title: In vivo visualization and functional analysis of collective endothelial cell migration
Creators: name: Hubert, Marvin
Publisher: name: University of Muenster, Institutional Repository MIAMI
Year: 2024
Subjects: subject: 570 Biology

description: Kollektive Endothelzellwanderung ist ein essentieller Prozess während der Embryonalentwicklung, welcher präziser interzellulärer Kontrolle bedarf. Die Adhäsion zwischen migrierenden Nachbarzellen wird durch Zellverbindungsproteine wie den Adherens junctions reguliert. In Endothelzellen ist dies vor allem das vaskuläre endotheliale (VE) Cadherin.
In dieser Arbeit fokussiere ich mich auf die Rolle von VE-cadherin als Regulator der kollektiven Endothelzellwanderung in der embryonalen Entwicklung der Kardinalvene im Zebrafisch und in kultivierten humanen Endothelzellen. VE-cadherin agiert in beiden Modellen als Mediator für Zellpolarisation und Migration. Im Detail werden diese Prozesse durch die direktionale Kontrolle von Aktin Polymerisierung und die Kontraktion von Aktomyosin über Aktin Regulatoren wie Rock und RAC1 reguliert. Während bisherige Arbeiten die Kontrolle von Aktin Regulatoren durch VE-cadherin nur in vitro gezeigt haben, zeigt diese Studie nun eine Aktivierung in vivo.
description: Summary ..... 1
1. Introduction ..... 2
1.1 The cardiovascular system ..... 2
1.2 The zebrafish (Danio rerio) as a model to study vascular development in vivo ..... 3
1.3 Formation of the cardiovascular system in the zebrafish embryo ..... 5
1.4 The development of the zebrafish common cardinal veins (CCVs) ..... 8
1.5 Single- and collective cell migration ..... 9
1.6 The role of the adherens junction molecule VE-cadherin during collective CCV EC migration ..... 12
1.7 The function of Actin regulators during directional cell cluster migration ..... 16
1.8 Aim of the study ..... 20
2. Material and Methods ..... 21
2.1 Material ..... 21
2.1.1 Buffers and solutions ..... 21
2.1.2 Primer ..... 22
2.1.3 Restriction endonucleases ..... 23
2.1.4 Antibodies and reagents ..... 24
2.1.5 Morpholinos ..... 25
2.1.6 Inhibitors for pharmacological treatments ..... 25
2.2 Methods ..... 26
2.2.1 Molecular biology methods ..... 26
2.2.2 Zebrafish Methods ..... 34
2.2.3 Cell culture methods ..... 38
2.2.4 Pharmacological treatments for zebrafish embryos and cell culture ..... 42
2.2.5 Confocal microscopy of HUVECs and in vivo imaging of zebrafish embryos ..... 42
2.2.6 Time-correlated single-photon-counting fluorescence lifetime microscopy (TCSPC-FLIM) ..... 43
2.2.7 Quantifications and statistical analysis ..... 43
3. Results ..... 45
3.1 Directionality and polarization of collective CCV EC migration are regulated by VE-cadherin in zebrafish embryos ..... 45
3.2 VE-cadherin controls CCV EC migration via the RhoA-Rock pathway ..... 54
3.3 Human umbilical vein endothelial cell migration and polarization are regulated by VE-cadherin through RAC1 signaling ..... 69
3.4 Overexpression of activated Rac1 in CCV ECs partially rescued the compromised cell migration phenotype caused by VE-cadherin deficiency ..... 83
3.5 Generation of a Rock activity sensor in the zebrafish endothelium ..... 86
4. Discussion and Outlook ..... 91
5. References ..... 99
6. Appendix ..... 117
6.1 Abbreviations ..... 117
6.2 Additional data ..... 121
6.2.1 Tables ..... 121
6.2.2 Figures ..... 121
6.2.3 Time-lapse movies ..... 123
6.3 Curriculum Vitae of Marvin Hubert ..... 136
6.4 Oath pursuant ..... 138
6.5 Acknowledgements ..... 139