Glucose homeostasis is fundamental for all living organisms. In vertebrates, the hormone insulin regulates the metabolism of carbohydrates, fats and proteins. In order to sustain the glucose homeostasis, the pancreatic β-cells, which produce and secrete insulin, must coordinate their efforts to secrete the right amounts of insulin required by the organism. In vitro studies, have suggested that a subpopulation of β-cells, referred to as “hub-cells”, coordinate islet Ca2+ dynamics during insulin secretion. However, it is unclear whether the hub-cell model pertains to an in vivo scenario, where the islet is densely vascularized and innervated. In this thesis, we employed the genetically-encoded calcium indicator GCaMP6, confocal imaging and optogenetics, to characterize the Ca2+ dynamics of the zebrafish β-cells in vivo. We found that pancreatic β-cells present endogenous Ca2+ spikes in vivo under basal conditions. These Ca2+ spikes are rapidly suppressed after lowering glucose levels via insulin administration. In addition, the temporal inhibition of blood flow decreases the Ca2+ spikes, suggesting that β-cells are systemically connected. Furthermore, β-cells show a synchronized response to a pericardial glucose injection. Specifically, we found that Ca2+ spikes originate and emanate from a subset of β-cells that are the first to respond to a glucose stimulus. We define these cells as “leader-cells”. We tested if these cells could coordinate the islet in vivo by employing 2-photon laser ablation. Whereas ablation of control cells had no significant effect on the amplitude and duration of the subsequent Ca2+ spikes responses, ablation of leader cells led to a reduction in the Ca2+ response. Furthermore, we developed systems for optogenetic interrogation of β-cells in vivo. We show that the light-gated Cl- ion pump halorhodopsin (NpHR) can be applied to inhibit β-cell depolarization in the zebrafish. We also present the optically orthogonal system of the red Ca2+ indicator K-GECO1 in combination with the blue-shifted channelrhodopsin CheRiff to activate individual β-cell in vivo. Using these new tools, we provide examples where the activation of individual β-cells showed heterogeneous potential to trigger influx of Ca2+ in the rest of the β-cells. Overall, our results led us to propose a hierarchical model of islet coordination. In contrast to the majority of β-cells, which occupy the bottom of the hierarchy since they present low capability to recruit other cells, the leader cells occupy the top levels, being capable to coordinate a majority of the islet’s β-cells.:List of figures xii
List of Tables xiii
1. Introduction 1
1.1. Diabetes and insulin 1
1.2. The endocrine pancreas 2
1.3. The diabetes pandemic 4
1.4. β-cell development in zebrafish and mammals 4
1.5. β-cells function and heterogeneity 6
1.6. β-cell coordination 8
1.7. Genetically-encoded calcium indicators 10
1.8. Genetically-encoded optogenetic actuators 13
1.9. Models to study In vivo β-cell coordination 16
2. In vivo β-cell Ca2+ dynamics 19
2.1. β-cells present endogenous Ca2+ spikes in vivo, which are not present ex vivo 19
2.2. Insulin injection reduces endogenous β-cell Ca2+ activity 22
2.3. Pharmacological inhibition of β-cell Ca2+ spikes interferes with glucose control 24
2.4 Transient blood flow interruption decreases β-cell calcium spikes 26
2.5 Glucose bolus leads to a synchronous response of β-cells 29
3. Leader β-cells coordinates Ca2+ dynamics in vivo 32
3.1. High speed 2D and 3D imaging reveals “leader” β-cells 32
3.2. Pan-islet response to glucose is impaired after leader β-cells ablation 41
4. Optically orthogonal toolset for in vivo optogenetics and Ca2+ imaging 46
4.1. Development of optogenetics actuators systems in zebrafish β-cells 46
4.2. Red fluorescent calcium reporters in zebrafish β-cells 47
4.3. In vivo temporal optogenetic silencing of β-cells 50
4.4. In vivo temporal optogenetic silencing of a subset of β-cells can inhibit the islet response 52
4.5. In vivo temporal optogenetic activation of β-cells 55
5. Discussion and future directions 61
5.1. β-cell calcium spikes are systemically influenced 61
5.2. First responder β-cells are present in vivo 64
5.3. Leader β-cells coordinate Ca2+ influx in vivo 66
5.4. β-cell optogenetic interrogation shows heterogeneous potential of individual β-cells for islet coordination 68
6. Materials and methods 75
6.1. Zebrafish strains and husbandry 75
6.2. Transgenic lines generation 76
6.3. Glucose measurements 77
6.4. Pericardial injection of glucose and insulin 77
6.5. Live imaging 77
6.6. Fast whole islet live imaging 78
6.7. Selective two-photon laser ablation of leader cells in the zebrafish islet. 78
6.7. Selective one-photon optogenetic interrogation of β-cells in the zebrafish islet. 79
6.8. Islet blood flow imaging 80
6.9. Mechanical heart stop 80
6.10. Immunostaining 80
6.11. TUNEL assay 81
6.12 Image analysis of GCaMP6s fluorescence intensity from in vivo imaging. 82
6.13 Quantification of GCaMP6s fluorescence intensity 82
6.14 Spatial drift correction images. 83
6.15 Statistical analysis 84
7. References 85
8. Annexes 90
9. Acknowledgments 97 / Die Glukosehomöostase ist für alle lebenden Organismen von grundlegender Bedeutung. Bei Wirbeltieren reguliert das Hormon Insulin den Stoffwechsel von Kohlenhydraten, Fetten und Proteinen. Um die Glukosehomöostase aufrechtzuerhalten, müssen die β-Zellen der Bauchspeicheldrüse, welche Insulin produzieren und absondern, ihre Bemühungen koordinieren, um die richtigen Mengen an Insulin zu sekretieren, die der Organismus benötigt. In-vitro-Studien haben gezeigt, dass eine Subpopulation von β-Zellen, die als „Hub-Zellen“ bezeichnet werden, die Insulinsekretion der Inseln koordiniert. Es ist jedoch unklar, ob sich die Hub-Cell-Theorie auf ein in-vivo-Szenario bezieht, bei dem die Insel dicht vaskularisiert und von Neuronen innerviert ist. In dieser Arbeit verwendeten wir den genetisch kodierten Calcium-Indikator GCaMP6, konfokale Bildgebung und Optogenetik, um die Ca2+-Dynamik der Zebrafisch-β-Zellen in vivo zu charakterisieren. Wir fanden heraus, dass Pankreas-β-Zellen in vivo unter basalen Bedingungen endogene Ca2+-Spitzen aufweisen. Diese Ca2+-Spitzen werden nach Senkung des Glukosespiegels durch Insulinverabreichung schnell unterdrückt. Darüber hinaus verringert die zeitliche Hemmung des Blutflusses die Ca2+-Spitzen, was darauf hindeutet, dass β-Zellen systemisch verbunden sind. Darüber hinaus zeigen β-Zellen eine synchronisierte Reaktion auf die perdikale Glukoseinjektion. Insbesondere fanden wir heraus, dass Ca2+-Spitzen von den β-Zellen hervorgerufen werden, die zuerst auf den Glukosestimulus reagieren. Wir definieren diese Zellen als 'Leader-Zellen'. Wir haben in vivo durch den Einsatz einer 2-Photonen-Laserablation getestet, ob diese Zellen die Insel koordinieren können. Während die Ablation von Kontrollzellen keinen signifikanten Einfluss auf die Amplitude und Dauer der nachfolgenden Ca2+-Spitzenreaktionen hatte, führte die Ablation von Leader-Zellen zu einer signifikanten Verringerung der GCaMP-Reaktion. Darüber hinaus haben wir Systeme für die optogenetische Abfrage von β-Zellen in vivo entwickelt: Wir zeigen, dass die lichtgesteuerte Cl—Ionenpumpe Halorhodopsin (NpHR) angewendet werden kann, um die Depolarisation von β-Zellen in vivo zu hemmen. Wir präsentieren auch das optisch orthogonale System des roten Ca2+-Indikators K-GECO1 in Kombination mit dem blauverschobenen Channelrhodopsin CheRiff, um einzelne β-Zellen in vivo abzufragen. Unter Verwendung dieser neuen Werkzeuge liefern wir Beispiele, bei denen die Aktivierung einzelner β-Zellen ein heterogenes Potenzial für die Auslösung des Ca2+-Einstroms in die übrigen β-Zellen in vivo zeigte. Insgesamt bietet diese Studie Hinweise darauf, dass eine Untergruppe von β-Zellen ein hohes Potenzial zur Koordination der Ca2+-Dynamik der Insel in vivo aufweist.:List of figures xii
List of Tables xiii
1. Introduction 1
1.1. Diabetes and insulin 1
1.2. The endocrine pancreas 2
1.3. The diabetes pandemic 4
1.4. β-cell development in zebrafish and mammals 4
1.5. β-cells function and heterogeneity 6
1.6. β-cell coordination 8
1.7. Genetically-encoded calcium indicators 10
1.8. Genetically-encoded optogenetic actuators 13
1.9. Models to study In vivo β-cell coordination 16
2. In vivo β-cell Ca2+ dynamics 19
2.1. β-cells present endogenous Ca2+ spikes in vivo, which are not present ex vivo 19
2.2. Insulin injection reduces endogenous β-cell Ca2+ activity 22
2.3. Pharmacological inhibition of β-cell Ca2+ spikes interferes with glucose control 24
2.4 Transient blood flow interruption decreases β-cell calcium spikes 26
2.5 Glucose bolus leads to a synchronous response of β-cells 29
3. Leader β-cells coordinates Ca2+ dynamics in vivo 32
3.1. High speed 2D and 3D imaging reveals “leader” β-cells 32
3.2. Pan-islet response to glucose is impaired after leader β-cells ablation 41
4. Optically orthogonal toolset for in vivo optogenetics and Ca2+ imaging 46
4.1. Development of optogenetics actuators systems in zebrafish β-cells 46
4.2. Red fluorescent calcium reporters in zebrafish β-cells 47
4.3. In vivo temporal optogenetic silencing of β-cells 50
4.4. In vivo temporal optogenetic silencing of a subset of β-cells can inhibit the islet response 52
4.5. In vivo temporal optogenetic activation of β-cells 55
5. Discussion and future directions 61
5.1. β-cell calcium spikes are systemically influenced 61
5.2. First responder β-cells are present in vivo 64
5.3. Leader β-cells coordinate Ca2+ influx in vivo 66
5.4. β-cell optogenetic interrogation shows heterogeneous potential of individual β-cells for islet coordination 68
6. Materials and methods 75
6.1. Zebrafish strains and husbandry 75
6.2. Transgenic lines generation 76
6.3. Glucose measurements 77
6.4. Pericardial injection of glucose and insulin 77
6.5. Live imaging 77
6.6. Fast whole islet live imaging 78
6.7. Selective two-photon laser ablation of leader cells in the zebrafish islet. 78
6.7. Selective one-photon optogenetic interrogation of β-cells in the zebrafish islet. 79
6.8. Islet blood flow imaging 80
6.9. Mechanical heart stop 80
6.10. Immunostaining 80
6.11. TUNEL assay 81
6.12 Image analysis of GCaMP6s fluorescence intensity from in vivo imaging. 82
6.13 Quantification of GCaMP6s fluorescence intensity 82
6.14 Spatial drift correction images. 83
6.15 Statistical analysis 84
7. References 85
8. Annexes 90
9. Acknowledgments 97
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:76222 |
| Date | 11 October 2021 |
| Creators | Delgadillo Silva, Luis Fernando |
| Contributors | Speier, Stephan, Gavalas, Anthony, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | German |
| 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 |
| Relation | https://doi.org/10.1038/s42255-019-0075-2 |
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