Funktionsanalyse des Vezf1 Gens während der

Funktionsanalyse des Vezf1 Gens während der
Embryonalentwicklung der Maus
Dissertation
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.)
vorgelegt der
Mathematisch-Naturwissenschaftlich-Technischen Fakultät
der Martin-Luther-Universität Halle-Wittenberg
von Herrn Frank Kuhnert
geboren am 01.04.1969 in Hovestadt
Gutachter:
1. Prof. Dr. Dr. Thomas Braun, Halle
2. Prof. Dr. Elmar Wahle, Halle
3. Prof. Dr. Heidi Stuhlmann, San Diego
Tag der Verteidigung: 9. Januar 2003
urn:nbn:de:gbv:3-000005384
[http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000005384]
Hiermit versichere ich, daß die vorliegende Dissertation eigenständig verfasst und keine
anderen als die angegebenen Hilfsmittel verwendet wurden. Die Passagen der Arbeit, die
anderen Schriften entnommen sind, wurden unter Angabe der Quelle kenntlich gemacht.
Frank Kuhnert
Palo Alto, 17 Juli 2002
TABLE OF CONTENT
1. INTRODUCTION ...................................................................................................... 1
1.1 Embryonic vascular development ........................................................................... 1
1.1.1 Endothelial lineage specification and vasculogenesis ..........................................................1
1.1.3 Angiogenic remodeling and maturation................................................................................5
1.1.4 Development of the lymphatic system ..................................................................................9
1.2 Cloning and Characterization of Vezf1 .................................................................. 11
1.2.1 Expression of Vezf1 during murine embryonic development.............................................13
1.3 Significance and Specific Aims ............................................................................ 14
2. EXPERIMENTAL PROCEDURES........................................................................ 17
2.1 General molecular biology techniques .................................................................. 17
2.2 Computer programs .............................................................................................. 17
2.3 Structural and expression analysis of Vezf1........................................................... 17
2.3.1 RNA in situ hybridization....................................................................................................17
2.3.2 Northern blot analysis ..........................................................................................................18
2.3.2 Cloning of the 5’ genomic region of the Vezf1 gene ..........................................................19
2.3.4 Analysis of the Vezf1 gene structure ...................................................................................19
2.3.5 cDNA library screening .......................................................................................................19
2.3.6 RLM-RACE .........................................................................................................................20
2.3.7 Construction of Vezf1 promoter analysis constructs..........................................................20
2.3.8 Transient transfection assays ...............................................................................................20
2.4 Analysis of Vezf1 function by gene inactivation .................................................... 21
2.4.1 Generation of the Vezf1 locus targeting constructs............................................................21
2.4.2 ES cell growth, transfection, infection and selection..........................................................21
2.4.3 Genotyping of embryos and ES cells ..................................................................................22
2.4.4 Generation of mutant mice ..................................................................................................23
2.4.5 Whole mount b-galactosidase staining of embryos............................................................23
2.4.6 Whole mount embryo PECAM-1 staining..........................................................................23
2.4.7 Histological Analysis ...........................................................................................................24
2.4.8 Immunohistochemistry ........................................................................................................24
2.4.9 Cultivation of megakaryocytes from embryonic livers ......................................................25
2.4.10 Vessel Morphometry..........................................................................................................25
2.4.11 RT-PCR analysis................................................................................................................25
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2.4.12 Hematopoietic precursor colony assay..............................................................................27
2.5 Overexpression of Vezf1 during embryonic development ...................................... 27
2.5.1 Generation of the Vezf1 expression construct .....................................................................27
2.5.2 Generation of Tie2VIL transgenic mice..............................................................................28
2.5.3 Genotyping of transgenic mice............................................................................................28
3. RESULTS ................................................................................................................. 29
3.1 Structural and expression analysis of Vezf1........................................................... 29
3.1.1 Reevaluation of the Vezf1 cDNA structure .........................................................................29
3.1.2 Reexamination of the Cloning of Vezf1 ..............................................................................31
3.1.3 Expression analysis of Vezf1 ...............................................................................................31
3.1.4 Cloning of the 5’ genomic region of the Vezf1 gene ..........................................................34
3.1.5 Analysis of the Vezf1 gene structure ...................................................................................34
3.1.6 Identification of the Vezf1 transcriptional start site ............................................................36
3.1.7 Characterization of the 5’ flanking and first intron region of the Vezf1 gene....................37
3.1.8 Functional analysis of Vezf1 promoter and intronic sequences..........................................39
3.2 Analysis of Vezf1 function by gene inactivation .................................................... 43
3.2.1 Targeted disruption of the Vezf1 gene in mouse ES cells...................................................43
3.2.2 Analysis in a mixed genetic background.............................................................................46
3.2.3 Analysis in the C57BL/6 background .................................................................................47
3.2.3.1 Vascular remodeling defects and hemorrhaging in E9.5 Vezf1-/- embryos ....................49
3.2.3.2 Analysis of E12.5 Vezf1-/- embryos ..................................................................................52
3.2.3.3 Normal smooth muscle cell maturation and recruitment in Vezf1-/- embryos................52
3.2.3.4 Normal megakaryocyte development in Vezf1
3.2.3.5 Vascular malformations in E13.5 Vezf1
+/-
-/-
embryos .............................................54
mutant embryos............................................55
3.2.3.6 Lymphatic hypervascularization in E13.5 Vezf1+/- mutant embryos ..............................58
3.2.3.7 Analysis of E12.5 Vezf1+/- mutant embryos.....................................................................59
3.2.3.8 Gene expression analysis..................................................................................................60
3.2.4 Analysis of the Vezf1 KO phenotype in the 129/Sv background .......................................63
3.2.4.1 Expression analysis of endothelial and hematopoietic markers in embryoid bodies .....64
3.2.4.2 Hematopoietic progenitor assays......................................................................................65
3.3 Vascular overexpression of Vezf1 ......................................................................... 67
4. DISCUSSION ........................................................................................................... 70
4.1 Vezf1 is a widely expressed transcription factor .................................................... 70
4.2 Zneu1 ................................................................................................................... 74
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4.3 Vezf1 is a critical regulator of vascular development ............................................. 75
4.3.1 Vezf1, a function in hemangioblast differentiation ? ..........................................................75
4.3.2 Vezf1 function in angiogenic remodeling and the maintenance of vascular integrity .......77
4.3.3 Vezf1, a negative regulator of lymphatic development.......................................................80
4.3.4 Vascular overexpression of Vezf1 .......................................................................................82
5. REFERENCES......................................................................................................... 84
6. APPENDIX............................................................................................................. 104
6.1 Abbreviations ..................................................................................................... 104
6.2 Curriculum vitae................................................................................................. 106
6.3 Presentations and publications ............................................................................ 107
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