Roles of HMG-box transcription factors in the pancreas development of the mouse (mus musculus, Linnaeus, 1758) [Elektronische Ressource] / vorgelegt von Oleg Lioubinski

Oleg Osenkov

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• Publié par: universitat_hamburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 8 Mo

Extrait

Roles of HMG-box Transcription Factors
in the Pancreas Development of the Mouse
(mus musculus, Linnaeus, 1758)
Dissertation
zur Erlangung des Doktorgrades
des Fachbereiches Biologie
der Universität Hamburg
vorgelegt von
Oleg Lioubinski
aus Novosibirsk, Russland
Hamburg, April 2004Table of Contents
SUMMARY...................................................................................................................................................1
I INTRODUCTION...............................................................................................................................4
1.1 TRANSCRIPTION FACTORS........................................................................................................... 4
1.1.1 Eukaryotic Gene, Regulation of Transcription ...................................................................... 4
1.1.2 Transcription Factors.............................................................................................................. 5
1.2 STRUCTURE, EXPRESSION AND KNOWN DEVELOPMENTAL ROLES OF THE HMG BOX
TRANSCRIPTION FACTORS IN MAMMALS................................................................................................. 6
1.2.1 SOX Transcription Factors.... 6
1.2.1.1 Expression and Functions of Sox Transcription Factors in Mammalian Embryonic
Development..................................................................................................................................... 8
1.2.2 TCF/LEF transcription factor family ..................................................................................... 9
1.2.2.1 Binding Partners of Tcf/Lef Factors ............................................................................ 10
1.3 THE WNT SIGNALING PATHWAY............................................................................................... 10
1.3.1 Functions of the Wnt Signaling Pathway.............................................................................. 12
1.3.2 Roles of the Canonical Wnt Signaling Pathway in Stem Cell Maintenance ....................... 13
1.4 MOUSE PANCREAS DEVELOPMENT AND FUNCTIONS............................................................... 14
1.4.1 Embryonic Development of the Mouse Pancreas................................................................. 15
1.4.1.1 Specification of the pancreatic domains and early development................................ 16
1.4.1.2 Growth of the Pancreatic Primordium......................................................................... 18
1.4.1.3 Specification and Differentiation of Endocrine and Exocrine Cell Types................. 19
1.5 RATIONALE AND HYPOTHESIS................................................................................................... 22
II RESULTS...........................................................................................................................................24
2.1 ANALYSIS OF THE ROLES OF SOX TRANSCRIPTION FACTORS IN MOUSE PANCREAS
DEVELOPMENT......................................................................................................................................... 24
2.1.1 Expression of Sox genes during Mouse Pancreas Development ......................................... 24
2.1.1.1 Sox Gene Expression Analysis by RT-PCR................................................................ 24
2.1.1.2 Sox Gene in situ Expression Analysis on Pancreatic Tissue Sections ....................... 26
2.1.2 Analysis of Sox Null Mutants for Their Pancreatic Phenotype ........................................... 30
2.2 ANALYSIS OF THE ROLES OF TCF/LEF TRANSCRIPTION FACTORS IN PANCREAS
DEVELOPMENT......................................................................................................................................... 32
2.2.1 Expression of Tcf/Lef genes during mouse pancreas development ..................................... 32
2.1.1.1 RT-PCR Expression Analysis of Tcf/Lef Genes. ........................................................ 32
2.1.1.2 LEF/TCF Gene Expression Analysis by in situ Hybridization on Pancreatic
Sections 332.2.2 Expression of Wnt Pathway Components during Mouse Pancreas Development .............. 35
2.2.3 Detection of Cells that Receive a Wnt Signal during Mouse Pancreas Development ........ 37
2.2.4 Creation of Modified Forms of TCF4................................................................................... 41
2.2.5 Testing the Modified Forms of TCF4 for Their Functionality in vitro................................ 44
+/tg +/tg2.2.6 Generation of pdx-dnTCF and pdx-caTCF Transgenic Mice ..................................... 46
+/tg +/tg2.1.1.1 Design of the Constructs Used for Generation of pdx-dnTCF and pdx-caTCF
Transgenic Mice ............................................................................................................................. 46
+/tg +/tg2.1.1.2 Generation of pdx-dnTCF and pdx-caTCF Transgenic Mice............................. 47
+/tg +/tg2.1.1.3 Analysis of Pancreata of the pdx-dnTCF and pdx-caTCF mice for the
Expression of the ß-galactosidase Gene. ....................................................................................... 48
+/tg +/tg2.1.1.4 Analysis of the pdx-dnTCF and pdx-caTCF Mice for the Expression of
Pancreatic Markers. ........................................................................................................................ 49
2.2.7 Bigenic Cre-LoxP System for Expression of dnTCF4 and caTCF4 Factors in Mouse
Pancreas.............................................................................................................................................. 50
dnTCF caTCF2.2.8 Generation of ROSA26 and ROSA26 Mice............................................................. 52
2.2.8.1 Construction of the pROSA-dnTCF4 and pROSA-caTCF4 Targeting Vectors.......... 52
dnTCF caTCF2.2.8.2 Generation of ROSA26 and ROSA26 mice .................................................... 53
2.2.9 In vivo Cre-mediated Activation of dnTCF4 Expression in the Pancreatic Islets .............. 55
III DISCUSSION................................................................................................................................56
3.1 CO-EXPRESSION OF SOX GENES IN OVERLAPPING DOMAINS OF THE DEVELOPING
PANCREAS................................................................................................................................................ 56
3.2 UNIQUE SOX EXPRESSION DOMAINS IN THE DEVELOPING PANCREAS ................................... 57
3.3 SCHWANN CELLS ARE DISPENSABLE FOR PANCREAS DIFFERENTIATION............................... 59
3.4 INHIBITION OR ACTIVATION OF THE CANONICAL WNT SIGNALING PATHWAY BY
EXPRESSION OF DOMINANT NEGATIVE OR CONSTITUTIVELY ACTIVE FORMS OF TCF4 IN MOUSE
PANCREAS................. 61
IV MATERIALS................................................................................................................................70
4.1 MATERIAL SOURCES.................................................................................................................. 70
4.2 LIST OF SOLUTIONS AND MEDIA ............................................................................................... 70
4.3 BACTERIAL STRAINS USED........................................................................................................ 71
4.4 BASIC VECTORS USED FOR CLONING ....................................................................................... 71
4.5 ES CELL LINE............................................................................................................................. 71
4.6 MOUSE STRAINS USED............................................................................................................... 71
4.7 PROBES USED FOR IN SITU HYBRIDIZATION.............................................................................. 72
4.8 ANTIBODIES USED...................................................................................................................... 72
4.8.1 Primary Antibodies................................................................................................................ 724.8.2 Secondary Antibodies............................................................................................................ 73
4.9 OLIGONUCLEOTIDES .................................................................................................................. 73
V METHODS.........................................................................................................................................74
5.1 ISOLATION AND PURIFICATION OF PLASMID DNA................................................................... 74
5.1.1 Analytical Scale Purification of DNA (Minipreps) .............................................................. 74
5.1.2 Large Scale Purification of DNA (Maxipreps)..................................................................... 74
5.1.3 Determination of DNA and RNA Concentration.................................................................. 74
5.1.4 Purification of DNA by Phenol Extraction and Ethanol Precipitation ............................... 75
5.2 PRODUCTION, PURIFICATION, AND CLONING OF DNA FRAGMENTS ...................................... 75
5.2.1 DNA Digests Using Restriction Enzymes ............................................................................. 75
5.2.2 Hybridization/Annealing of Synthetic Oligonucleotides................................................