Cell turnover is a common feature of many organs in all animals and is required to maintain organ structure and function. It is achieved by a tightly regulated balance between cell death and cell division, which can be re-adjusted in response to injury and nutrient availability. How the balance between dying and dividing cells is coordinated has however remained unclear. Planarians represent an important model for studying cell turnover in adult animals, because all tissues undergo continuous cell turnover and a single stem cell type – the neoblast – is the exclusive source of all new cells. Moreover, planarians change their body size proportionally and reversibly depending on the nutritional status: feeding induces rapid and transient neoblast proliferation that results in animal growth, while starvation increases the rate of cell death, leading to de-growth. Importantly, also during starvation neoblasts keep proliferating at a basal-level. The hypothesis I addressed with my thesis research is that planarian energy metabolism might be a central mediator of cell turnover, particularly proliferation control and growth. I approached this hypothesis at several levels, including the characterization of the planarian energy metabolism and energy stores, the dependency of proliferation on the diet, and genetic requirements of proliferation control during starvation and feeding.
I found that planarians have orthologs of key enzymes of most animal metabolic pathways, but, surprisingly, seem to lack fatty acid synthase. This suggests that planarians are likely not only auxotrophic for cholesterol, but also for fatty acids. I described that planarians store energy as triacylglycerols (TAGs, stored in lipid droplets) and glycogen, with the intestine as the main storage organ. Interestingly, the amount of TAGs and glycogen changes with size and is higher for larger animals, suggesting a regulatory interplay with the known size-dependency of growth/degrowth rates. Further, we demonstrated that the energy stores are the physiological basis of Kleiber’s law that describes the near-universal scaling between metabolic rate and body mass. I further showed that proliferation occurs in three different modes, one during starvation when proliferation is maintained at basal levels and two after feeding, an initial proliferation mode (at three hours after feeding), which is diet independent and a later proliferation (at 24 hours after feeding), which is diet dependent. The two feeding-induced proliferation modes differ not only in their diet-dependencies, but also in their gene expression profiles, as assessed by RNA-sequencing. To identify genes involved in proliferation regulation, I assessed the requirements of different candidate genes in all three proliferation modes in a small-scale RNA interference screen. This screen revealed that insulin signaling, TORC1 and FGFR are involved in regulating basal proliferation during starvation and – most interestingly –that AMP-activated protein kinase (AMPK)-depleted animals showed increased proliferation during starvation at levels characteristic of recently fed animals. This result uncovered AMPK as a modulator that adjusts the neoblast proliferative activity to the nutritional state, potentially independently of TOR.
In sum, my work shows how energy metabolism and storage are coordinated with proliferation and growth in planarians and identified AMPK as a central modulator that adjust proliferation to cellular energy states. I discuss potential mechanisms by which AMPK modulates proliferation and putative links between AMPK and cell death, the second process of cell turnover. The energy state as the central mediator of cell turnover and the key players and mechanisms that my work revealed in planarians might also apply across different species:Chapter 1
1. Introduction 1
1.1 Cell turnover is a crucial process for tissue homeostasis 1
1.2 Cell division 2
1.2.1 Control mechanisms of cell division 2
1.2.1.1 Cell cycle machinery 2
1.2.1.2 Organization of the cell cycle control system – cell-cycle intrinsic regulation by Cdk-cyclin complexes 3
1.2.1.3 External control of cell cycle progression 4
1.2.1.4 Metabolic control of cell cycle progression 6
1.2.2 Metabolic requirements of proliferating cells 10
1.2.2.1 The energy stores 11
1.3 Cell death 13
1.4 Suggested mechanisms that coordinate cell death and division and their caveats 14
1.5 Planarians as a model to study cell turnover 16
1.6 Planarian body anatomy 18
1.7 Planarian stem cell system 19
1.7.1 Neoblasts form a heterogeneous population 19
1.7.2 Neoblast proliferative activity 21
1.7.3 Neoblast cell cycle machinery 22
1.7.4 Regulation of neoblast proliferative activity 22
1.8 Cell death in planarians 23
1.9 Mechanisms that coordinate the rate of dividing and dying cells in planarians still remain elusive 24
1.10 Scope of the thesis 24
Chapter 2
2. Planarian energy metabolism and the regulation of planarian growth dynamics 26
2.1 Introduction 26
2.2 Part 1: Planarian energy metabolism 27
2.2.1 The metabolic machinery of S. mediterranea 27
2.2.2 Planarian energy stores 30
2.2.2.1 Visualization of lipid and glycogen storage compartments in planarians 30
2.2.2.2 Investigation of feeding-dependent changes in lipid and glycogen stores 31
2.3 Part 2: Role of planarian organismal energy stores in regulating their growth and degrowth dynamics 36
2.3.1 Background information about known aspects of growth and degrowth dynamics in planarians 36
2.3.1.1 Growth and degrowth arise mainly from changes in cell number 36
2.3.1.2 Growth and degrowth rates are size dependent 37
2.3.2 Energy stores increase disproportionately with size and strongly contribute to the size-dependent dry mass increase 38
2.3.3 Metabolic rate and energy intake are unlikely causes of the size-dependency of the energy stores 41
2.4 Summary and Discussion 43
2.4.1 Part 1: First insights into planarian energy metabolism 43
2.4.1.1 Core planarian metabolic pathways 43
2.4.1.2 Characterization of planarian energy stores 44
2.4.2 Part 2: Implications of size-dependent behavior of planarian energy stores 44
2.4.2.1 Role of energy stores as the physiological origin of Kleiber’s law in planarians 44
2.5 Outlook 46
Chapter 3
3. Towards understanding a systems-level regulation of neoblast proliferative activity 48
3.1 Introduction 48
3.2 Assay development for quantitative determination of proliferating cells 50
3.3 Food quantity and quality affect the later proliferation phase, but not the initial response to feeding 53
3.4 Deep sequencing time course provides insights into gene-expression changes in response to feeding 56
3.5 Discussion 59
3.5.1 Evidence for feeding-induced neoblast regulation at the G0/G1-to-S transition 59
3.5.2 Three distinct modes of neoblast proliferation 59
3.5.3 Early and late proliferation modes show distinct transcriptional profiles 59
3.5.4 Implications from feeding and gene expression profiling experiments 60
3.5.4.1 Potential explanations for diet dependence of the late proliferation mode 60
3.5.4.2 Potential mechanisms of diet-independent early proliferation response 61
3.5.5 Summary and Outlook 61
Chapter 4
4. Towards identifying the mechanisms underlying the regulation of neoblast proliferation 63
4.1 Introduction 63
4.1.1 Chosen gene candidates and their known role in proliferation 64
4.2 RNAi-mediated depletion of candidate genes to test their regulatory role in proliferation 67
4.2.1 Assay design and optimization for the functional RNAi screen 67
4.2.2 Results of small-scale RNAi screen 69
4.3 AMPK - a potential integrator of neoblast proliferation to the nutritional state of the animal 73
4.3.1 AMPK and LKB1 knockdown increases proliferation during starvation 73
4.3.2 AMPK depletion-phenotype of increased proliferation during starvation seems to be TOR independent 73
4.4 Discussion 76
4.4.1 Evidence for a mechanism that regulates basal proliferation during starvation 76
4.4.2 AMPK integrates neoblast activity in response to feeding 77
4.4.2.1 Implications of my observations 77
4.4.2.2 Possible experiments to test the role of AMPK during the regulation of proliferation 78
4.4.3 AMPK potentially regulates proliferation independently of TOR 79
4.4.4 An evolutionarily conserved stem cell switch? 80
4.4.5 Summary and Outlook 80
Chapter 5
5. Discussion and Outlook 81
5.1 Cell-autonomous roles of AMPK in proliferation regulation 83
5.1.1 Independent regulation of ribosomal translation elongation as a potential modulator of neoblast proliferation 83
5.1.2 AMPK might regulate cell cycle progression directly 85
5.1.3 AMPK might regulate symmetric versus asymmetric cell division 85
5.2 Cell non-autonomous roles of AMPK in proliferation regulation 86
5.2.1 AMPK might modulate the release of lipid stores 86
5.3 Possible role of AMPK in regulation of autophagic cell death 87
5.4 AMPK as a potential modulator of cell turnover that couples cell proliferation and cell death to the animal’s energy state 88
5.5 Summary and Outlook 89
Materials and Methods 91
List of Figures 106
List of Tables 107
Acknowledgments 108
References 110
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:72521 |
| Date | 27 October 2020 |
| Creators | Frank, Olga |
| Contributors | Roy, Richard, Stewart, Francis, Rink, Jochen C., Technische Universität Dresden, Max-Planck-Institut für molekulare Zellbiologie und Genetik |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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