Analysis of in vivo functions of Memo in embryonic and mammary gland development Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Patrick Kaeser aus Fribourg, Schweiz Basel, 2007 Genehmigt von der Philosophisch-Naturwissenschaftichen Fakultät Auf Antrag von Prof. N. Hynes Prof. P. Matthias Prof. M. Affolter Basel, den 08 Oktober 2007 Table of Contents 1 Introduction ..................................................................................................................... 10 1.1 Part I: Cardiovascular development ........................................................................ 10 1.1.1 Molecules involved in early mesodermal differentiation of endothelial cells 12 1.1.1.1 Flk-1/VEGF................................................................................................. 12 1.1.1.2 Fibroblast growth factor.............................................................................. 13 1.1.2 The blood islands ............................................................................................ 13 1.1.3 The hemangioblast .......................................................................................... 13 1.1.4 Endothelial proliferation................................................................................. 14 1.1.5 Assembly of blood vessels .............................................................................. 15 1.1.5.1 Vasculogenesis............................................................................................ 15 1.1.5.2 Angiogenesis ............................................................................................... 16 1.1.5.2.1 Sprouting angiogenesis......................................................................... 17 1.1.5.2.2 Non-sprouting angiogenesis or intussusception.................................... 18 1.1.5.3 Molecules involved in vasculogenesis and angiogenesis............................ 19 1.1.5.3.1 Growth factors and their receptors........................................................ 19 VEGF .................................................................................................................. 20 PDGF................................................................................................................... 20 FGFs.................................................................................................................... 21 Flt-1 or VEGFR1................................................................................................. 22 Flk-1 or VEGFR2................................................................................................ 22 Tie-2 (receptor for angiopoietin-1 and angiopoietin-2) ...................................... 23 1.1.5.3.2 Extracellular matrix............................................................................... 24 Fibronectin .......................................................................................................... 24 Collagens............................................................................................................. 25 1.1.5.3.3 Cell adhesion molecules........................................................................ 25 Vascular endothelial cadherin ............................................................................. 25 Integrins (α5β1 and αvβ3)...................................................................................... 26 1.1.5.4 Endothelial cell migration ........................................................................... 27 1.1.5.5 Molecules involved in endothelial cell migration....................................... 28 1.1.5.5.1 VEGF/Flk-1........................................................................................... 28 1.1.5.5.2 Fibronectin ............................................................................................ 28 1.1.5.5.3 Integrin αvβ3 .......................................................................................... 28 1.1.6 Vascular remodeling....................................................................................... 29 1.1.7 Remodeling, patterning and maturation.......................................................... 30 1.1.7.1 Molecules involved in vessel maturation and patterning............................ 32 1.1.7.1.1 PDGF..................................................................................................... 32 1.1.7.1.2 TGF-β.................................................................................................... 32 1.1.7.1.3 Tie-2, angiopoietin-1 and angiopoietin-2.............................................. 33 1.1.7.1.4 Tie-1 ...................................................................................................... 34 1.1.7.1.5 Extracellular matrix molecules.............................................................. 34 1.1.8 References ....................................................................................................... 36 1.2 Part II: Apoptosis and involution of the mammary gland....................................... 49 1.2.1 Generalities...................................................................................................... 49 1.2.1.1 History of apoptosis: p53, Bcl-2, apoptosis in C.elegans............................ 49 1.2.1.2 Apoptosis vs necrosis.................................................................................. 51 1.2.1.3 Apoptosis is required for development and homeostasis............................ 53 1.2.1.4 Different forms of apoptosis ....................................................................... 53 1.2.2 Intrinsic apoptosis pathway: role of the mitochondrion.................................. 53 1.2.2.1 The Bcl-2 family and its 3 subfamilies ....................................................... 54 1.2.2.2 Model for the mode of action between Bcl-2 family members................... 59 1.2.3 Extrinsic or receptor-activated apoptotic pathway.......................................... 60 1.2.4 Bcl-2 family and the cell cycle........................................................................ 61 1.2.5 Role of BH3-only subfamily members in oncogenesis................................... 62 1.2.6 Therapeutic modulators of apoptosis .............................................................. 63 1.2.7 Involution in the mammary gland: a 2-phase process..................................... 63 1.2.7.1 The first phase of involution ....................................................................... 65 1.2.7.1.1 LIF-Stat3-cEBP-IGFBP-5 axis............................................................. 65 1.2.7.1.2 C/EBPδ.................................................................................................. 67 1.2.7.1.3 Akt......................................................................................................... 67 1.2.7.1.4 Death receptor pathway......................................................................... 68 1.2.7.1.5 TGF-β3.................................................................................................. 69 1.2.7.2 The second phase of involution................................................................... 71 1.2.7.2.1 A process dependant on proteases......................................................... 71 1.2.7.2.2 Adipocyte differentiation...................................................................... 73 1.2.7.2.3 Phagocytosis.......................................................................................... 74 1.2.8 References ....................................................................................................... 74 2 Aim of this thesis............................................................................................................. 85 3 Results ............................................................................................................................. 86 3.1 Part I: Memo is required for vascular integrity during mouse embryonic development as shown by knockout study.......................................................................... 86 3.1.1 Abstract ........................................................................................................... 86 3.1.2 Introduction ..................................................................................................... 86 3.1.3 Results ............................................................................................................. 87 3.1.4 Discussion ....................................................................................................... 95 3.1.5 Materials and methods .................................................................................... 99 3.1.6 Tables and Figures........................................................................................ 104 3.1.7 References ..................................................................................................... 125 3.2 Part II: Suppression of cell-cell contact followed by apoptosis and loss of integrity of mammary alveoli in mice with Memo deletion ............................................................ 128 3.2.1 Abstract ......................................................................................................... 129 3.2.2 Introduction ................................................................................................... 129 3.2.3 Results ..........................................................................................................