Thesis
Reference
Genetic control of the cellular remodeling driving head-regeneration in hydra
CHERA, Simona
Abstract
Afin de caractériser les événements moléculaires et cellulaires impliqués dans la régénération de l'hydre, j'ai utilisé l'ARN interférence afin d'éteindre de façon spécifique l'expression génique. Cette approche a permis de caractériser trois phases distinctes dans la régénération de la tête. Immédiatement après amputation, le gène Kazal1, un inhibiteur de serine-protéase, permet la survie cellulaire en prévenant une autophagie excessive. Simultanément, la voie de signalisation MAPK/CREB en synergie avec la voie de signalisation Wnt joue un rôle clé dans la régénération de la tête en induisant l'apoptose de cellules différenciées et la prolifération de cellules souches dans le bourgeon de régénération. Plus tard, le gène Parahox Gsx/cnox-2 contrôle la prolifération et la différenciation de nouvelles cellules nerveuses apicales et la formation de la tête. En conclusion, nous avons montré que, dans sa version "sauvage", la régénération hydre est une variante de la régénération épimorphique.
CHERA, Simona. Genetic control of the cellular remodeling driving head-regeneration in hydra . Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3991
URN : urn:nbn:ch:unige-22785
DOI : 10.13097/archive-ouverte/unige:2278
Available at:
http://archive-ouverte.unige.ch/unige:2278
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES
Département de Zoologie et Biologie Animale Dr. Brigitte GALLIOT Prof. Pierre SPIERER ______________________________________________________________
Genetic control of the cellular remodeling driving head-regeneration in hydra
THÈSE
présentée à la Faculté des Sciences de l`Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Simona CHERA
de
Bucarest (Roumanie)
Thèse N° 3991
GENÈVE
Atelier d'impression de la Section de Physique
2008
3
Reme rciem ents:
Je tiens à remercier Brigitte Galliot pour sa tolérance et sa disponibilité.
Merci à toutes les personnes qui m’ont aidée pendant cette thèse: Luiza Ghila, Renaud de Rosa, Marijana Miljkovich-Licina, Kevin Dobretz, Manon Quiquand, Han Petry, Virginie Voeffray, Lizbeth Muster, Wanda Buzgariu, Gaspare Benenati, Pierre Heuze, Philippe Jean et Fadi Hamdan.
Un grand merci à ma famille, spécialement ma maman.
Merci aux membres du jury: Prof Dr. Jeremy Brockes, Prof. Dr. Konrad Basler, Prof. Dr. Ivan Rodriguez, Prof. Dr. Jean-Claude Martinou, Prof. Dr. Pierre Spierer et Dr. Brigitte Galliot.
Acknowled g em ents:
I’ve always wanted to be a scientist. My family is morally responsible for this “obsession”. I would like to thank my mother, father, brother, and grandparents for rising me exactly how I am. I owe to my parents my cynical sense of humor, my troubleshooting skills, the capacity to scientifically argue my points of view and obviously much more. My mother was the first one to know that I’ll be a biologist. I’m also grateful to her for supporting me to switch from physics to biology and to all my physics teachers for making me wanting to do so. I also thank my biology teachers and professors for showing me how interesting biology can be.
Retrospectively thinking, I don’t think I knew what it really means to do biological research until I’ve started to work in Brigitte’s lab. Well, I might have had an idea about how things should be (mostly from
“virus-kills-all” B-class movies) but, before Geneva, it didn’t seem realistic. In the lasts years I’ve learned a lot, not only about science but also about everything else. The “scientific niche” within University of Geneva had an incredible motivational role by facilitating contact with great scientists and providing access to extremely interesting seminars. Therefore I am extremely grateful to Brigitte for giving me the opportunity of being a part of this dynamic environment. Furthermore, I would like to thank her for the help and support during these years, as well as for her patience.
Brigitte’s laboratory is a lively ecosystem where work is fun and fun is work. I would like to thank the past and future members of the lab: Luiza Ghila, Renaud de Rosa, Marijana Miljkovich-Licina, Kevin Dobretz, Manon Quiquand, Han Petry, Virginie Voeffray, Lizbeth Muster, Wanda Buzgariu, Gaspare Benenati Pierre Heuze, Philippe Jean, Fadi Hamdan for keeping up this atmosphere and for the stimulating scientific discussions. In addition, special thanks to the members of the NCCR Frontiers in Genetics Bioimaging Platform and especially Dr. Christoph Bauer for his assistance and advises over these years.
Furthermore, I thank the members of my thesis committee for their time and interest in my thesis: Prof Dr. Jeremy Brockes, Prof. Dr. Konrad Basler, Prof. Dr. Ivan Rodriguez, Prof. Dr. Jean-Claude Martinou, Prof. Dr. Pierre Spierer, and to my thesis supervisor and coordinator Dr. Brigitte Galliot.
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ABBREVIATIONS:
β-Cat – β-Catenin
AEC – apical epithelial cap AIF – apoptosis inducing factor APC – adenomatous polyposis coli ATF – activating transcription factor BrdU – bromodeoxyuridine
CBP – CREB binding protein Ci – Cubitus intrerruptus (Ci) CK1 – casein kinase 1 Cos2 – kinesin Costal2
COUP-TF – chicken ovalbumin upstream promoter transcription factor
CRE – cAMP-response element CREB – CRE-binding protein CREM – CRE modulator Dkk – Dickkopf
dsRNA – double stranded RNA ECM – extracellular matrix EGF – epidermal growth factor
ERK – extracellular signal-regulated kinases EST – expressed sequence tag
FGF – fibroblast growth factor
Fgfr – fibroblast growth factor receptor Fu – Fused
GFP – green fluorescent protein
GSK-3β – Glycogen synthase kinase 3β HA – head-activator
Hh – Hedgehog
hpa – hours post-amputation Hv – Hydra vulgaris
JNK - Jun N-terminal kinase KID – kinase-inducible domain LRP – LDL-receptor-related protein MAPK – mitogen-activated protein kinase MMP – matrix metalloproteinase
MSK1 – mitogen- and stress-activated protein kinase
NE – nuclear extracts NGF – nerve growth factor Pax7 – paired box gene 7
PCNA – proliferation cell nuclear antigen P-CREB – phospho-CREB
PI3 – phosphatidyl inositol 3 PKA – protein kinase A PKC – protein kinase C Ptc – Patched
Rb – retinoblastoma RNAi – RNA interference
RSK – ribosomal protein S6 kinase Smo – Smoothened
SPINK – Serine Protease Inhibitors Kazal Sufu – Supressor of fused
Tcf - T cell-specific transcription factor TSP – thrombospondin
TUNEL – Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling WCE – whole cell extracts
WE – wound epidermis
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REMERCIEMENTS………...3
ABBREVIATIONS……….4
1. RÉSUMÉ EN FRANÇAIS………...……….7
2. SUMMARY……….9
3. PROLOGUE ... 11
3.1 Regeneration – A Journey in the Dark ... 11
3.2 Hydra – The Shadow of the Past ... 11
3.2.1 Hydra – the perfect “lab rat” ... 11
3.2.2 Hydra – the key phylogenetic position ... 12
3.2.3 Hydra – the cell biology... 12
3.2.4 Hydra – the extreme developmental plasticity ... 12
3.2.5 Hydra – the molecular biology ... 13
3.2.5.1 Hydra in the Genomic Era………13
3.2.5.2 Hydra - the Molecular Tool Set ... 13
3.3 The Structure of the Introduction ... 13
4. INTRODUCTION ... 15
4.1 The Hydra Model System ... 15
4.1.1 Morphology and Cellular Biology of Hydra Polyps………..15
4.1.1.2 Epithelial Cells... 16
4.1.1.3 Interstitial Cells... 18
4.1.1.4 General conclusions about hydra cell cycle ... 20
4.1.2 Homeostasis and Development... 21
4.1.2.1 Two pairs of opposed gradients maintain the homeostatic dynamic equilibrium ... 21
4.1.2.2 Budding... 23
4.1.2.3 Sexual reproduction ... 24
4.1.2.4 Regeneration in hydra – generalities ... 24
4.1.2.5 Reaggregation ... 26
4.2 Cellular and Molecular Mechanisms Underlying Animal Regeneration ... 27
4.2.1 Blastema-dependent regenerating systems ... 28
4.2.1.1 The Wound-Healing Phase... 28
4.2.1.2 The Blastema Formation Phase ... 29
4.2.2 Hydra Morphallactic Regeneration ... 32
4.2.2.1 Morphallaxis and Hydra Regeneration ... 32
4.2.2.2 Cellular Modifications and Biological Activities Observed during Hydra Head Regeneration ... 33
4.2.3 Key developmental pathways involved in animal regeneration ... 36
4.2.3.1. Role of thrombin in wound healing and blastema formation... 36
4.2.3.2. The FGF signaling pathway and blastema proliferation ... 37
4.2.3.3. The CREB transcription factor and the initiation of regeneration... 39
4.2.3.4. The Wnt signaling pathway in regeneration ... 41
4.2.3.5 The Involvement of the Hedgehog Pathway in Animal Regeneration ... 45
4.3 Objectives of this work ... 47
4.3.1. RNA interference applied to Hydra... 47
4.3.2 Cellular and molecular analysis of hydra head-regeneration... 47
4.3.2.1 Role of the MAPK/CREB signaling pathway during early head-regeneration ... 47
4.3.2.2 Cellular remodeling during early head-regeneration ... 48
6 4.3.2.3 Neurogenesis during early-late head-regeneration - investigating the cellular and developmentalregulation of
the hydra gsx homologue (cnox-2) ... 48
4.3.2.4 Comparative analysis of regeneration and budding ... 48
5. RESULTS………...49
CHAPTER 1 – Serine protease inhibitors are required for surviving the amputation stress…...51
CHAPTER 2 – Asymmetrical activation of the MAPK/CREB pathway during hydra regeneration……...69
CHAPTER 3 – Cellular characterization of the MAPK/CREB signaling pathway….…………...….77
CHAPTER 4 – Morphallaxis No More! Apoptosis and blastema formation during hydra head-regeneration....93
CHAPTER 5 – Neurogenesis and Regeneration…...………..131
CHAPTER 6 – Tracing back the early steps of neurogenesis in the first-evolved nervous system…….147
CHAPTER 7 – Developmental plasticity of hydra polyps…...………...169
CHAPTER 8 – RNAi and developmental plasticity………...………..183
CHAPTER 9 – Budding and Regeneration two faces of the same coin?...193
6. GENERAL DISCUSSION………209
6.1 Surmounting the amputation stress………211
6.2 A complex cellular remodeling to initiate head regeneration in hydra……...………..212
6.3 The Neurogenic Dependent Phase of Regeneration……….………...213
6.4 A New View on Hydra Regeneration………..……….214
6.5 Regeneration and Budding…………..………..215
6.6 Perspectives………...………..….216
7. BIBLIOGRAPHY……….…………..217
8. ANNEX..……….227
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1. R é sum é en fran ç ais
Grâce à sa petite taille, sa croissance rapide, sa position clé dans l’arbre du vivant et son organisation cellulaire simple mais extrêmement dynamique, l’hydre est un excellent modèle pour l’étude de la régénération. Ce travail a utilisé une méthode récemment mise au point dans le laboratoire permettant d’éteindre de façon fiable, spécifique et sûre l’expression génique par l’ARN interférence (ARNi). Cette méthode combinée aux méthodes d’analyses cellulaires dynamiques et quantitatives que j’ai développées au cours de ce travail, fournit une approche fonctionnelle performante qui affine considérablement les recherches portant sur l’hydre et renforce donc sa valeur en tant que système modèle. Le principal sujet de mon travail de doctorat était de caractériser les événements cellulaires et moléculaires qui soutiennent la régénération de la tête chez l’hydre. Le sujet étant ambitieux, nous nous sommes concentrés sur quatre questions distinctes qui présentent un intérêt général pour comprendre les mécanismes qui rendent la régénération animale possible :
1) Comment l’hydre surmonte le stress dû à l’amputation ?
2) Comment s’établit l’activité organisatrice de la tête dans la moitié amputée de la tête ? 3) Quelle est la contribution de la neurogénèse au cours de la régénération de la tête ? 4) Quelles sont les différences cellulaires et moléculaires entre bourgeonnement et régénération ?
Après une introduction dédiée au modèle de l’hydre et aux notions clé liées à la régénération animale, ce travail de thèse est structuré de manière à suivre de près l’analyse successive de ces questions biologiques. Dans la première partie de l’introduction (Chapitre 4.1), j’ai décrit les spécificités anatomiques et cellulaires du polype adulte : son organisation tissulaire en deux couches, ses lignages cellulaires, les particularités du cycle cellulaire et les programmes morphogénétiques. Dans une seconde partie, j’ai présenté les différents modes de régénération identifiés dans le monde animal ainsi que quelques voies de signalisation impliquées dans la régénération (Chapitre 4.2). Les résultats que j’ai obtenus au cours de mon travail de thèse sont présentés en neuf chapitres correspondant à des travaux publiés (7) ou en cours de publication (2). Ces chapitres englobent quatre thèmes principaux:
Un contrôle étroit de l’autophagie est nécessaire au maintien de l’homéostasie de l’hydre et à sa régénération: Dans le premier chapitre (Chapitres 1, 7 & 8 - Résultats) nous illustrons comment l’inhibition par ARNi du gène Kazal1, codant pour un inhibiteur de serine–
protéase, affecte l’homéostasie de l’hydre ainsi que son potentiel de régénération. Dans le contexte homéostatique, nous avons montré que les hydres cessent de bourgeonner, puis suite à une inhibition prolongée du gène Kazal1, ne sont plus capables de survivre et meurent. L’analyse cellulaire de ces animaux révèla la formation d’autophagosomes dans les cellules glandulaires et épithéliales de l’endoderme, reflétant une autophagie massive. Au cours de la régénération, le même phénotype cellulaire est observé mais avec une apparition fortement accélérée, une vague immédiate d’autophagie étant induite par l’amputation dans le bourgeon de régénération. Suite à une inhibition prolongée, les hydres ne survivent pas au stress de l’amputation plus de quelques heures. Ce phénotype cellulaire est similaire à celui décrit chez les malades porteurs d’une mutation du gène SPINK1 (Serine Protein Inhibitor Kazal-type) ; ces individus souffrent d’une pancréatite chronique dû à une autophagie trop importante. Les souris mutées pour le gène correspondant (Spink3) présentent également une autophagie massive du pancreas exocrine puis du tube digestif avoisinant, qui se développe dès la naissance. Toutes ces données suggèrent que l’inhibiteur de sérine protease Kazal1 joue une rôle clé lors du stress que l’hydre doit surmonter après l’amputation.
Il s’agit de la première étude chez l’hydre qui mette en évidence l’importance de la cyto- protection, suggèrant la nécessité d’un programme d’auto-préservation pendant le processus de régénération. Il s’agit également de la première démonstration que la mutation de gènes orthologues puisse induire le même phénotype cellulaire chez l’hydre et chez l’homme, qui pourtant ont divergé il y a environ 700 millions d’années.
L’initiation de la régénération : Chez l’hydre, pendant les premières heures qui suivent la bissection, les cellules du bourgeon de régénération sont sujettes à d’importantes transformations menant à l’établissement d’une nouvelle activité organisatrice de la tête. Dans la section Résultats (Chapitres 2, 3, 4) nous montrons que la voie de signalisation MAPK/CREB qui inclut les kinases ERK et RSK, le facteur de transcription CREB et le co- activateur CBP, en synergie avec la voie de signalisation canonique Wnt joue un rôle clé dans ce processus. Dans le Chapitre 4.2 nous montrons que la phosphorylation de CREB est en
8 effet nécessaire à la régénération de la tête. Afin d’identifier les fonctions cellulaires de ces trois composants pendant les premières phases de la régénération, nous avons d’abord détecté par le biais d’expériences d’immunohistochimie la co-expression de RSK, CREB et CBP dans tous les types cellulaires, incluant les cellules épithéliales endodermales et ectodermales, les cellules souches interstitielles, les nématoblastes et les neurones sensoriels et ganglionnaires (Chapitre 3 - Résultats).
Puis dans le Chapitre 4 - Résultats nous décrivons les analyses immunocytologiques qui ont mis en évidence une apoptose précoce, transitoire mais massive de la moitié des cellules situées dans la partie la plus superficielle du bourgeon de régénération de la tête. Cette apoptose semble restreinte à la seule lignée cellulaire interstitielle. Au cours des premières 45 minutes suivant la bisection, ces cellules apoptotiques surexpriment la protéine Wnt3 tandis que les cellules situées dans la région juste en dessous de la zone d’apoptose entrent en phase S comme l’ont révélé les marquages systématiques au Bromo-deoxyuridine (BrdU), puis rapidement se divisent de façon synchrone. Les cellules qui cyclent sont des cellules interstitielles dont la β-catenin est transloquée dans le noyau après 90 minutes de régénération. Lorsque l’induction des gènes RSK, CREB et CBP dont les transcrits apparaissent normalement dans les bourgeons de régénération rapidement après amputation, est inhibée, l’apoptose et la prolifération cellulaire sont abolies, conduisant soit à la mort des animaux soit à une régénération retardée de plusieurs jours. De même lorsque les gènes Wnt3, CREB et β-catenin sont éteints, l’activation de la voie Wnt dans le bourgeon de régénération n’est plus observée.
Ces données suggèrent que l’apoptose des cellules de la lignée interstitielle, c’est à dire des progéniteurs, des cellules nerveuses différenciées et des nématoblastes, est un pre-requis à l’organisation du blastème de régénération et à la mise en place de l’activité organisatrice de la tête. Comme les cellules épithéliales sont supposées porter l’activité morphogénétique chez l’hydre, nous discutons comment l’apoptose des neurones en particulier permet la modification des échanges d’information qui ont lieu entre les lignées cellulaires myoepithéliales et interstitielles. Nous proposons que cette modification du flux des informations fournisse un contexte transitoirement favorable à la réactivation du programme de développement qui aboutira à la formation d’une nouvelle tête (Chapitres 4, 7, 8 - Résultats).
L’analyse fonctionnelle du gène cnox-2 (Gsx) démontre la nécessité d’une néo- neurogénèse au stade semi-tardif afin de différencier une nouvelle tête : Après bisection au niveau central de l’animal, le modelage de la tête pendant la régénération précède l’apparition des rudiments tentaculaires et commence environ 20 heures après l’amputation.
Dans le chapitre 5 - Résultats nous montrons que cette période est marquée par une prolifération cellulaire intense dans le bourgeon de régénération suivie par la différentiation de nouvelles cellules nerveuses apicales. Nous avons identifié un marqueur des progéniteurs neuronaux, cnox2, orthologue du gène ParaHox nommé Gsx chez les deuterostomes et Ind chez la Drosophile. Ce gène est exprimé dans les cellules interstitielles apicales au moment où la néo-neurogénèse démarre. Nous avons montré que l’inhibition du gène cnox2 engendre une diminution du nombre de neurones apicaux ce qui corrèle avec l’efficacité de la régénération. Ce résultat a également été confirmé chez les mutants sf-1, dépourvu de neurones lorsqu’ils sont exposés à une température restrictive. Dans cette conditon, ces mutants n’expriment plus le gène cnox2 et sont déficients pour la régénération. Ces données suggèrent l’importance de la néo-neurogénèse pour le modelage d’une nouvelle tête.
Régénération versus bourgeonnement : Dans le chapitre 9 - Résultats nous avons voulu savoir si la reproduction asexuée observée au cours du bourgeonement et la régénération après amputation s’appuyaient sur des mécanismes similaires, voire identiques. Dans ce but nous avons comparé les patrons d’expression de huit gènes régulateurs différents qui ont été étudiés à différentes périodes de la régénération et du bourgeonnement, ainsi que les zones de prolifération. Les résultats suggèrent que l’initiation de ces deux processus repose sur des remodelages moléculaires et cellulaires vraisemblablement différents, alors que la progression observée aux phases semi-tardives et tardives est assez similaire sans être toutefois identique et implique des mécanismes communs.
Dans la dernière partie de ma thèse consacrée à la discussion, je compare les données présentées dans ce travail aux principaux modes de régénération animale et je montre comment ces nouveaux résultats modifient notre conception de la régénération de la tête chez l’hydre, c’est-à-dire comment ils remettent en question la vue classique de la régénération morphollactique. Du coup la régénération de la tête de l’hydre apparaît, dans sa
9 version « sauvage », comme une variante de la régénération épimorphique observée après amputation de la patte du triton ou de la nageoire du poisson-zèbre. Enfin je tente de dresser l’inventaire des différentes questions biologiques qui peuvent maintenant être abordées grâce au système modèle de l’hydre.
2. Summary
The main purpose of my PhD project was to characterize the cellular and molecular events that are underlying hydra head regeneration. As this is a rather ambitious endeavour that covers quite a large range of topics we focused our attention on four distinct questions concerning head-regeneration:
1) How hydra overcome the amputation stress?
2) How the organizer activity is established in the head-regenerative half?
3) What is the contribution of neurogenesis during head regeneration?
4) What are the differences between budding and regeneration?
The thesis is structured to follow closely these biological questions.
Hydra is an excellent model system for studying regeneration due to its small size, fast population growth, key phylogenetic position and simple cellular organization. Moreover, a wide range of functional tools recently became available, unlocking the access to refined molecular biology (see Prologue). The biological interest of the hydra model system such as the bilayered tissue organization, the cell lineages, the cell cycle particularities and the morphogenetic programs are detailed in the first part of the introduction (see Introduction, Chapter 4.1). In the second part, I present the key notions regarding regeneration in the animal kingdom as well as a “selected few” signalling pathways involved in regeneration (see Introduction, Chapter 4.2). The results are presented in 9 chapters, which correspond to published (7) and submitted (2) papers. These chapters cover four main themes:
A tight control of autophagy is required during hydra homeostasis and regeneration: In the first chapter of the Results section, we illustrate how silencing the Kazal1 gene, which encodes for a serine-protease inhibitor, affects hydra homeostasis and regeneration. We showed that upon prolonged silencing Kazal1(RNAi), animals are not able to survive and they die. The cellular analysis of these animals revealed a transient widespread autophagic event, taking place in the endodermal-epithelial cells of the regenerating half. This phenotype is similar to the one present in humans or mice that lost the related Kazal serine-protease inhibitors SPINK1 and Spink3, that suffer from chronic pancreatitis due to extensive autophagy. All these data suggest a key role for the hydra Kazal1 serine-protease inhibitor in surmounting the amputation stress in hydra. This is the first study in hydra that highlights the importance of cyto-protection, and it might suggest the requirement for a self-preservation program in the regeneration process per-se.
The initiation of regeneration: In hydra, within the first hours that follow bisection, the myoepithelial cells of the regenerating tip undergo dramatic transformations that lead to the establishment of a «de novo» head-organizer activity. In the Results section (Chapter 2, 3, 4) we will show that the CREB pathway, including the RSK kinase, the CREB transcription factor and the CBP co-activator, along with the canonical Wnt pathway play a key role in this process. In Chapter 2 we show that CREB phosphorylation is indeed required for head- regeneration. When we investigated the cellular and developmental function of the three members of this pathway during early regeneration, we first noticed in immunohistochemistry experiments that RSK, CREB and CBP were co-expressed in all cell types, including endodermal and ectodermal epithelial cells, interstitial stem cells, dividing nematoblasts and sensory neurons (see Chapter 3 - Results). We then describe in Chapter 4 - Results a massive, early but transient apoptosis that takes place in head-regenerating tips, as detected in immunocytological experiments tracing the RSK, CREB and CBP proteins. This apoptosis appears to be restricted to a single cell lineage, i.e. the interstitial cell lineage. We showed that in the first 45 minutes after bisection, these apoptotic cells over-express the Wnt protein.
Moreover, the region underneath the apoptotic stump is an active proliferating zone, as
10 revealed by the BrdU pulse labelling experiments. These BrdU positive cells are interstitial cell progenitors, which synchronously translocate beta-catenin into the nucleus after 90 minutes of regeneration. Interestingly, when the RSK, CREB, CBP genes were silenced upon RNA interference, apoptosis was no longer observed and all animals died or head-regeneration was significantly delayed. Also interestingly, when Wnt, CREB and beta-catenin are silenced, the Wnt over-production in apoptotic cells and beta-catenin translocation in cycling cells doesn’t take place in either one of the contexts.
These data suggest that apoptosis of the interstitial cell lineage is a prerequisite to the setting up of the head organizer activity developed by the myoepithelial cells and also for the organisation of the regenerative blastema. We will discuss how apoptosis likely modifies the cross-talk between those distinct cell lineages, i.e. myoepithelial versus interstitial, hence providing a context that is favourable for the reactivation of the developmental program.
The requirement for de novo neurogenesis during hydra head-regeneration: The head patterning during hydra head-regeneration precedes the formation of the tentacle rudiments and starts at around 16 hours post-amputation. In the Chapter 5 - Results we show that this period is marked by an intensive cell proliferation in the regeneration tip followed by the de novo differentiation of the new neurons. We identified a marker for the neuronal progenitors, the hydra Gsx/Ind homolog, cnox2. This gene is expressed in the apical interstitial cells at the moment when de novo neurogenesis begins. We showed that silencing the cnox2 gene leads to a decrease in the numbers of apical neurons that correlates with the efficiency of regeneration. This result was also confirmed in the temperature sensitive nerve free mutant sf- 1 all the interstitial cell lineage in which the cnox2 expression is abolished and regeneration is deficient. All these data suggest the importance of de-novo neurogenesis during head- patterning.
Regeneration versus budding: In Chapter 9 – Results we analyzed the similarities between the initiation and progression of regeneration and budding by coupling the expression patterns of eight different genes regulatory genes along with cellular studies. These suggested that the initiation of these two processes are likely different, whereas the latter phases are likely similar if not identical and thus rely on common mechanisms.
In the last section, I will discuss the way the data presented in this work modify our concepts about hydra head-regeneration, namely how they challenge the classical view of morphallactic regeneration. Finally I address several fundamental biological questions that can now be investigated with the help of the hydra model system.
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3. PROLOGUE
“ There and Back Ag ain ”
“ This book is largely concerned with Hydra and from its pages a reader may discover much of their character and little of their history” 1
As someone with a taste for statistics and basic “MicrosoftWord” skills could easily find out, the most used two words in this work are “hydra” (164x) and “regeneration” (205x). The reason for this recurrence is the obvious interest of the author in these two matters and especially in the link between them. Therefore I decided to start this work by a chapter that will present a concise list of arguments explaining the choice of hydra as a model system for regeneration. Further information will also be found in the next chapters of this introduction.
3.1 Regeneration – A Journey in the Dark
Almost all metazoan phyla include organisms that are able to undertake adult regeneration.
As it will be presented in this work, simple organisms like hydra or planarians are able to regenerate their main body axis, while more complex metazoans like fish or salamander regenerate multi-tissues structures such as limbs, jaws, tails, fins and hearts. This widespread distribution of the regenerative potential in the animal kingdom suggests that regeneration is an ancient feature of organisms that was either maintained or lost during evolution (Sanchez- Alvarado, 2000). Interestingly, in mammals, the regenerative potential decreases during development, the adults retaining only a limited capacity of tissue regeneration. It is probably safe to claim that the interest of the general public for regenerative research is based on the prospect of inducing regeneration in humans. From this perspective, the importance of deciphering the mechanisms of regeneration is rather obvious, considering its impact on biology and medicine. As the regeneration research is still in its infancy, the perspective of inducing mammalian regeneration lays far away in the future. In order to reach this objective, several key questions need to be answered: What are the cellular and molecular mechanisms of regeneration and especially what are the mechanisms that lead to the reactivation of the developmental program in a regenerative model system? What features are distinguishing the regenerative from the non-regenerative model systems? How could the developmental programs be reactivated in non-regenerative systems? Most studies are addressing these questions by dissecting the regenerative programs in animals with high potential of regeneration like hydra.
3.2 Hydra – The Shadow of the Past
The heterogeneity of the regenerative capacities among metazoans generates an unexpected paradox: most of the classical embryological/genetical systems such as Xenopus, chick, Drosophila, C. elegans or mouse are not really suitable for the study of regeneration, as they posses very reduced or no regenerative potential. Therefore, the regeneration field implemented “non-standard” model systems like newt, axolotl, planarians or hydra. In this work we make use of the hydra model system to study the putative firstly evolved cellular and molecular mechanisms of regeneration. Here we will present some of the most notable features of this model system and their impact on the regeneration research.
3.2.1 Hydra – the perfect “lab rat”
Due to its small size and quick population growth, Hydra is easy and inexpensive to cultivate in laboratory conditions. In appropriate feeding conditions a large number of animals can be
1 based on J.R.R Tolkien, The Fellowship of the Ring
12 generated in a very short time by a form of asexual reproduction, named budding. We took advantage of these features and designed a simple method for synchronizing the hydra population by detaching the mature buds from the parental polyp. This method proved to be extremely useful for the reliable statistical analysis of cellular and molecular mechanisms during regeneration, due to the consistency of the results (see Results, Chapter 4).
Is the hydra polyp immortal? This seems to be a ridiculous question but, in fact, the exact lifespan of the hydra polyp is unknown. The same polyps were followed in laboratory conditions during several years showing very little signs of aging or decrease in the budding activity (Martinez, 1998). Could it be that this “immortality” is the reason for hydra’s astonishing capacities of regeneration or, conversely, continuous regeneration makes hydra
“immortal”? This is a very interesting question that our laboratory tries to address by studying the hydra telomerase activity in homeostatic and regeneration conditions (not the subject of this work).
Moreover, during the past decades several hydra strains were isolated. For example, the AEP sexual strain allows the artificial induction of the sexual cycle of the polyp (Martin et al., 1997) and therefore offers the possibility of generating transgenic hydra, a method that was recently developed by the group of Thomas Bosch (Wittlieb et al., 2006). In addition, the regeneration deficient strain reg16 and the heat-shock inducible strains sf1 and A10 are extremely useful in studying the modulations in the regenerative process. Therefore, the low costs of generating large numbers of individuals in short time, the possibility of synchronising hydra cultures and the “immortal” state of the polyp makes hydra an ideal system to maintain in laboratory conditions.
3.2.2 Hydra – the key phylogenetic position
In addition to the maintenance advantages presented above, hydra has a basal phylogenetic position that enforced it as a key model for deciphering the ancestral role of the conserved regulatory genes involved in developmental processes of bilaterians. Consequently, dissecting its mechanisms of regeneration provides insights on the most ancient regenerative program available. Moreover, the quest for developmental regulatory genes revealed an astonishing level of conservation between these genes and their vertebrates’ homologues (Galliot, 2000;
Holstein et al., 2003). Thus, hydra allows us to either map the original regenerative function of the genes that were subsequently recruited for implementing other tasks in more evolved organisms or, conversely, to confirm the conserved function of a gene among the animal kingdom. In this work we will present several examples of gene functions, conserved along evolution from hydra to men (see Results, Chapter 5).
3.2.3 Hydra – the cell biology
Due to its small size and simple organisation, hydra offers the possibility of studying cellular processes at the entire organism level, and not only at the levels of organs and tissues. In addition, the tissue architecture and cellular biology of hydra was extensively studied in the last 50 years. The precise quantitative and qualitative characterisation of the cellular types and their tissue distribution is well known. The limited but still diverse number of cellular types makes hydra a great model for studying lineage tracing and interactions during regeneration.
In vitro culture of hydra cell is not possible this being considered a terrible limitation of the system. However, this inconvenience finally turned to be an advantage as it forced the field to develop the maceration method (David, 1973). This method, established more then 40 years ago, proved to be an incredible useful tool in the quantitative characterisation of the regenerative cellular events.
3.2.4 Hydra – the extreme developmental plasticity
In hydra, the developmental programs are never turned off and the polyp maintains a permanent growth state, behaving like a perpetual embryo. The homeostatic organisation is maintained by a well-controlled equilibrium between cell gain and cell loss. The tissues from the upper and lower body column are continuously transformed into head and foot tissues, whereas the tissues of the central part of the body column move towards the growing bud.
Hydra also displays a unique developmental plasticity among multicellular adult organisms,
13 being able to regenerate, bud and even reaggregate following dissociation. Moreover, the cell populations and their regulatory interactions involved in all these morphogenetic processes were intensively studied in the past decades, providing a solid cellular biology background for the future research. These studies suggest a key role for the epithelial cells during hydra morphogenetic processes, tightly tuned by the interstitial cell compartment (see Introduction).
3.2.5 Hydra – the molecular biology
Despite its apparent simplicity, almost all the main signalling pathways are already present in hydra. Since the first cloning of the homeobox genes (Schummer et al., 1992), many of the genes belonging to these pathways were cloned and showed to be involved in hydra developmental processes (for review see (Galliot, 2000; Holstein et al., 2003). Moreover, the function of some of these genes products was confirmed in heterologous contexts like achete scute and nanos in Drosophila, goosecoid, wnt and Dickkopf1/2/4 in Xenopus, Csk and STK in yeast, and COUP-TF in mammalian cells.
3.2.5.1 Hydra in the Genomic Era
In addition to the deliberate cloning of developmental significant genes, the EST-sequencing projects provided the sequence of many well-known regulatory genes that were further analysed at cellular and developmental level. Recently, a large-scale (www.hydrabase.org) and a small-scale (National Institute of Japan) Hydra EST projects were completed.
Furthermore, in late 2004, a Hydra genome project was initiated, the first draft of the genome being published in 2006.
3.2.5.2 Hydra - the Molecular Tool Set
The recent development of powerful molecular tools opened new avenues in the functional analysis of the hydra genes. The coupling of fluorescent gene expression patterns with antibody detection, dyes and cell cycle markers provided a more dynamic view of hydra gene regulations during homeostasis and development. Moreover, gain-of-function assays are possible in hydra by injecting GFP-reporter constructs in the eggs (Wittlieb et al., 2006) or electroporating intact animals and stumps (Lohmann and Bosch, 2000; Miljkovic et al., 2002).
Gene knockdown became available by electroporation of either anti-sense oligonucleotides or dsRNA. In addition to these strategies, our group developed a highly efficient and nontoxic method of silencing genes by feeding hydra with bacteria producing dsRNA. This method has the advantage of silencing progressively the gene of interest by repeating the feedings until the gene is completely knocked-down. Furthermore, several laboratories are implementing microarrays for large-scale studies in hydra, such as the screen for cell-type specific markers (http://hydra.lab.nig.ac.jp/hydra/)(Hwang et al., 2007).
3.3 The Structure of the Introduction
The first chapter of the introduction will address the basic cellular biology of hydra polyp with emphasis on the three different cell populations and their dynamic during morphogenetic processes. This knowledge is required for better understanding the key cellular events taking place during homeostasis, as well as budding and regeneration. In this chapter I will first discuss the morphology and function of the hydra cell lineages and their uncommon cell cycle.
The myo-epithelial cell lineages are responsible for shaping the bi-layered structure of the body column. In addition to their structural role, the endodermal myo-epithelial cell lineage is also involved in the digestion of food. Apart from these two types of myo-epithelial cells, all other cells – such as neurons, gland cells, gametes and mechano-receptor cells – derive from a third cell lineage, i.e. the interstitial cell lineage. Although this cell lineage is not contributing structurally to the animal shape, its role in fine-tuning the myo-epithelial cell lineages during homeostasis as well as morphogenetic events was demonstrated. The cell cycle of hydra cells is uncommon, being characterized by a very short G1 phase and a variable and unusually long G2 phase. This feature of hydra cell cycle allows a rapid cellular response following amputation. Moreover, almost all epithelial cells seem to proliferate in the hydra polyp in homeostatic conditions. This perpetual growth state implies the existence of constrains involved in the maintenance of polyp morphology. These mechanisms, which rely on anti- parallel morphogenetic gradients, will be briefly described in the second part of this chapter. In
14 addition to the active maintenance of the homeostatic state, hydra can undergo different morphogenetic programs, such as sexual cycle, budding, regeneration or even reaggregation.
These four morphogenetic programs will be succinctly described in Chapter 4.1, in order to provide a better view about the dynamic of the hydra polyp. In conclusion, in hydra, both homeostasis and development require massive cellular reorganisation that involves migration, tissue remodelling, cell proliferation and cell death.
In the second chapter of the introduction, the general cellular and molecular basis of regeneration will be briefly addressed. The concepts of epistatic and morphallactic regeneration are discussed, along with the classical views of hydra regeneration and its position in the general regenerative context. The epistatic regeneration consists of three phases: the wound healing phase, the blastema formation and the differentiation of the missing structure. The cellular and molecular basis of each of these phases and especially the blastema formation phase are of critical importance for the normal progression of regeneration and they are very much studied in the regeneration field. Interestingly, the classical view considers hydra regeneration as morphallactic, i.e. taking place in the absence of cell division and, as a consequence, without forming a blastema. This view is now challenged by the new data presented in the Results section of this work. The fundamental concepts of regeneration will be followed by a selection of several transduction pathways which are playing a key role during regeneration and which seem to be shared by the different regenerative model systems. Furthermore, in the Results section of this work I will describe recent advances that demonstrate through functional analyses of cell specific-genes how the cross-talk between different cell populations promotes cellular and developmental plasticity in hydra.
15
4. INTRODUCTION
4.1 The Hydra Model System
Hydra belongs to Cnidaria, a phylum that contains animals with radial symmetry exclusively found in aquatic environments. The fossil evidences of embryos and larvae place the cnidarian emergence in Ediacaran period (last period of Neoproteozoic Era, preceding the Cambrian Period of Paleozoic) around 610 millions years ago (Chen et al., 2002; Chen et al., 2000; Hofmann et al., 1983) (see Figure 1).
Figure 1. The Cnidaria position in the Animal Kingdom.
The Cnidaria phylum is divided in four distinct classes: Anthozoa (corals), Scyphozoa (jellyfish), Cubozoa (box jelly fish) and Hydrozoa (hydroids); all known since Ordovician (Robertson, 1985). Cnidarians can live either exclusively as polyps (Anthozoa) or they can alternate a polyp and medusa stage (Medusozoa). Among hydrozoans Hydra is a fresh-water animal, which lost the medusa stage and lives exclusively as polyp.
In the firs part of this chapter I shall focus on hydra cellular organization and more precisely on the three cell lineages and their interactions. Subsequently I shall briefly describe the classical views concerning the maintenance of the homeostatic state, as well as the main morphogenetic processes known in hydra.
4.1.1 Morphology and Cellular Biology of Hydra Polyps
The knowledge of hydra cellular architecture and dynamic in homeostatic conditions is extremely important for the study of regeneration. As it will be described in the Results Section of this work (Chapter 4 – Results section), during early regeneration the tissue organization is lost and the cells undergo morphological and functional modifications, which are essential for the normal progression of the process.
The typical cnidarian polyp is an apico-basal polarized tube, which encompasses a gastric cavity with a single opening surrounded by tentacles. At the opposite side cnidarians develop
16 a special structure named basal disc, which is used to attach to the surfaces. Traditionally, cnidarians are considered as diploblastic animals lacking the mesodermal structures.
Nevertheless, recent studies on medusa molecular markers and tissues organization are challenging this idea and suggest the tripoblastic origin of cnidarians (Seipel and Schmid, 2005, 2006) and even their bilateral symmetry (Finnerty et al., 2004)
The cellular architecture of the body wall consists in two distinct myo-epithelial cell layers, the endoderm and ectoderm, separated by an extracellular matrix named mesoglea. The endodermal and ectodermal myo-epithelial cells have different origins. In addition to these two cell populations, hydra presents a third cell line named the interstitial cell line (see Figure 2).
Figure 2.The tissue architecture of hydra polyp and the main cell types.
The different hydra cell types were characterized by using a specific maceration procedure which completely dissociates the tissue, but maintains intact the morphology of the cells (David and Campbell, 1972). In addition to the qualitative analysis, this technique provides quantitative results concerning the relative distribution of each cell type. A refined version of the maceration procedure was applied in this work for the characterization of the cellular events taking place in the regeneration stump in the first hours of hydra regeneration.
4.1.1.2 Epithelial Cells A. Morphology
Despite their common epithelial origin, the endodermal and ectodermal myo-epithelial cells represent two completely distinct cell lineages and fulfill different functions in hydra. The ectodermal myo-epithelial cells are shaping the outer layer of the polyp with a protective role.
They are big cuboidal or columnar cells (40-60 µm in length) with a large nucleus (12-14 µm) located either central or shifted to the basal side. Inside the nucleus there are one or two clearly shaped nucleoli. On the basal side (proximal to mesoglea) the ectodermal cells expand two muscle processes (David, 1973; Lentz, 1966)(see Figure 3A).
The typical body column morphology of the ectodermal cells is changing at the extremities of the polyp. In the apical part, the ectodermal epithelial cells of the tentacles incorporate 10 to 20 nematocytes and form the specialized battery cells used for attacking and paralyzing the prey. At the opposite basal end, the ectodermal cells are transformed into glandular epithelial cells filled with granular material that are shaping the basal disk.
The endodermal myo-epithelial cells are outlining the gastric cavity being involved in the digestion of the food in homeostatic conditions. They are elongated columnar cells (80-130
17 µm), with short muscle processes in the basal part (proximal to mesoglea) and flagella like structures in the apical part. The large spherical nucleus (12-14 µm) is located central and contains only one nucleolus. In well-fed animals the apical part of the endodermal cells is filled with granules and digestive vacuoles (see Figure 3B). The basal part (proximal to mesoglea) usually contains one large vacuole surrounded by a thin layer of cytoplasm. At the apical and basal extremities of the polyp the endodermal cells undergo slight morphological changes.
Figure 3.The morphology of the Hydra myo-epithelial cells : A) ectodermal cell; B) endodermal cell. Red - anti-tubulin; Blue - DAPI; bar 5 µm
B. Epithelial cells dynamics
As hydra tissue grows permanently, the polyp size and cellular framework is dynamically maintained by a well-regulated turnover of the cells. The steady state is preserved by at least two fine-tuned mechanisms. The first one involves the formation of a new hydra from the parental body using the excess of cells resulted by proliferation. This process accounts for most of the cell loss (Otto and Campbell, 1977) and represents the asexual form of hydra reproduction named budding that will be detailed later in this chapter. The second mechanism involves the passive displacement of the epithelial cells to the extremities of the polyp where they terminally differentiate. Due to the permanent displacement wave caused by the continuous proliferation, these cells will be sloughed at the apical or basal ends of the animal (Campbell, 1967a, b). Another consequence of this passive displacement is that epithelial cells need to continuously assess their relative position in the organism, indicating the presence of permanently active patterning mechanisms. In addition to these two size control mechanisms, apoptosis was shown to contribute in the regulation of the polyp cell number and distribution (Cikala et al., 1999).
The requirement for such a variety of size-maintaining mechanisms is easy to understand, considering that almost all epithelial cells in the body column are continuously proliferating (Campbell, 1965, 1967a). Indeed, the continuous [3H]thymidine labeling experiments have demonstrated that more than 90% of the epithelial cells in the body column of hydra are proliferating. Moreover, in the body column region (without head and foot) no localized growth zone was characterized, the epithelial cell proliferation taking place randomly at a rate comparable with the one of the polyp growth, which suggests a permanent state of self- renewal (David and Campbell, 1972). By contrast, the epithelial cells from the tentacles and foot region are arrested in the G2 phase of the cell cycle (Dubel et al., 1987). Therefore, in the body column of intact polyps, the stem cells are permanently dividing and their progeny is displaced to the basal and apical extremity where they will differentiate.
Surprisingly, besides proliferation, the epithelial stem cells of hydra are also able to perform several physiological functions such as food digestion (in the endoderm) or protection by maintaining the osmoregulation (in the ectoderm). This is a totally uncommon situation as, in higher metazoans, the epithelial stem cell function is restricted to proliferation. The fact that in hydra the same epithelial cells are simultaneously capable of proliferation and physiological functions (like protection and digestion) suggests that stem cells were initially able to perform both functions. This dual capacity was lost later in favor of separated abilities between stem and differentiated cells (Bode, 2003).
In addition to the classical [3H]thymidine labeling, other methods for studying cell cycle are available in hydra, such as BrdU labeling (Dubel et al., 1987; Holstein et al., 1991; Miljkovic- Licina et al., 2007; Schaller et al., 1989). Interestingly, the BrdU labeling experiments proved
18 that although the proliferating cells are randomly distributed along the body column, the distribution of S phase epithelial cells is not uniform alongside the animal (Holstein et al., 1991). This apparently contradicting result is explained by the different experimental design used in the [3H]thymidine and BrdU assays. The continuous [3H]thymidine experiments were done by repeated injection of radioactive thymidine at intervals of 8 hours (shorter than the S phase of the epithelial cells) for a total of 5 days. At the end the labeled population contained all the cycling epithelial cells, no matter of their cell cycle phase. By contrast, the BrdU staining was done only for one hour followed by immediate fixation and, as such, labeling only the S-phase cells. The BrdU experiment showed that the head and body column have a similar BrdU labeling index, in contrast with the peduncle zone in which the index is twice lower (Holstein et al., 1991). The variation in the distributions of S phase cells along the body column was recently confirmed by flow-cytometry studies (Ulrich and Tarnok, 2005).
C. The epithelial cell cycle
Epithelial cells have a cell cycle period, which can vary between 40 and 85 hours. The duration of each phase of the cell cycle was estimated by [3H]thymidine experiments and flow- cytometry (Ulrich and Tarnok, 2005). The G1 phase is unusually short or almost absent, followed by a 12-15 hours S phase. The G2 is atypically long and variable, ranging from 24 hours to as long as 72 hours. This is uncommon for animal cells, which usually have a fix and short G2. It was assumed that in hydra the G2 period replaced the G1 period (David and Campbell, 1972), this switch allowing a prompt response to morphogenetic signals by rapid increase in proliferation. The mitotic period is 1.5 hours (David and Campbell, 1972). Due to this very long cell cycle, the epithelial cells are protected against short exposure to cell cycle inhibitors or heat shocks. This property is extensively used in the hydra field for generation of conditional mutants named “epithelial hydra” which will be described later in this chapter and which are extremely useful in the study of regeneration.
4.1.1.3 Interstitial Cells
The interstitial cell lineage is not forming a layer by itself but instead the cells are scattered among the epithelial cells. The interstitial cells are multipotent stem cells, which can generate both somatic cells and male/female gametes. The somatic derivatives are the nerve cells, nematocytes (mechano-receptor cells) and secretory cells (see Figure 4).
Figure 4.The interstitial cell lineage in hydra.
19 Their progenitors all undergo several cell divisions before they become differentiated into the mature cellular type. This interstitial cell differentiation pathway is typical for hydrozoans but surprisingly not for most cnidarian species, where nerve cells and nematocytes appear to derive from epithelial stem cells (Holstein and David, 1990). The interstitial cells and their derivatives comprise for about 80% of the total cells of the polyp.
A. Morphology
Interstitial cells
Interstitial cells are preferentially distributed in the ectodermal layer where they form clusters.
Based on size and morphology, they can be subdivided in two distinct populations: the large interstitial cells and the small interstitial cells.
The large interstitial cells (12-20µm) have a lemon-shape and a medium sized nucleus (8-12 µm). They are usually found in pairs due to the presence of cytoplasmic bridges. The small interstitial cells are spherical with a size of 7-12 µm and a small-condensed nucleus (4-7 µm).
They are usually forming large nests (see Figure 5A).
Figure 5.The morphology of the hydra interstitial cells and their derivatives. Red is anti-tubulin, blue is DAPI; bar 5 µm
Nematoblast and Nematocytes
Nematoblasts and nematocytes terms designate two distinct stages of the same pathway.
Nematoblasts are differentiating mechano-receptor cells, progenitors of nematocytes. They are small spherical cells, located in the ectoderm, which through successive synchronous cell divisions, assemble in a syncitial aggregate of 8 to 16 cells (see Figure 5B). These cells will start to differentiate a capsule and form the mature nematocytes (Rich and Tardent, 1969).
Nematocytes are classified in four different classes: stenoteles, desmonemes, holotricohus and atrichous isorhiza according to the type of capsule they posses. The capsule choice is influenced by the position along the body axis where they matured (Fujisawa et al., 1986).
Mature nematocytes have a small-condensed nucleus and the specific nematocyst capsule (see Figure 5C). The capsule contains anions and toxins at a very high osmotic pressure which are released when a special structure named cnidocil is stimulated (Lindgens et al., 2004). Thanks to these two structures, cnidocil and nematocyst, nematocytes are receptor- effecter units which function autonomously as the release of the capsule is not regulated by the nervous system (Aerne et al., 1991) despite having synaptic connections to nerve cells (Westfall, 1996).
Nerve cells
Although cnidarians together with Ctenophora (combjellies) are the first in the animal kingdom to develop a nervous system, they already possess several distinct classes of nerve cells.
Morphologically, at least two nerve cell types could be distinguished: ganglia cells and sensory cells (see Figure 5D). Ganglia cells have a small cell body that initiates two or more branched cytoplasmic processes. The nucleus is small, with condensed granular chromatin surrounded by granular cytoplasm. Sensory cells have an elongated ovoidal cell body with a long single cytoplasmic process at one end and with a globular cytoplasmic extension at the other end. The nucleus resembles with the one of the ganglia cells.
20 Gland cells (Secretory cells)
The precursor of the gland cells is probably a large interstitial cell located in the ectoderm, which migrates into the endoderm during differentiation (Bode et al., 1987; Smid and Tardent, 1984). Gland cells produce digestive enzymes and together with the neighboring epithelial cells are involved in the digestion of the food. They are oval cells (25-35 µm) with an eccentric nucleus and cytoplasm filled with large vacuoles and granules. Similar to endodermal epithelial cells there are 2 flagella at the apical end. Gland cell morphology show morphological changes connected with their position along the body axis (David, 1973).
B. Interstitial cells dynamics
Although interstitial stem cells are practically immobile, grafting experiments showed that the interstitial progenitor cells display rapid motility in wounded polyps (Fujisawa et al., 1990;
Khalturin et al., 2007). Migration of interstitial cells was also demonstrated in intact polyps although at slower rates (Teragawa and Bode, 1995). These cells are progenitors of gland cells, mechanoreceptor cells or neurons. Mature nerve cells are not distributed randomly along the body column of the polyp. They are populating preferentially certain regions like the basal disk or the head, where they display six fold more abundant. This pattern is a direct consequence of the migration of the neuronal precursors from the gastric region to the extremities of the animal where they get differentiated (David and Hager, 1994). Beside the active migration, there is also a passive displacement of the differentiated cells to the extremities of the polyp.
The interstitial and epithelial cell lineages dynamically interact and, as a consequence of this crosstalk, the differentiation patterns and migration rates of interstitial cells are regulated (Minobe et al., 1995). In addition, it was shown that epithelial cells could indirectly influence the decision of the interstitial cells to enter the differentiation pathway in response to the amount of nutrients (Hoffmann and Kroiher, 2001).
C. The interstitial cell cycle
The outcome of interstitial cells multipotency and various differentiation programs is the wide range of different stage progenitors and un-differentiated cells scattered along the body column. By this date, there are very few markers available to distinguish between the different interstitial cell progenitors. However, a detailed analysis of several key markers involved in the differentiation of the neuronal progenitors will be presented in Chapters 5 and 6 of the Results Section. The descriptive cellular morphology still remains the main method of distinguishing between these sub-populations. Cell cycle shows also distinct patterns associated with specific morphological features. For example, it has been demonstrated that the small interstitial cells are cycling faster than the big interstitial cells, having a homeostatic cell cycle of 16 hours (Campbell and David, 1974).
Similar to the epithelial cells, the interstitial cells seem to also have a very short G1 phase, although the accuracy of the measurement was not the same. The S phase was calculated to range from 9 to 13 hours for nests of one, two and four cells, with a mean of 11 hours. The G2 phase for the four, eight and sixteen cell nests is around 3-4 hours and very constant, as oppose to the G2 phase of single and paired cell nests which seems to be very variable and considerably much longer than their S phase. The [3H]thymidine experiments suggested the presence of at least two different subpopulations of paired cells, one with G2 around 6 ± 2 hours and another with G2 around 14 ± 8 hours. Single interstitial cells also showed a huge variability of G2 phase. The M phase was estimated at one hour for all types of interstitial cells. The total cell cycle length is varying from 16 hours in nests of four and sixteen interstitial cells to 27 ± 8 hours in pairs and single interstitial cells, all these values being calculated in homeostatic conditions (Campbell and David, 1974).
4.1.1.4 General conclusions about hydra cell cycle
There are three main results, which can be concluded from the above experiments:
The first one is that all hydra cells in homeostatic conditions are characterized by a very short G1 phase and a long and variable G2 phase (see Figure 6). As described earlier, this characteristic might be necessary for a rapid cellular feedback in response to stress. Indeed, the plasticity of hydra cell cycle was confirmed by recent results obtained by our group, in
21 which interstitial cells are undergoing a very fast initial cell cycle during first hours of head- regeneration (see Chapter 4 of the Results section).
The second result came as a confirmation of the morphological heterogeneity of the interstitial cells by suggesting the existence of at least two classes of interstitial cells with different cell cycle lengths. It is still not known whether these two classes represent distinct interstitial stem cells populations or distinct phases during the differentiation of a single cell population.
The third and probably the most striking result is that nearly all hydra cells are proliferating.
The main consequence of this endless proliferation is the continuous growth state of the animal. How the hydra polyps are able to maintain their size and morphology, what are the putative mechanisms involved in this regulation and how these mechanisms influence their regeneration potential are very important questions for this work and they will be extensively discussed during the following chapters.
Figure 6.Comparative schemes of the epithelial (left) and the interstitial (right) cell cycle. The schemes are depicting the proportional length in time of each cell cycle phase.
4.1.2 Homeostasis and Development
In the second part of this chapter, I will illustrate the amazing developmental plasticity of hydra polyps by briefly presenting several morphogenetic programs in hydra. The following pages are not intended to be a complete analysis of these processes, but rather an overview of hydra developmental potential. A deep conceptual and molecular analysis of hydra regeneration and its position in the regenerative field will be provided in the next chapter of this introduction (see Chapter 4.2).
4.1.2.1 Two pairs of opposed gradients maintain the homeostatic dynamic equilibrium
A stringently regulated dynamic equilibrium between cell gain and cell loss is responsible for the maintenance of the homeostatic state in hydra. The new cells produced by cell proliferation are differentiating either during the active migration (neuronal progenitors and nematocytes) or the passive displacement (epithelial cells) to the apical and basal poles of the polyp where, for a short period, they form the differentiated structures. Subsequently, these cells are displaced by the continuous influx of younger cells, which are reaching the extremities and then sloughed off from the polyp. This dynamic growth state of hydra requires the existence of stable patterning mechanisms, needed to maintain hydra morphology. It was stipulated that at the core of hydra patterning mechanisms are the morphogenetic gradients.
The first clues about hydra morphogenetic gradients came from transplantation experiments.
At the beginning of the last century, Elena Brown showed, 15 years prior to Spemann’s experiments, that head or basal disc tissues are able to act as an organizer, their transplantation into a host polyp inducing the formation of the according secondary axis (Browne, 1909). Subsequent transplantation studies proved the existence of two pairs of gradients: the head-activation/head inhibition pair and the foot-activation/foot inhibition pair,
22 with an anti-parallel orientation along the body axis of the animal (Wolpert et al., 1972). For example, the competence of head inhibition varies in a graded manner along the body column. The transplantation of an apical region into a host at three different levels showed an increase in the induction of the secondary axis that was directly proportional with the distance from the head (see Figure 7A). In addition, if an upper body column explant is introduced into the middle of the body column of an intact polyp host, this will generate a secondary axis with a frequency of 20%. If the explant is introduced in a similar position in decapitated animals, the frequency of head induction will increase to 87% (MacWilliams, 1983b; Webster, 1971)(see Figure 8). These experiments prove the presence of a head inhibitory gradient. As previously shown, the head tissue is the best to induce secondary axis when transplanted.
This capacity is also retained in the upper body column tissue but the frequency of head induction drops proportionally according to the distance from the head (MacWilliams, 1983a).
This result suggested the existence of a head-activation gradient (see Figure 7B).
Figure 7. Schematic representation of experiments illustrating the gradients of head inhibition (A) and head activation (B) (after (Bode and Bode, 1984)).
Figure 8. Inhibition of head formation by the host head. A) Transplantation of the graft into an intact polyp; B)Transplantation of the graft into a decapitated polyp (after (Bode and Bode, 1984)).
