Esm1 modulates spinal cord vascularization in Vegfaa gain-of-function zebrafish models Zur Erlangung des akademischen Grades einer DOKTORIN DER NATURWISSENSCHAFTEN (Dr. rer. nat.) von der KIT-Fakultät für Chemie und Biowissenschaften des Karlsruher Instituts für Technologie (KIT) genehmigte DISSERTATION von M.Sc. Silvana Maria Knapp 1. Referent/in: Prof. Dr. Ferdinand le Noble 2. Referent/in: Prof. Dr. Martin Bastmeyer Tag der mündlichen Prüfung: 6. Februar 2020 Acknowledgements I thank Ferdinand le Noble for great support during the last years. I could freely pursue my own ideas and could independently work on interesting projects. Your dedication to science and constant enthusiasm is impressive and exemplary. Special thanks go my academic colleagues, especially to Dietmar Gradl, Laëtitia Préau, Anne Ramms and Andria Michael, who accompanied me most of my time as a PhD student. You were always motivating, supportive and helpful. I am grateful for all the help I got from every single member of the lab. Special thanks go to Esther Fuchs, Karolin Rahm and Stefanie Kalb. I really valued your great assistance in all matters concerning experiments. The entire time of my PhD, my family and my partner were always encouraging me. You were cheering me up during difficult times, kept me same and showed me what really matters in life. Table of Contents Table of Contents 1 Summary................................................................................................................................ 6 2 Zusammenfassung ............................................................................................................... 7 3 Introduction ........................................................................................................................... 9 3.1 The vascular system ........................................................................................................ 9 3.1.1 The development of vascular network .......................................................................... 9 3.1.2 Architecture of blood vessels ..................................................................................... 10 3.1.3 From endothelial precursors to tubular structures: vasculogenesis ............................ 11 3.1.4 Expanding the vessel network: angiogenesis ............................................................. 12 3.1.5 Arteries and veins: differences between the tubular systems ..................................... 13 3.1.6 The vascular system makes difference between health and disease ......................... 14 3.2 The zebrafish vascular system ...................................................................................... 15 3.2.1 The zebrafish is a great model organism ................................................................... 15 3.2.2 Development of the zebrafish trunk vasculature ........................................................ 16 3.2.3 Development and function of the zebrafish spinal cord .............................................. 21 3.2.4 Friendship between nerves and blood vessels ........................................................... 21 3.3 The VEGF family – main effectors of vascular guidance ................................................ 22 3.3.1 The VEGF ligands ..................................................................................................... 23 3.3.2 The VEGF receptors .................................................................................................. 24 3.3.3 Clinical suitability of VEGFR-2, VEGFR-1 and VEGF-A ............................................. 27 3.4 Endothelial cell-specific molecule 1 ............................................................................... 28 3.4.1 Proteoglycans are important modulators of cellular responses .................................. 28 3.4.2 Endocan – a distinctive proteoglycan ......................................................................... 29 3.4.3 Endocan and its role in pathological conditions .......................................................... 30 3.4.4 VEGF-A signaling and ESM1 expression are linked to each other ............................. 31 3.5 Aim of the work .............................................................................................................. 31 4 Results ................................................................................................................................. 33 4.1 Esm1 is expressed during early zebrafish development and levels are increased upon loss of flt1. ................................................................................................................................. 33 4.2 Esm1 mRNA is expressed in developing and developed ISVs ....................................... 34 4.3 Esm1 promoter activity in the vasculature and cells of the spinal cord ........................... 37 4.3.1 The esm1 promoter is active in the endothelium of various regions ........................... 37 3 Table of Contents 4.3.2 Active esm1 promoter in developing and developed trunk vasculature ...................... 38 4.3.3 Esm1 promoter activity in flt1 mutants is comparable to that in the wildtype and is present in the spinal cord vasculature ................................................................................... 39 4.3.4 The esm1 promoter is preferentially active in arterial endothelial cells ....................... 41 4.3.5 Neurons of the spinal cord show esm1 promoter activity ........................................... 43 4.4 Altered esm1 levels influence degree of ectopic sprouting at the neuro-vascular interface in the zebrafish trunk ................................................................................................................. 43 4.4.1 Loss of esm1 rescues hypersprouting in flt1 mutants ................................................. 44 4.4.2 Number of tertiary sprouts is decreased in esm1 mutants upon flt1 knock down ....... 49 4.4.3 Esm1 gain-of-function promotes vascular sprouting ................................................... 50 4.5 The effects of esm1 on tertiary sprouting was recapitulated in another Vegfaa GOF model, the vhl mutant ................................................................................................................ 53 4.6 The development of the trunk vasculature is not distinctively altered upon loss of esm1 56 5 Discussion ........................................................................................................................... 59 5.1 Gene expression pattern of esm1 .................................................................................. 59 5.1.1 Esm1 gene expression is enhanced in Vegfaa GOF models ..................................... 60 5.1.2 Esm1 is expressed in the vasculature of zebrafish embryos ...................................... 60 5.1.3 Esm1 is active preferentially in arterial endothelial cells and subset of neurons ......... 62 5.1.4 BAC transgenesis as an alternative tool to study gene expression ............................ 62 5.2 Esm1 alone does not influence the vascular architecture in the trunk ............................ 63 5.3 Esm1 has minor effect on ISV morphology and endothelial cell proliferation ................. 63 5.4 Spinal cord vascularization is modulated by Esm1 when Vegfaa/Kdrl signaling is highly active 64 5.5 Esm1 affects binding efficiency of Vegfaa to Kdrl .......................................................... 66 5.6 Using Esm1 for treating vascular conditions in medicine ............................................... 67 6 Material and Methods .......................................................................................................... 69 6.1 Material ......................................................................................................................... 69 6.1.1 Transgenic and mutant zebrafish lines used .............................................................. 69 6.1.2 Solutions and buffer ................................................................................................... 70 6.1.3 Enzymes, chemicals and kits ..................................................................................... 71 6.1.4 Oligonucleotides ........................................................................................................ 72 6.1.5 Tools for mutant generation ....................................................................................... 74 6.1.6 Plasmids .................................................................................................................... 74 6.1.7 Online tools and softwares ......................................................................................... 75 4 Table of Contents 6.2 Methods ........................................................................................................................ 76 6.2.1 Ethics statement ........................................................................................................ 76 6.2.2 Zebrafish methods ..................................................................................................... 76 6.2.3 Molecular biological methods ..................................................................................... 77 6.2.4 Staining methods ....................................................................................................... 80 6.2.5 BAC Recombineering ...............................................................................................