoan LC3/ATG8 gene families and RNA-‐

seq profiles of LC3A/B, LC3C, GABARAP and GABARAPL2 genes in H.

vulgaris.

Figure S5: Different sensitivity to MG132 in Ho_CS, Ho_CR and Hv.

Figure S6: Transcriptomic analysis of 75 autophagy genes in aging Ho_CS Figure S7: RNA-‐seq profiles of 75 genes involved in autophagy in mammals

Figure S8: Alignment of vertebrate and non-‐vertebrate p62/SQSTM1 protein sequences

Figure S9: Phylogenetic and expression analysis of p62/SQSTM1 and WIPI2 in Hydra Figure S10: Anti-‐aging roles of Rapamycin in Ho_CS Hydra

Figure S11: Alignment of vertebrate and non-‐vertebrate WIPI2 protein sequences

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Discussion

Figure 1: The MitoQ and Tiron molecules

Figure 2: Current model of ROS-‐apoptosis crosstalk during AR and BR in Hydra

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List of Tables

Chapter 3

Table-‐S1A: Sequence Accession Numbers of the H. vulgaris (Hv) and H. oligactis (Ho_CS, Ho_CR) genes involved in or related to autophagy.

Table-‐S1B: Sequence Accession Numbers of the H. vulgaris (Hv) and H. oligactis (Ho_CS, Ho_CR) genes involved in proliferation and differentiation of interstitial cell (i-‐cell) lineages.

Table S2: Sequences of the primers used to build the mCherry-‐GFP-‐LC3A autophagy sensor

Table S3: Sequences of the siRNA primers used to silence p62/SQSTM1 and WIPI2

Discussion

Table 1: Biological effects of pharmacological and genetical inhibition of ROS metabolism in Hydra

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List of Abbreviations

AR -‐ Apical Regeneration

Ask1 -‐ Apoptosis signal-‐regulating kinase 1 ATP -‐ Adenosine TriPhosphate

AT-‐rich -‐ Adenine -‐ Thymine rich BMP -‐ Bone Morphogenetic Proteins BR -‐ Basal Regeneration

Ca²⁺ -‐ Calcium ions

cAMP -‐ cyclic Adenosine MonoPhosphate Cat -‐ Catalase

CBP -‐ CREB binding protein CG-‐rich -‐ Cytosine -‐ Guanine rich cNeoblast -‐ Clonogenic Neoblast

cpYFP -‐ circularly permuted yellow fluorescent protein

CRE -‐ cAMP response element

CREB -‐ cAMP Response Element Binding Protein

Cul3 -‐ Cullin 3

DAMP -‐ Damage-‐Associated Molecular Pattern

DIAP1 -‐ Drosophila Inhibitor of APoptosis 1 DNA -‐ DeoxyriboNucleic Acid

Dpp -‐ Decapentaplegic (morphogen) DrICE -‐ Death related ICE-‐like caspase Dronc -‐ Death regulator Nedd2-‐like caspase Dsp-‐1 -‐ Dorsal switch protein 1

DUOX -‐ Dual oxidase

eESC -‐ epidermal Epithelial Stem Cells Gal4 -‐ Galactose-‐induced gene 4 Gal80 -‐ Galactose-‐induced gene 80

Gal80^TS -‐ Galactose-‐induced gene 80 (thermo sensitive)

gESC -‐ gastrodermal Epithelial Stem Cells GTP -‐ Guanosine triphosphate

H2O2 -‐ hydrogen peroxide Hid -‐ Head involution defective HO^.-‐ Hydroxyl radical

Ho_CR -‐ Hydra oligactis Cold Resistant Ho_CS -‐ Hydra oligactis Cold Sensitive Hpa -‐ hours post-‐amputation

Hsp70 -‐ Heat shock protein 70 hyIAP -‐ Hydra Inhibitor of APoptosis

i-‐cells -‐ Interstitial stem cells Il1 – Interleukin1

Jak/Stat -‐ Janus kinase/Signal Transduces and Activator of Transcription proteins JNK -‐ c-‐Jun N-‐terminal kinases

Keap1 -‐ Kelch-‐like ECH-‐associated protein 1 MAPK -‐ Mitogen-‐activated protein kinase mpa -‐ minutes post amputation

mtROS -‐ mitochondrial Reactive Oxygen Species

Myd88 -‐ Myeloid differentiation primary response 88

NAC -‐ N-‐Acetyl Cysteine

NADPH -‐ Nicotinamide Adenine Dinucleotide Phosphate (reduced form) NCBI -‐ National Center for Biotechnology Information

NFE2F/Nrf2 -‐ Nuclear factor erythroid 2-‐

related factor 2

NOD -‐ Nucleotide-‐binding Oligomerization Domain

NOX -‐ NADPH oxidase

O2-‐. -‐ superoxide radical anion pCREB -‐ Phospho CREB pH3 -‐ phospho Histone 3

Pi3K -‐ Phosphoinositide 3 Kinases PUFA -‐ PolyUnsaturated Fatty Acids

RHO-‐1 -‐ Ras HOmolog gene family -‐ member 1

RNAi -‐ RiboNucleic Acid interference ROCK -‐ Rho-‐associated protein kinase ROS -‐ Reactive Oxygen Species RSK -‐ Ribosomal S6 Kinase

siRNA -‐ small interfering Ribonucleic Acid SOD -‐ SuperOxide Dismutase

SPINK -‐ Serine Protease INhibitor Kazal-‐type TOP:dsGFP -‐ TCF optimal promoter : destabilized green fluorescent protein TRL – Toll like receptors

UAS -‐ Upstream activating sequence Wg – wingless

Z-‐VAD -‐ Carbobenzoxy-‐valyl-‐alanyl-‐aspartyl-‐

[O-‐methyl]-‐ fluoromethylketone

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INTRODUCTION

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I. Hydra, a versatile model for studying wound healing, regeneration, aging and autophagy

I.1. Hydra anatomy and morphology

Hydra is a species that belongs to Cnidaria, a phylum that in the metazoan kingdom occupies a sister group position to Bilateria. Hydra exclusively resides in fresh water and exhibits a polyp-‐like structure with an apical to basal axis, a head-‐like structure on the apical side and a basal disc also named foot on the basal side. The Hydra head consists of a dome shaped structure named hypostome surrounded by a ring of tentacles (Figure 1, left). At the tip of the hypostome, an opening serves as a mouth/anus that leads to the gastric cavity where food digestion takes place. The tentacles, which contribute to the feeding process, are mainly made of large epithelial cells, the battery cells that contain terminally differentiated mechano-‐sensory cells named nematocytes. These stinging cells differentiate highly specialized structures filled with venom called nematocysts. Upon contact with the prey, the nematocysts get explosively discharged into the target, releasing the venom that paralyzes the prey. On the basal side, Hydra polyps differentiate a basal disk, i.e. a structure lined with mucous cells that secrete a mucous which keeps the animal attached to the substrates present in its natural habitat (for example, lily pads at the surface of ponds, stones in the rivers, wooden sticks). In the laboratory conditions, animals are maintained in glass or plastic dishes to which their basal disk does attach as well.

Hydra are made of two cell layers, the inner one that lines the gastric cavity, called gastrodermis or endoderm, and the outer one that plays a protective role, named epidermis or ectoderm. These two layers that are single-‐cell thick are separated by a collagen-‐containing extracellular structure called mesoglea (Figure 1, right). Both of these layers are filled with epithelial cells mostly referred as endodermal and ectodermal epithelial stem cells (ESC) (Bosch, 2007; Galliot, 2012), which actually correspond to gastrodermal and epidermal stem cells respectively (Buzgariu et al., 2018). These two stem cell types are unipotent, meaning that they self-‐renew and only provide epithelial cells that acquire distinct and specific features at the extremities. ESCs constantly self-‐renew along the body column while passively moving towards the budding region as well as the apical and basal extremities of the animal where they get terminally differentiated to be finally sloughed off (Hobmayer

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et al., 2012). A third population of stem cells, the interstitial stem cells, often shortly named i-‐cells, provide all the other cell types, including the germ cells when the animal become sexual, the gland cells that populate the gastrodermis and all cells of the nervous system, making them a classic example of multipotent stem cells (Figure 1, right). The nerve cells form in Hydra diffused nervous system. I-‐cells are located in the central part of animal body column, where they are ‘’squeezed’’ into the epidermal ESCs.

Figure 1. Anatomy of a Hydra polyp

Hydra displays a tube-‐shaped (left) terminated at its apical side by a dome structure named hypostome, encircled with tentacles and a peduncle region that precedes the basal disc at its basal side. Polyps are made of two cellular layers, the epidermis (green) and the gastrodermis (light-‐red) which are separated by an extra-‐cellular matrix named mesoglea. Epidermis and gastrodermis are predominantly made of epithelial cells that are specific to each layer and cannot replace each other. The third stem cell lineage, called interstitial stem cells (yellow) can differentiate into different cell types, such as gland cells located in the gastrodermis (purple), nematoblasts (magenta), precursors to nematocytes and nerve cells (not shown).

I.2. Hydra homeostasis and developmental properties

Hydra is characterized by a highly dynamic homeostasis. These animals represent a balance between an intense sustained proliferation of stem cells in its central part and cell death at the extremities where old cells are discarded. Both endodermal and ectodermal ESCs belong to the group of slow self-‐renewing cells that have a cell cycle of approximately 3-‐4 days while i-‐cells have a faster cell cycle, lasting for 24-‐30 hours (Holstein and David, 1990). Similarly to mammalian embryonic stem cells, Hydra stem cells are characterized with very short G1 phase and pausing during the G2 (Savatier et al., 1994; Fluckiger et al., 2006), however there is still a difference between ESCs and i-‐cells. While ESCs pause in G2-‐phase up-‐to 2.5 to 3.5 days (Holstein et al., 1991), i-‐cells have a shorter G2 phase, which varies between 4 and 18

Epidermis Gastrodermis

Tentacles

Basal disk Hypostome

Peduncle

Mesoglea

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h (Holstein and David, 1990), when regularly fed. Additionally they have a specific behavior pattern in different zones along the body column of the Hydra. While in the central part of the animal they constantly proliferate, ESCs get passively displaced towards the apical and basal extremities, where cell cycling stops and they get terminally differentiated to mostly battery cells or mucous cells respectively, except of few epithelial cells that remain un-‐differentiated in the hypostome (Dübel, 1989). I-‐

cells behave a bit differently, since a large amount of them migrate in the form of progenitors toward extremities, where they get differentiated to nerve cells, nematocytes or gland cells. It is important to stress out that epithelial cells usually differentiate before mitosis during the G2 phase; i-‐cells perform this in the G1/G0 phase, as a post-‐mitotic event (Buzgariu et al., 2014). When they reach the apical or basal extremities, these terminally differentiated cells get expelled from the animal via the cell death process.

Hydra is a very attractive model system due to the simplicity of its maintenance; they grow quite rapid, doubling time being around 3.5 days together with possibility to culture massive numbers of clonally derived animals. Animals need a constant temperature of ‘’wine cellar’’ that is 18°C, and to be fed with, freshly hatched Artemia nauplii as described by (Bosch, 2007; Bossert and Galliot, 2012). Taken together, Hydra provides a simple, sustainable and useful research model that is used by about 20 laboratories around the world. Beside its dynamic homeostasis, Hydra is characterized by a series of developmental properties that are unusual in adult organisms as described in the next part.

Budding and sexual reproduction

Hydra polyps can propagate either asexually in the process called budding, or sexually via gametogenesis. Budding is one additional consequence of the dynamic tissue turnover during homeostasis. In normal conditions when animals are regularly fed, a small bud starts to grow recruiting the cells in a specific region of the parental animal, which is located in the lower bottom of the polyp, called budding zone.

Budding usually has the similar cycle of 3-‐4 days where a small bud appears, grows and finally de-‐attaches from the parental polyp. Hydra has a way to preserve its genetic material even in the very harsh conditions. When there is a lack of food, or the

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temperature of water is lower, polyps can undergo sexual differentiation. Some animal can have testis and change to oocyte, however the opposite is occurring more frequently. In the end the fertilized egg develops into the embryo that can stay for some time in resting stage covered with protective, chitinous shell called thecae (Bossert and Galliot, 2012).

Aging and autophagy

First aging experiments conducted in Hydra were done in the 1950s when different species were followed over several years. The Belgian biologist Paul Brien compared three different Hydra: H. vulgaris, H. oligactis and H. viridisima. He found that when animals are kept at native conditions, at 18°C and regularly fed, they continue to reproduce by budding and there is no sign of aging or how he called it at the time

‘’exhaustion’’. However, if he would challenge these ‘’cozy’’ conditions for the animals by transferring them from 18°C to 10°C, H. oligactis, but not H. vulgaris started to show very interesting behavior. They stopped to bud and turn to sexual type of reproduction, which as explained in the previous paragraph, is as sign of stress and a sort of a defensive mechanism in Hydra. After the polyps reached sexual maturity, they started to show a type of degeneration that is very similar to an aging phenotype (Brien, 1953). This results were confirmed and further characterized by more recent studies where it was shown that H. oligactis can be induced to age, having a maximum lifespan of 120 days post-‐induction, i.e. transfer to 10°C (Yoshida et al., 2006).

Additional insight came from our laboratory where two distinct strains of H. oligactis were shown to behave differently. One of them, now called Cold resistant (Ho_CR), does not show aging phenotype when induced for gametogenesis, while the other named Cold sensitive (Ho_CS) does (Tomczyk et al., 2015; Tomczyk et al., 2017).

Induction of autophagy is one of the main cellular strategies for survival during the harsh conditions, such as lack of food, and is lately being linked with the induction of cell death, since the macroautophagy is activated via the same signaling pathways that also controls apoptosis (Codogno and Meijer, 2005; Das et al., 2012). Autophagy was not heavily studied in Hydra, with the exception being the laboratory of B. Galliot.

Similarly to the pancreatic autophagy phenotype observed in the mutation of SPINK1 and SPINK3 genes in human, Hydra Kazal1, a cytoprotective protein, shows a role in

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prevention of excessive autophagy (Chera et al., 2006). Kazal1 silencing leads to the lower budding rate and excessive cell death in the homeostatic context, followed by massive autophagy upon amputation (Chera et al., 2006). Interestingly when compared with starvation in control animals that accumulate autophagic vacuoles mostly in eESCs, during Kazal1 RNAi gESCs are the one showing massive numbers of autophagosomes (Chera et al., 2009a). Previously autophagy was studied in Hydra with the use of the tools that provide a static view of autophagy such as biochemical methods and mostly immunohistochemistry, using the antibodies to show the autophagosome formation (Chera et al., 2006; Buzgariu et al., 2008; Chera et al., 2009a). In the past years, we managed to develop an autophagy sensor to monitor the autophagic flux in live, intact adult animals. As a side project of my doctoral studies I developed this tool derived from the existing elegant method used in mammalian cells (Pankiv et al., 2007). This tool, presented in Chapter 3 of the Result section of this thesis, now opens the possibility us to quantify autophagy in Hydra.

Re-‐aggregation and regeneration

A valuable strategy to investigate patterning in adult Hydra polyps is a procedure called re-‐aggregation. Another interesting trait of Hydra that shows how resilient these animals are; re-‐aggregation can be performed under controlled laboratory conditions, where polyps are dissociated to the single cell level. Later on, a cell suspension is centrifuged and subsequently aggregates formed. After 20-‐30 hours they will form a hollow sphere, and finally develop into one or more individual polyps after several days (Gierer et al., 1972; Technau et al., 2000).

As a regeneration model, Hydra is known for 280 years since the Swiss scientist Abraham Trembley (during 1740s) discovered that it can regenerate every missing part when cut. From then Hydra is used by scientists to uncover the biological mechanisms that support such an efficient regenerative program. In the 18th century, it was not clear whether cnidarian polyps, i.e. corals, hydra, would be plant or animals. To solve that question, Trembley decided to cut the polyp and monitor whether it regenerates. Once it became clear to him that Hydra was both regenerating and behaving as an animal (not aware that actually Antonie van Leeuwenhoek described this specie as animals 40 years ago), he realized that he had made a major

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discovery, that of whole body regeneration in the animal kingdom. This discovery launched the whole field of regenerative biology. Since then, Hydra got established as a potent model to study how animals can efficiently repair wounds and regenerate every missing part of their body. Despite decades of work on dissecting its regenerative program, much of it is still not understood. In the next section, our current knowledge on the cellular and molecular basis of Hydra regeneration will be summarized.

I.3. Cellular basis of Hydra regeneration

Sources of cells that are used for regeneration vary greatly in different regenerative animals. At the moment it is accepted that there are three main mechanisms that provide new cells during regeneration: (1) Stem cell activation, where resident stem cells start to divide and produce more cells like itself, following by differentiation into the required cellular types (Figure 2A, top) (Weissman et al., 2001). Clonogenic Neoblasts (cNeoblasts) in planarian regeneration (Wagner et al., 2011) and i-‐cell progenitors in Hydra mid-‐gastric apical regeneration (Chera et al., 2009b) are a good example of stem cell activation. (2) De-‐differentiation is a process where differentiated cells temporarily lose their differentiated characters, re-‐enter the cell cycle and produce cells that can now act as progenitor cells that continue to proliferate for a while to form a blastema and subsequently differentiate to form the missing structure (Figure 2A, middle) (Jopling et al., 2011). Cellular de-‐

differentiation is a main source for regeneration in zebrafish heart (Jopling et al., 2010), but also in bone regeneration in zebrafish fin (Knopf et al., 2011). (3) New cells can be a result of a process called trans-‐differentiation, during which a cell changes a state from one cell type to another, and this can occur without cell division (Figure 2A, bottom) (Jopling et al., 2011). Trans-‐differentiation is much less common than the previously mentioned mechanisms. Some invertebrates such as jellyfish have high trans-‐differentiation potential, but this is heavily reduced in vertebrate regeneration (Shen et al., 2004). Although not naturally occurring, but rather induced, common examples of this mechanism are the formation of the lens of the eye in the chick (Eguchi and Okada, 1973; Araki and Okada, 1977), or newt where pigmented

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epithelial cells can de-‐differentiate and then re-‐differentiate into missing lens cells (Jopling et al., 2011).

Additionally, stem cells can be multipotent or be restricted for their contribution to the novel regenerated structure. Planarian cNeoblast are an example of classical pluripotent stem cells, while Hydra’s i-‐cells are undifferentiated multi-‐potent stem cell that when needed can provide many different cellular types, such as nematocytes, nerve or gland cells (Nishimiya-‐Fujisawa and Kobayashi, 2012) (Figure 2B), while for example in salamander and axolotl, limb regeneration is occurring in a much more restricted fashion (Figure 2C). Axolotl regenerates its limb using different stem cells that show lineage restriction, and there is no contribution of, for example, muscle cells to epidermis regeneration (Kragl et al., 2009).

Figure 2. Sources of new cells in regeneration

(A) Stem cells can have three distinct action patterns during regeneration: activation (top), de-‐differentiation (middle) and trans-‐differentiation (bottom). (B) cNeoblasts (S.

mediterranea) and i-‐cells (Hydra) show multi-‐potency, while in axolotl, muscle, skeleton or Schwan cells are lineage-‐restricted during regeneration (C). Scheme after (Tanaka and Reddien, 2011)

It is important to state that apical and basal regeneration in Hydra are very different.

While apical regeneration results in the formation of a complex head structure, basal regeneration results in a simpler structure, the foot. Also apical regeneration is simpler to follow, since it is visually easier to monitor the morphological changes such as the appearance of tentacle rudiments (Bode, 2003), especially with kinetics-‐

type experiments, and thus it was studied much more. On the level of cellular

A B

C

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remodeling, Hydra apical regeneration can be divided into four different phases:

early, early-‐late, late and very late (Figure 3) (Galliot, 2013).

During the immediate phase (up to 2 hours post amputation; hpa) (Figure 3, top-‐

left), when the wound-‐healing process is launched, several important events take place. I-‐cells, located in epidermis undergo apoptosis while gastrodermal ESCs lose their typical morphology. In the early phase, between 2 and 12 hpa (Figure 3, bottom-‐left), apoptotic i-‐cells are engulfed by the gastrodermal ESCs, that now transiently lost their epithelial organization, which they re-‐gain in the early-‐late phase (Figure 3, top-‐right). Something similar to these cellular changes can be seen during the Hydra re-‐aggregation process (Murate et al., 1997). After the wound is successfully healed during the earlier phases, the late phase is characterized by a visible re-‐construction event, with the appearance of tentacle rudiments that become visible from 40 hpa (Figure 3, bottom-‐right) (Galliot, 2013).

Figure 3. Phases of cellular remodeling during Hydra apical regeneration

Hydra successfully performs the wound healing process during immediate to early phases in regeneration. ESCs in gastrodermis are shown in gray with red nuclei, and i-‐

cells as green spots in white epidermal ESCs. I-‐cells that undergo apoptosis are shown as stars, which are later being engulfed by gESCs that transiently lost their epithelial organization (bottom-‐left). First regeneration visual markers can be seen during the late phase (bottom-‐right), where tentacle rudiments appear, followed by formation of hypostome (Explained in details in the text). Scheme after (Galliot, 2013)

For some time, it was considered that Hydra undergoes only mophallaxis – a regenerative program that does not rely on cell proliferation (Bosch, 2007). However,

Immediate (0-2 hpa) Early (2-12 hpa) Early-late (>16 hpa) Late (>40 hpa)

Hydra, a model for studying the role of injury-induced ROS signalling during regeneration and monitoring the autophagy flux in live animals

Thesis

Reference

Hydra, a model for studying the role of injury-induced ROS signalling during regeneration and monitoring the autophagy flux in live animals

Hydra, a Model for Studying the Role of Injury-induced ROS

Signalling during Regeneration and Monitoring the Autophagy Flux in Live Animals

TABLE of CONTENTS

Introduction

Results Chapter 1

Chapter 2

Chapter 3

Discussion

Chapter 3

Discussion

INTRODUCTION

A B

C