Aus dem Institut für Anatomie und Zellbiologie Abteilung Anatomie II der Albert-Ludwigs-Universität Freiburg im Breisgau und From the Department of Biochemistry and Molecular Biology MD Anderson Cancer Center, Houston, Texas, USA Detailed analysis of Lmx1b expression in multiple organs and its role in dorso-ventral patterning with emphasis on myogenic precursor cells INAUGURAL-DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg im Breisgau Vorgelegt 2003 von Heiko Schweizer geboren in Freiburg im Breisgau Dekan: Prof. Dr. Josef Zentner Erstgutachter: Prof. Dr. Beate Brand-Saberi Zweitgutachter: Prof. Dr. Randy Johnson Jahr der Promotion: 2005 In Gedenken meiner lieben Mutter gewidmet Table of Contents A1 GERMAN SUMMARY (DEUTSCHE ZUSAMMENFASSUNG) ........................... 1 A2 ENGLISH SUMMARY................................................................................................. 2 1. INTRODUCTION ......................................................................................................... 3 1.1 Lmx1b – a LIM homeodomain transcription factor....................................................................... 4 1.1.1 Structure and properties of Lmx1b...................................................................................................... 4 1.1.2 Expression of Lmx1b in mouse and chick ........................................................................................... 5 1.1.3 Effects of gain-of-function and loss-of-function of Lmx1b.................................................................. 7 1.1.4 Lmx1b is involved in the Nail-patella syndrome (NPS) ...................................................................... 8 1.1.5 Lmx1b expression is affected in transgenic mice and by overexpression of other limb genes............ 9 1.1.6 Downstream targets of Lmx1b........................................................................................................... 10 1.1.7 Induction of Lmx1b ........................................................................................................................... 10 1.2 General limb development.............................................................................................................. 12 1.2.1 Axis formation .................................................................................................................................. 13 1.2.2 The proximo-distal axis of the limb .................................................................................................. 14 1.2.3 The anterior-posterior axis of the limb .............................................................................................. 15 1.2.4 The dorso-ventral axis of the limb .................................................................................................... 15 1.2.5 Genetic interactions along the three axes .......................................................................................... 17 1.2.6 Establishment of the dorso-ventral boundary and boundaries in general.......................................... 18 1.2.7 Muscle formation .............................................................................................................................. 20 1.3 Mammary gland development ....................................................................................................... 26 1.3.1 Fetal development ............................................................................................................................. 26 1.3.2 Postnatal development....................................................................................................................... 27 1.3.3 Pubertal and postpubertal development............................................................................................. 27 1.3.4 Adult development including pregnancy and involution................................................................... 27 1.3.5 The cellular composition of mammary glands .................................................................................. 28 1.4 Kidney development........................................................................................................................ 29 1.4.1 WT-1: Wilms’ tumor suppressor gene............................................................................................... 29 2. MATERIALS AND METHODS................................................................................ 31 2.1 Animals............................................................................................................................................. 31 2.1.1 Mice .................................................................................................................................................. 31 2.1.2 Chick and quail ................................................................................................................................. 32 2.2 Microsurgery and interspecific grafting experiments.................................................................. 33 2.2.1 Isolation of mouse embryos .............................................................................................................. 33 2.2.2 Dissection of mammary glands ......................................................................................................... 33 2.2.3 Quail to chick grafting experiments .................................................................................................. 34 2.3 Cell culture and in vitro culture of embryos................................................................................. 40 2.3.1 Cell culture ........................................................................................................................................ 40 2.3.2 Micromass culture............................................................................................................................. 43 2.3.3 Whole embryo culture....................................................................................................................... 46 2.4 Molecular biology............................................................................................................................ 48 2.4.1 General methods................................................................................................................................ 48 2.4.2 Large plasmid DNA preparation ....................................................................................................... 49 2.4.3 Preparation of digoxygenin labeled probes ....................................................................................... 50 2.4.4 Genotyping of transgenic mice by Southern blotting and PCR......................................................... 51 2.5 Whole mount and histological analysis.......................................................................................... 56 2.5.1 X-gal staining .................................................................................................................................... 56 2.5.2 Immunohistochemistry...................................................................................................................... 61 2.5.3 In situ hybridization histochemistry.................................................................................................. 64 2.6 Photography and imaging............................................................................................................... 68 2.7 Solutions, buffers and antibodies ................................................................................................... 69 3. RESULTS..................................................................................................................... 72 3.1 Lmx1b 3’LacZ mice ......................................................................................................................... 72 3.1.1 Genotyping........................................................................................................................................ 72 3.1.2 Phenotype, expression level and localization.................................................................................... 73 3.1.3 Intercrosses of Lmx1b 3’LacZ/LacZ X Lmx1b KO +/- mice............................................................. 74 3.2 Detailed analysis of Lmx1b expression in Lmx1b 3’LacZ transgenic mice ................................. 75 3.2.1 Expression of Lmx1b in the murine limb .......................................................................................... 75 3.2.2 Expression of Lmx1b during mammary gland development ............................................................. 80 3.2.3 Lmx1b expression in the kidney ........................................................................................................ 84 3.2.4 Lmx1b expression in other organs and in adults................................................................................ 85 3.2.5 Brief summary of Lmx1b expression................................................................................................. 87 3.3 Induction of Lmx1b and boundary formation .............................................................................. 88 3.3.1 Lmx1b positive cells maintain a homogenous distribution in a micromass culture........................... 88 3.3.2 Ventral proximal mouse limb mesenchyme does not initiate Lmx1b expression when grafted ..........