Hematopoiesis in the adult organism is maintained by a complex, hierarchically organized cascade of differentiating cells that ultimately originate from adult hematopoietic stem cells (HSCs). HSCs have been extensively studied in perturbative settings (e.g transplantation assays), which are well suited for exploring cell potential but tell very little about cell behaviour in a physiological state. Novel \textit{in situ} techniques have made it possible to observe hematopoiesis in its natural environment, yet many questions about native HSC behaviour remain controversial. It is yet unclear whether and to what extent HSCs contribute to steady-state blood production, and whether they represent a reserve population that can be activated upon emergency. As, to date, common definitions of HSCs comprise cells of heterogeneous function, incoherent results are likely linked by another fundamental question: what is the identity of the HSC population residing on the apex of the hematopoietic hierarchy? To answer these questions is essential to gain a deeper understanding of fatal human diseases like leukemia or aplastic anemia. We describe an integrative approach combining non-invasive experimental methods with mathematical inference to uncover underlying pathways and differentiation patterns of steady state hematopoiesis. By this, we identify a population of Sca-1\textsuperscript{hi} CD201(EPCR)\textsuperscript{hi} HSCs as the hithertho elusive apex population, and show that they contribute marginally, but continuously, to native steady-state hematopoiesis. Further, we clarify the architecture of a shortcut route of thrombopoiesis, which emanates directly from HSCs and links them trough a previously undefined CD48\textsuperscript{-/low} megakaryocyte progenitor population directly to platelets. Finally, we provide an extensive analysis of the dynamic label propagation and proliferation behaviour of hematopoietic stem and progenitor cells in prototypical stress situations mimicking blood loss, infection and inflammation. We find that apex stem cells do not directly contribute to emergency hematopoiesis and are only activated upon severe myeloablation and chronic inflammation. Further, innate immune training did not influence HSC contribution in response to reoccurring stimulation. In sum, we demonstrate that primitive HSCs do neither represent a major contributor to steady-state blood production, nor a reservoir for emergency supply. We thus question current dogma of hematopoiesis, and argue that the primary function of HSCs in the adult organism remains yet to be discovered.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190 / Die Blutbildung in erwachsenen Organismen wird durch eine komplexe, hierarchisch organisierte Kaskade von sich fortwährend differenzierenden Zellen aufrechterhalten, und hat ihren Ursprung in adulten h\'amatopoietischen Stammzellen (HSZs). HSZs wurden ausgiebig unter unphysiologischen Extrembedingungen (z.B. Transplantation) untersucht, in welchen sich viel über das maximale potential einer Zelle, aber wenig über ihr Verhalten in ungestörtem Zustand sagen lässt. Obwohl durch moderne \textit-Techniken mittlerweile h\'amatopoietische Differenzierung in ihrer ursprünglichen Umgebung beobachtet werden kann, bleiben etliche Fragen zum nativen Verhalten von HSZs offen. So ist es noch unklar, ob und in welchem Ausmaß HSZs überhaupt zur stationären Blutbildung beitragen, oder ob sie eine Reservepopulation darstellen, die im Notfall aktiviert werden kann. Da bis dato gebr\'auchliche Definitionen von HSZs verschiedene Sub-Populationen mit heterogener Funktionalität umfassen, sind die inkohärenten Ergebnisse wahrscheinlich mit einer anderen grundlegenden Frage verknüpft: Was ist die wahre Identit\'at der HSZ-Population, die an der Spitze der h\'amatopoetischen Hierarchie steht? Die Beantwortung dieser Fragen ist essenziell für ein besseres Verst\'andnis von fatalen humanen Krankheiten, wie beispielsweise Leuk\'amie oder aplastische An\'amie. Wir beschreiben einen integrativen Ansatz, der nicht-invasive experimentelle Methoden mit mathematischer Modellierung vereint, um die zugrundeliegenden Pfade und Differenzierungsmuster der station\'arer H\'amatopoese zu ergründen. Auf diese Weise können wir eine Population von Sca-1\textsuperscript{hi} und CD201(EPCR)\textsuperscript{hi} HSZs als die bisher unbekannte Apex-Population identifizieren, und zeigen, dass diese Zellen geringf\'ugig, aber kontinuierlich, zur nativen H\'amatopoese beitragen. Darüber hinaus klären wir die Architektur eines direkten thrombozytischen Differenzierungspfades, welcher unmittelbar von HSZs abzweigt und diese über eine neu definierte CD48\textsuperscript{-/lo} Megakaryozyten-Vorläuferpopulation direkt mit Thrombozyten ver- bindet. Schließlich liefern wir eine umfassende Analyse des dynamischen Differenzierungs- und Proliferationsverhaltens von hämatopoetischen Stamm- und Vorläuferzellen in prototypischen Stresssituationen, die Blutverlust, Infektion und Entzündung nachahmen. Wir zeigen, dass primitive Stammzellen nicht direkt zur Notfall-Hämatopoese beitragen und nur bei schwerer Myeloablation und chronischer Entzündung aktiviert werden. Ebenfalls hat das Training des angeborenen Immunsystems keinen Einfluss auf die Aktivität von HSZs bei wiederholter Stimulation. Unsere Ergebnisse belegen, dass primitive HSZs weder einen wesentlichen Beitrag zur stationären Blutbildung leisten, noch ein Reservoir für die Notfallversorgung darstellen. Wir stellen somit das derzeitige Dogma der Hämatopoese in Frage und argumentieren, dass die primäre Funktion der HSZ im erwachsenen Organismus noch zu entdecken ist.:Contents Abstract iv Zusammenfassung vii List of Figures xii List of Tables xiii List of Abbreviations xv 1 Introduction 1 1.1 Hematopoiesis and the hematopoietic system 1 1.1.1 Developmental origins 1 1.1.2 The hematopoietic hierarchy 3 1.2 Hematopoietic stem cells 5 1.2.1 Characteristics of hematopoietic stem cells 7 1.2.2 Identification of hematopoietic stem and progenitor cells 7 1.2.3 HSC heterogeneity 11 1.3 Novel genetic tools to analyse native hematopoiesis 12 1.3.1 Conditional gene targeting 12 1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15 1.4.1 Models of native Hematopoiesis 16 1.4.2 Models of HSC quiescence 23 1.4.3 Aged hematopoiesis 25 1.5 Hematopoietic stem cell behaviour in stress response 26 1.5.1 Inflammation and Infection 27 1.5.2 Myeloablation 30 1.5.3 Innate immune training 32 2 Aim of Thesis 35 3 Material and Methods 37 3.1 Materials 37 3.1.1 Antibodies 37 3.1.2 Buffer and Solutions 38 3.1.3 Chemicals and Reagents 39 3.1.4 Cultivation Media 40 3.1.5 Kit Systems 41 3.1.6 Software 41 3.2 Mice 42 3.2.1 Animal housing and husbandry 42 3.2.2 Mouse strains 42 3.3 Genotyping of mice 43 3.4 Treatments of mice 46 3.4.1 Reporter induction 46 3.4.2 Hematopoietic stress induction 46 3.5 Hemograms 48 3.6 Transplantation and in vivo repopulation assays 49 3.6.1 Preconditioning and transplantation of recipients 49 3.6.2 Competitive repopulation assay 50 3.6.3 Limiting dilution assay 50 3.6.4 Transplantation into sublethally irradiated hosts 51 3.7 Cell preparation for transplantation and flow cytometry 51 3.7.1 Bone marrow 51 3.7.2 Splenocytes 51 3.7.3 Peripheral blood 51 3.7.4 Peritoneal cells 52 3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52 3.8 Flow Cytometry 53 3.8.1 Cell staining 54 3.8.2 Cell counting 55 3.8.3 Cell identification and gating 55 3.8.4 Chimerism analysis 57 3.8.5 Cell sorting 58 3.9 Cytokine detection assay 58 3.10 in vitro single cell expansion assay 59 3.11 Sequencing 61 3.11.1 Single cell RNA sequencing 61 3.11.2 Bulk RNA sequencing 62 3.11.3 Single cell and bulk transcriptome analysis 62 3.12 Mathematical modelling 62 3.13 Data Normalization 63 3.14 Statistical Analysis 64 4 Results 67 4.1 Identification of an apex HSC population 67 4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68 4.1.2 ES HSCs reside at the top of the hierarchy 71 4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76 4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77 4.2.2 The myeloid lineage diverges within phenotypic HSCs 80 4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82 4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82 4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88 4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92 4.4 Ageing modulates the dynamics of fate mapping 95 4.5 HSCs in times of crisis: Contribution to stress recovery 99 4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100 4.5.2 Severe myeloablation provokes HSC activation 105 4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107 4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113 4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118 4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120 5 Discussion 123 5.1 Labels matter: What is a true HSC? 123 5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124 5.1.2 ES HSCs reside at the top of the hierarchy 126 5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127 5.1.4 The myeloid lineage diverges within phenotypic HSCs 130 5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132 5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132 5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135 5.3 Fundamental dynamics of label propagation are preserved in aged mice 136 5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138 5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139 5.4.2 Severe myeloablation provokes HSC activation 143 5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145 5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149 5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150 6 Conclusion 153 References 155 Acknowledgements 185 Appendices 187 Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189 Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that targets joints, resulting in in permanent disability. Synovial macrophages have been implicated in the pathogenesis of RA; however, their exact origins and functions remains unclear. In this study, we show evidence that synovial macrophages are mostly derived from embryonic origin during normal development. Macrophages are derived from either hematopoietic stem cells (HSC) or erythro-myeloid progenitors (EMP), and it is postulated that different subpopulations of synovial macrophages may have distinct functions contributing to either homeostasis or inflammation. To investigate the phenotypes of synovial macrophage populations and characterize their lineage-specific functions in arthritic joints, we utilized both cell lineage-tracing and K/BxN serum-transfer arthritis mouse models. Utilizing Flt3Cre;Rosa26LSL-YFP mice to label HSC-derived cells, we demonstrated that there is minimal HSC contribution to synovial macrophage populations during homeostasis. Use of RankCre;Rosa26LSL-YFP and Cx3cr1CreERT2;Rosa26LSL-tdTomato mice to label EMP-derived cells corroborated the finding that the EMP compartment maintains the largest contribution to synovial macrophage populations during normal development. Analysis of macrophages in Csf1rMericreMer;Rosa26-LSLtdTomato mice provided definitive prove that synovial macrophages derived from yolk-sac EMP precursors in adult mice. Use of serum transfer arthritis (STA) mice demonstrated that while most macrophages in the inflamed synovium were EMP-derived, there was a marked increase in HSC-derived cells compared to those present in homeostasis. Although this study has contributed to eluding that the heterogeneity of synovial macrophages in both homeostasis and inflammatory arthritis (IA) is complex and lineage-specific, further studies are needed to clearly define lineage-specific functions of macrophages in synovial tissues and in IA.

La peau est un organe très exposé à l’environnement et fournit la première ligne de défense contre de nombreux pathogènes. Cette fonction est remplie dans l’épiderme murin par les cellules de Langerhans (LCs) et les cellules T dendritiques de l’épiderme (DETCs). Alors que le développement de ces cellules a bien été étudié, peu d’expériences ont été effectuées sur leur renouvellement en condition homéostatique chez des animaux adultes sans manipulations. Nous avons alors développé un système de traçage cellulaire par fluorescence multicolore pour étudier l’homéostasie des LCs et des DETCs. Cette approche de «fate mapping» m’a permis de mettre en évidence un modèle dans lequel le réseau adulte des LCs est formé d’unités prolifératives adjacentes composées de LCs en division et leurs cellules filles. Nous avons identifié que les cellules en division étaient majoritairement représentées par la fraction la plus immature des LCs, suggérant que ces LCs peuvent régénérer leur réseau grâce à une capacité de prolifération limitée. Lors d’une inflammation importante, les LCs sont renouvelées par des progéniteurs issus de la moelle osseuse et s’organisent également en unités de prolifération. Je me suis ensuite intéressé à l’homéostasie des DETCs. Ce réseau est formé de la même manière par des unités prolifératives de DETCs. Un modèle de greffe de peau nous a permis de montrer que les DETCs semblent renouveler les cellules disparues dans une zone restreinte. En conclusion, mes travaux de thèse ont permis de révéler les dynamiques cellulaires qui régissent l’homéostasie des cellules immunitaires de l’épiderme. / The skin is an organ very much exposed to the environment and supplies the primary line of defence against several pathogens. In the mouse model epidermis, this function is fulfilled by Langerhans’ cells (LCs) and dendritic T cells (DETCs). While LCs and DETCs development have thoroughly been studied, few experiences have been carried out concerning the renewal of these cells through homeostatic conditions in adult “nonmanipulated” animals. Then we have designed a new system of fate mapping, by way of multi-coloured fluorescence to study the LCs and DETCs homeostasis. This method of fate mapping allowed me to highlight a model in which the adult network of LCs is made up of adjacent proliferating units, made of dividing LCs and of their daughter cells. We have identified that the dividing cells were mainly represented by the most immature fraction of LCs, suggesting that these LCs can renew their network thanks to a limited ability to proliferate. During significant inflammation, LCs are renewed by progenitors coming from the bone marrow and organize themselves in proliferation units as well. I also took an interest in the homeostasis of DETCs. In the same way as for the LCs, this network seems to be made up of DETCs proliferating units. A model of skin graft led us to show that the DETCs seem to renew the missing cells in a restricted area. As a conclusion, my research work allowed me to reveal the cellular dynamism which governs the homeostasis of the epidermis’ immune cells.

Les Cellules Folliculaires Dendritiques (FDC) régulent l'homéostasie des lymphocytes B et sont indispensables à la mise en place des réponses immunes humorales. Les FDC s'organisent, au sein des organes lymphoïdes secondaires, en réseaux tridimensionnels denses, nécessaires à leur fonctionnement. Les études s'intéressant aux FDCs, empruntent classiquement des approches in vitro ou ex vivo, peu adaptées à la nature de ce type cellulaire. Au cours de mon travail de thèse, nous avons utilisé plusieurs systèmes de 'multicolor fate mapping' dans le but de déchiffrer in situ les mécanismes à l'origine du développement initial, et du remodelage des réseaux de FDCs en contexte inflammatoire. Nous avons démontré que les FDCs provenaient de la prolifération clonale et de la différentiation des Cellules Marginales Réticulaires (MRC), un autre sous-type cellulaire stromal résidant près des sinus sous-capsulaires ganglionnaires, et dont les fonctions étaient à ce jour, inconnues. Lors des réponses immunes, nous avons prouvé que les FDCs nouvellement formées, ne dérivaient ni du recrutement de progéniteurs circulants ni de la prolifération de FDCs matures, mais plutôt de la prolifération clonale des MRCs, suivie de leur différentiation en FDCs. Au-delà de l'étude de la biologie des FDCs, notre travail a révélé une fonction importante des MRCs dans le soutien de la plasticité des réseaux de FDCs. / Follicular Dendritic Cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods may thus be of little interest to understand the genuine immunobiology of FDCs in their native habitat. Herein, we utilised various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of Marginal Reticular Cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. In contrast, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

Taste buds are chemosensory endorgans embedded in the oral epithelium composed of cells that undergo continuous replacement. Mature taste cells live on average 10-14 days and are replaced by new cells when they die. However, the mechanism by which taste cells are produced and integrated into the taste bud as mature taste cells remains unknown. Previous studies approached this issue from either cell cycle gene expression properties or lineage tracing of precursor cells. In our study, we apply a new fate mapping technique that combines these two ideas. This technique, Mosaic Analysis with Double Markers, allows for simultaneous gene knockout and subsequent tracking of single cells. This allows us to study the potency of precursor cells supplying the taste bud while analyzing how gene function regulates the maturation pathway these taste cells take. The following experiments illustrate the initial phase of this investigation.