Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1485 Organ-specific mechanisms of vascular development in the mesentery YANG ZHANG ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0406-9 UPPSALA urn:nbn:se:uu:diva-356488 2018 Dissertation presented at Uppsala University to be publicly examined in Rudbeck Hall, Dag Hammarskjölds v 20, Uppsala, Friday, 5 October 2018 at 13:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Wiebke Herzog (University of Muenster c/o Max Planck Institute for Molecular Biomedicine). Abstract Zhang, Y. 2018. Organ-specific mechanisms of vascular development in the mesentery. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1485. 73 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0406-9. Understanding how the vascular systems are formed has significant clinical importance. General mechanisms underlying vascular development have been extensively studied during the past decades. However, the mechanisms regulating the development and function of the blood and lymphatic vessels in specific organs are poorly understood. The aim of this thesis was to investigate lymphatic vascular development in the mesentery, which is a fold of peritoneum that attaches the intestine to the abdominal wall, and contains arteries, veins, lymphatic vessels, nerves and lymph nodes. We found that mesenteric lymphatic vessels were formed through lymphvasculogenesis - coalescence of isolated lymphatic endothelial cell (LEC) clusters, rather than by lymphangiogenesis - sprouting from the veins or pre-existing lymphatic vessels. The lymphvasculogenic process was selectively sensitive to inhibition of the vascular endothelial growth factor receptor 3 (VEGFR3)/ phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling pathway. Using genetic lineage tracing, we uncovered that part of the mesenteric lymphatic vasculature was derived from cKit lineage cells likely originating from the blood-forming hemogenic endothelium of major arteries (Paper I). This is in contrast to the previously accepted dogma that all mammalian lymphatic vessels are of venous endothelial origin. By characterizing a mouse mutant lacking the non-venous-derived LEC progenitors we found that an alternative venous source of LECs could however compensate to build a functional mesenteric lymphatic vasculature (Paper IV). We further described in the developing mesentery that a transient loss of venous integrity, characterized by the formation of inter-endothelial cell gaps, was accompanied by extravasation of red blood cells, which were cleared by the developing lymphatic vessels. By studying mice with defective platelet function, we revealed a previously unappreciated role of platelets in maintaining the integrity of the remodeling embryonic blood vasculature and thus preventing excessive blood-filling of lymphatic vessels (Paper III). We also studied the mechanism of vessel maturation into functional lymphatic vessels, which involves smooth muscle cell recruitment. Analysis of mice with LEC-specific deletion of Pdgfb, encoding the platelet derived growth factor B (PDGFB), showed that LEC-autonomous PDGFB was required for the recruitment of smooth muscles cells that in turn control lymphatic vessel size and function (Paper II). Keywords: Lymphatic vasculature, mesentery, hemogenic endothelium, lymphvasculogenesis, endothelial integrity, platelet, blood-filled lymphatic vessel, morphogenesis and maturation, compensation Yang Zhang, Department of Immunology, Genetics and Pathology, Vascular Biology, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden. © Yang Zhang 2018 ISSN 1651-6206 ISBN 978-91-513-0406-9 urn:nbn:se:uu:diva-356488 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-356488) There is pleasure in recognizing old things from a new viewpoint. Richard Feynman To my family 致我的家人 List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Stanczuk, L., Martinez-Corral, I., Ulvmar, M.H., Zhang, Y., Laviña, B., Fruttiger, M., Adams, R.H., Saur, D., Betsholtz, C., Ortega, S., Al- litalo, K., Graupera, M., and Mäkinen, T. (2015) cKit lineage hemo- genic endothelium-derived cells contribute to mesenteric lymphatic vessels. Cell Reports 10: 1708-1721 II Wang, Y., Jin, Y*., Mäe M.A*., Zhang, Y., Ortsäter, H., Betsholtz, C., Mäkinen, T‡., and Jakobsson, L‡. (2017) Smooth muscle cell recruit- ment to lymphatic vessels requires PDGFB and impacts vessel size but not identity. Development 144, 3590-3601 (*These authors con- tributed equally to this work. ‡Authors for correspondence) III Zhang, Y., Daubel, N., Stritt, S., and Mäkinen, T. (2018) Transient loss of venous integrity during developmental vascular remodeling leads to red blood cell extravasation and clearance by lymphatic ves- sels. Development 145: pii: dev156745 IV Zhang, Y., Stritt, S., Martinez-Corral, I., Laviña, B., Betsholtz, C., and Mäkinen, T. (2018) Alternative lymphatic endothelial progenitor cells compensate for the loss of non-venous derived progenitors to form mesenteric lymphatic vessels. Manuscript Reprints were made with permission from the respective publishers. Contents Introduction ...............................................................................................11 Structure and function of the vascular system ........................................11 Development of the blood and lymphatic vasculatures ...........................16 Vasculogenesis .................................................................................16 Angiogenesis ....................................................................................18 Lymphangiogenesis ..........................................................................22 Hemogenic endothelium (HE)...........................................................30 Origins of endothelial cells ....................................................................31 BEC origins ......................................................................................31 LEC origins ......................................................................................33 The role of platelets in angiogenesis and lymphangiogenesis .................34 Aims of the thesis ......................................................................................37 Present investigations ................................................................................38 Paper I ..............................................................................................38 Paper II .............................................................................................39 Paper III ...........................................................................................41 Paper IV ...........................................................................................42 Outlook .....................................................................................................45 Acknowledgements ...................................................................................48 References .................................................................................................51 Abbreviations AGM Aorta-gonad-mesonephros ANG Angiopoietin BBB Blood-brain barrier BEC Blood endothelial cell bFGF Basic fibroblast growth factor BM Basement membrane BMP Bone morphogenetic protein CCBE1 Calcium-binding EGF-like domain 1 CCM1 Cerebral cavernous malformation 1 CCV Common cardinal vein CDK5 Cyclin-dependent kinase 5 CNS Central nervous system COUP-TFII COUP transcription factor 2 CV Cardinal vein CX Connexin DC Dendritic cell Dll4 Delta-like 4 EC Endothelial cell ECM Extracellular matrix EHT Endothelial-to-hematopoietic transition eNOS Endothelial nitric oxide EPH Ephrin receptor ERK Extracellular signal reguated kinase ETS E26 transforming-specific E-number Embryonic day GPVI Glycoprotein VI HDAC3 Histone-modifying enzyme histone deacetylase 3 HE Hemogenic endothelium HEC Hemogenic endothelial cell HEY Hairy- and enhancer of split-related with YRPW motif HSC Hematopoietic stem cell HSPC Hematopoietic stem and progenitor cell IAHC Intra-aortic hematopoietic cluster IHH Indian hedgehog ITAM Immunoreceptor tyrosine-based activation motif KLF Krüppel-like factor LEC Lymphatic endothelial cell LN Lymph node NICD Notch intracellular domain NPAS4L Neuronal PAS domain-containing protein 4-like protein NRP Neuropilin PAR3 Partitioning defective 3 PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PDPN Podoplanin PI3K Phosphoinositide 3-kinase PROX1 Prospero hemeobox 1 PLCγ2 Phospholipase Cγ2 P-number Postnatal day RASIP1 RAS-interacting protein 1 RBC Red blood cell ROS Reactive oxygen species SCF Stem cell factor SEMA Semaphorin SIM Structure illumination microscopy SLP76 Src homology 2 domain-containing leukocyte pro- tein of 76 kDa SMC Smooth muscle cell S1P Sphingosine-1-phosphate S1PR Sphingosine-1-phosphate receptor TJ Tight junction VE-cad VE-cadherin VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor vWF Von Willebrand factor 4-OHT 4-hydroxytamoxifen Introduction Structure and function of the vascular system The vascular system is composed of the blood vasculature and the lymphatic vasculature. The blood vasculature delivers nutrients, metabolites, oxygen, carbon dioxide, hormones, and blood cells to almost all