Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VII, 161 Bl. Ill., graph. Darst. |
| ISBN: | 9783000449925 |
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| 001 | BV041710699 | ||
| 003 | DE-604 | ||
| 005 | 20140415 | ||
| 007 | t | ||
| 008 | 140227s2014 ad|| m||| 00||| eng d | ||
| 020 | |a 9783000449925 |9 978-3-00-044992-5 | ||
| 035 | |a (OCoLC)873552944 | ||
| 035 | |a (DE-599)BVBBV041710699 | ||
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| 041 | 0 | |a eng | |
| 049 | |a DE-M49 |a DE-91 |a DE-12 | ||
| 100 | 1 | |a Meier, Florian |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |c Florian Meier |
| 264 | 1 | |c 2014 | |
| 300 | |a VII, 161 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Techn. Univ., Diss., 2014 | ||
| 546 | |a Zsfassung in dt. Sprache | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027157896&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-027157896 | ||
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| adam_text | INDEX
1 ABSTRACT 1
2 ZUSAMMENFASSUNG 3
3 INTRODUCTION
- THE VERTEBRATE BRAIN AND THE FORMATION OF THE NEURAL TUBE 5
3.1 THE VERTEBRATE BRAIN 5
3.2 THE FORMATION OF THE NEURAL TUBE 5
4 PARTI: IN VITRO GENERATION OF MESO-DIENCEPHALIC DOPAMINERGIC NEURONS 8
4.1 INTRODUCTION IN MDDA NEURONS AND PARKINSON S DISEASE 8
4.1.1 IN VIVO DEVELOPMENT OF MDDA NEURONS 9
4.1.1.1 THE INDUCTION OF THE MDDA PROGENITOR DOMAIN AND GENERATION OF
MDDA
PRECURSORS 9
4.1.1.2 WNTS AND THEIR EFFECT ON THE MDDA PRECURSOR DEVELOPMENT 11
4.1.1.3 THE DICKKOPF FAMILY AND ITS FUNCTION AS WNT/P-CATENIN SIGNALING
MODULATORS
12
4.1.1.4 IMPORTANT TRANSCRIPTION FACTORS FOR THE PROPER MDDA NEURON
DEVELOPMENT 13
4.1.1.5 SUBSTANTIA NIGRA PARS COMPACTA (SNC) AND VENTRAL TEGMENTAL AREA
(VTA) 16
4.1.2 CELL REPLACEMENT THERAPY AND IN VITRO CELL-MODELS FOR PARKINSON S
DISEASE 17
4.1.2.1 GENERATION OF MDDA NEURONS IN VITRO 17
4.1.2.2 REPROGRAMMING OF SOMATIC CELLS INTO IPS CELLS 18
4.1.2.3 DIFFERENTIATION OF PLURIPOTENT CELLS STEM INTO MDDA NEURONS 18
4.1.2.4 DIRECT REPROGRAMMING OF SOMATIC CELLS INTO DOPAMINERGIC NEURONS
19
4.1.2.5 PITX3 HETEROZYGOUS CELL-MODEL FOR SPORADIC PARKINSON S DISEASE
CASES...20
4.1.3 AIMS OF THE PROJECT IN VITRO GENERATION OF MDDA NEURONS 21
4.2 RESULTS 22
4.2.1 GENERATION OF A VECTOR FREE IPS CELL LINE 22
4.2.2 PITX3
GFP/
*
IPSC* LINE IS A REPORTER CELL LINE FOR THE DIFFERENTIATION INTO MDDA
NEURONS 25
4.2.3 WNT1 ADDITION INCREASES THE NUMBER OF TH
+
/PITX3
+
MDDA NEURONS
DIFFERENTIATED FROM MURINE PLURIPOTENT STEM CELLS 26
I
HTTP://D-NB.INFO/1048757684
4.2.3.1 WNT1 AND SEQUENTIAL FACTOR TREATMENT ON DIFFERENTIATING JM8
CELLS INCREASE
THE GENERATION OF TH
+
/PITX3
+
MDDA NEURONS 26
4.2.3.2 WNT1 TREATMENT AND LMXLA OVEREXPRESSION DO NOT ACT
SYNERGISTICALLY IN
THE GENERATION OF TH
+
/PITX3
+
NEURONS 31
4.2.4 ESTABLISHMENT OF A MONOLAYER PROTOCOL FOR THE DIFFERENTIATION OF
IPS CELLS INTO
MDDA NEURONS TO TEST THE EFFECT OF WNT/P-CATENIN SIGNALING ACTIVATION 32
4.2.4.1 INDUCTION OF THE NEURAL CELL FATE BY THE INHIBITION OF NODAL AND
BMP
SIGNALING PATHWAYS 33
4.2.4.2 INDUCTION OF THE MDDA NEURON CELL FATE AND DIFFERENTIATION INTO
MDDA
NEURONS 33
4.2.5 WNT/P-CATENIN SIGNALING AGONISTS INCREASE THE DIFFERENTIATION OF
IPS AND ES CELLS
INTO TH
+
/PITX3
+
MDDA NEURONS USING THE NEWLY ESTABLISHED PROTOCOL 34
4.2.5.1 TREATMENT WITH WNT/P-CATENIN SIGNALING AGONISTS PROMOTE THE
DIFFERENTIATION
OF PITXSF
3
* IPSC^S INTO TH
+
/PITX3
+
MDDA NEURONS 34
4.2.5.2 COMBINED DKK3 AND WNT1 TREATMENT PROMOTES THE DIFFERENTIATION OF
NESN-GFP ES CELLS INTO TH
+
/PITX3
+
MDDA NEURONS 37
4.2.6 HIGH LEVELS OF WNT/P-CATENIN SIGNALING RATHER INHIBIT TH
+
/PITX3
+
MDDA NEURON
DIFFERENTIATION OF PITX2F
SFP/
* IPSC^S IN VITRO 38
4.2.7 WNT/P-CATENIN SIGNALING AGONISTS INCREASE THE AMOUNT OF SNC-LIKE
MDDA
NEURONS AFTER DIFFERENTIATION OF P/ FX3
G
^
+
IPSC^S 42
4.2.8 REPROGRAMMING OF FIBROBLASTS INTO MDDA NEURONS 45
4.2.8.1 ESTABLISHMENT AND OPTIMIZATIONS OF THE REPROGRAMMING METHOD 45
4.2.8.2 IDENTIFICATION OF TF COMBINATIONS THAT MIGHT INDUCE THE
EXPRESSION OF PITX3
IN REPROGRAMMED MDDA NEURONS FROM FIBROBLASTS 49
4.3 DISCUSSION 56
4.3.1 GENERATION OF A VECTOR FREE REPORTER IPS CELL LINE AND THEIR
DIFFERENTIATION INTO
MDDA NEURONS 56
4.3.1.1 GENERATION OF A REPROGRAMMING-VECTOR FREE IPS CELL LINE 56
4.3.1.2 PITX3?
FP/
*
IPSC^S AS REPORTER CELL LINE FOR THEIR DIFFERENTIATION INTO MDDA
NEURONS 57
4.3.1.3 PITX$
S,P/
* IPSC^S AS A MODEL-CELL LINE FOR PD CASES CAUSED BY
POLYMORPHISMS IN THE PITX3 GENE 58
II
4.3.2 INFLUENCE OF WNT/|3-CATENIN SIGNALING ON THE DIFFERENTIATION OF
PLURIPOTENT STEM
CELLS INTO TH
+
/PITX3
+
MDDA NEURONS 58
4.3.2.1 WNT1 INCREASES THE AMOUNT OF MDDA NEURONS GENERATED BY 5 STAGE
DIFFERENTIATION PROTOCOL 59
4.3.2.2 ESTABLISHMENT OF A NEW MONOLAYER PROTOCOL FOR THE
DIFFERENTIATION OF MURINE
PLURIPOTENT CELLS INTO MDDA NEURONS 60
4.3.2.3 INFLUENCE OF WNT1, DKK3 AND GSK3|$ INHIBITION ON THE IN VITRO
DIFFERENTIATION INTO MDDA NEURONS 61
4.3.2.4 WNT/|3-CATENIN SIGNALING AGONISTS INCREASE THE GENERATION OF
MDDA NEURONS
OF THE SNC MDDA SUBTYPE 65
4.3.3 REPROGRAMMING OF FIBROBLASTS INTO MDDA NEURONS 66
4.3.3.1 GENERATION OF TH
+
/TUBB3
+
NEURONS AFTER ESTABLISHMENT OF THE
REPROGRAMMING PROCEDURE 66
4.3.3.2 GENERATION OF A FUNCTIONAL VIRUS, WHICH IS CAPABLE TO EXPRESS
THREE TFS...66
4.3.3.3 IDENTIFICATION OF A TF COMBINATION CAPABLE OF INDUCING PITX3
EXPRESSION IN
REPROGRAMMED TH
+
DA NEURONS 67
PART2: FGF/FGFR2 SIGNALING REGULATES THE SURVIVAL AND POSITIONING OF
BERGMANN GLIA CELLS
IN THE DEVELOPING MOUSE CEREBELLUM 72
5.1 INTRODUCTION 72
5.1.1 CEREBELLUM DEVELOPMENT 72
5.1.2 FGF SIGNALING IN THE CEREBELLUM DEVELOPMENT 74
5.1.2.1 FGF SIGNALING DURING THE PATTERNING PHASE OF THE CEREBELLUM 74
5.1.2.2 FGF SIGNALING AFTER THE PATTERNING PERIOD IN THE CEREBELLUM
DEVELOPMENT ..75
5.1.2.3 GRADIENTAL FGFR2 EXPRESSION DURING THE LATE EMBRYONIC CEREBELLUM
DEVELOPMENT 77
5.1.2.4 DISRUPTED CEREBELLAR STRUCTURE IN FGFR2 CKO MICE 77
5.1.3 AIMS OF THE PROJECT FGFR2 CKO IN THE CEREBELLUM DEVELOPMENT 78
5.2 RESULTS 79
5.2.1 FGFR2 MRNA IS DEPLETED AND FGFRL EXPRESSION IS REDUCED IN FGFR2
CKO
EMBRYOS 79
5.2.2 REDUCED NUMBERS AND MISPOSITIONING OF BG CELLS IN THE EGL ARE THE
PRIMARY
CEREBELLAR DEFECTS IN FGFR2 CKO MICE 79
III
5.2.3 REDUCED SURVIVAL OF MIGRATING CELLS WITHIN THE CBA BUT UNAFFECTED
PROLIFERATION
OF PC/BG PROGENITORS AND GCPS IN THE ABSENCE OF FGFR2 82
5.2.4 FGF TARGET GENE ACTIVATION IS ALMOST COMPLETELY ABOLISHED IN THE
CBA OF THE
FGFR2 CKO EMBRYOS 83
5.2.5 FGF9/FGFR SIGNALING INHIBITS THE MIGRATION OF BG CELLS IN
CEREBELLAR EXPLANTS IN
VITRO 85
5.3 DISCUSSION 89
5.3.1 DELETION OF FGFR2 DURING EMBRYONIC CEREBELLUM DEVELOPMENT 89
5.3.2 VARIABLE PENETRANCE OF THE CEREBELLAR PHENOTYPE IN FGFR2 CKO MICE
AND FGFRL
AS POSSIBLE GENETIC MODIFIER 90
5.3.3 REDUCED SURVIVAL OF BERGMANN GLIA IN FGFR2 CKO 91
5.3.4 FGFR2 AS ESSENTIAL STOP SIGNAL MEDIATOR FOR BERGMANN GLIA
MIGRATION 91
5.3.5 BERGMANN GLIA AS PRIMARY AFFECTED CELL TYPE IN FGFR2 MUTANTS 93
5.3.6 CONCLUSION 94
6 MATERIAL AND METHODS 95
6.1 MATERIAL 95
6.1.1 EQUIPMENT 95
6.1.2 DISPOSABLE MATERIAL 96
6.1.3 SOLUTIONS AND FACTORS FOR CELL CULTURE 96
6.1.4 CELL CULTURE MEDIA 98
6.1.5 SOLUTIONS FOR BACTERIA CULTIVATION 99
6.1.6 CHEMICALS 100
6.1.7 SOLUTIONS AND BUFFERS 101
6.1.8 RADIOACTIVE ISOTOPE 103
6.1.9 KITS 103
6.1.10 ENZYMES AND RESTRICTION ENZYMES 104
6.1.11 ANTIBODIES 105
6.1.11.1 PRIMARY ANTIBODIES 105
6.1.11.2 SECONDARY ANTIBODIES 106
6.1.12 PRIMERS 106
IV
6.1.12.1 PRIMERS FOR IPS CELL ANALYSIS 106
6.1.12.2 PRIMERS FOR CLONING 107
6.1.12.3 PRIMERS FOR GENOTYPING 107
6.1.13 QPCR: TAQMAN PROBES AND MASTER MIX 108
6.1.14 PLASMIDS 108
6.1.15 PLASMIDS WHICH CONTAIN THE SEQUENCES FOR RIBOPROBES 110
6.1.16 CELL LINES 111
6.1.17 BACTERIA STRAINS 111
6.1.18 MOUSE STRAINS AND LINES 112
6.1.19 SOFTWARE 112
6.2 METHODS 113
6.2.1 CULTURE OF PLURIPOTENT CELLS AND FIBROBLASTS 113
6.2.1.1 GENERAL ASPECTS OF CELL CULTURE 113
6.2.1.2 COATING OF TISSUE CULTURE DISHES/PLATES AND COVERSLIPS 113
6.2.1.3 PASSAGING OF CELLS 113
6.2.1.4 CRYO-PRESERVATION OF CELLS 113
6.2.1.5 THAWING OF CELLS STORED IN THE LIQUID NITROGEN 114
6.2.1.6 ES AND IPS CELL CULTURE 114
6.2.1.7 PREPARATION AND CULTIVATION OF MOUSE EMBRYONIC FIBROBLAST
CULTURES 114
6.2.2 DIFFERENTIATION OF ES/IPS CELLS 115
6.2.2.1 GENERAL ISSUES FOR DIFFERENTIATION OF PLURIPOTENT CELLS INTO
MDDA NEURONS....
115
6.2.2.2 DIFFERENTIATION OF IPS CELLS INTO THREE GERM LAYERS 115
6.2.2.3 DIFFERENTIATION OF ES/IPS CELLS INTO MDDA NEURONS ACCORDING TO
THE 5
STAGE PROTOCOL 115
6.2.2.4 ESTABLISHMENT OF A NEW MONOLAYER PROTOCOL FOR THE
DIFFERENTIATION OF
PLURIPOTENT CELLS INTO MDDA NEURONS 116
6.2.3 REPROGRAMMING OF FIBROBLASTS 117
6.2.3.1 REPROGRAMMING OF PITX3F
SFP/
*
FIBROBLASTS INTO IPS CELLS 117
6.2.3.2 REPROGRAMMING OF FIBROBLASTS INTO NEURONS 118
V
6.2.4 VIRUS GENERATION AND VIRUS-TITER DETERMINATION 118
6.2.5 CEREBELLAR EXPLANT CULTURES AND MIGRATION ASSAY 119
6.2.6 CHARACTERIZATION OF LIVING CELLS (IPS CELLS, MDDA NEURONS) AND
ANALYSIS OF THEIR
KARYOTYPE 120
6.2.6.1 FAC-SORTING OF PITX2F
S1P
*
MDDA NEURONS 120
6.2.6.2 TERATOMA ASSAY 120
6.2.6.3 GENERATION OF CHIMERIC MICE USING IPS CELLS 121
6.2.6.4 CHROMOSOME COUNTING 121
6.2.7 LUCIFERASE ASSAY 121
6.2.8 MUTANT MICE 122
6.2.9 MOLECULAR BIOLOGY 122
6.2.9.1 POLYMERASE CHAIN REACTION (PCR) 122
6.2.9.2 AGAROSE GEL ELECTROPHORESIS 123
6.2.9.3 ISOLATION OF DNA FRAGMENTS FROM AGAROSE GELS 123
6.2.9.4 ISOLATION OF GENOMIC DNA FROM TISSUES AND CELLS 123
6.2.9.5 ISOLATION OF TOTAL RNA 124
6.2.9.6 CDNA SYNTHESIS 124
6.2.9.7 GENOTYPING OF MICE 124
6.2.9.8 SCREENING FOR PREPROLL-ATTB VECTOR FREE IPS CELL LINES 124
6.2.9.9 DIGESTION OF DNA FRAGMENTS 125
6.2.9.10 LIGATION OF DNA FRAGMENTS INTO PLASMID- OR LENTIVIRAL-VECTORS
125
6.2.9.11 TRANSFORMATION OF CHEMICALLY COMPETENT BACTERIA 125
6.2.9.12 MINI-PREPARATION OF PLASMID DNA 125
6.2.9.13 MAXI-PREPARATION OF PLASMID DNA 125
6.2.9.14 PREPARATION OF GLYCEROL STOCK 126
6.2.9.15 CLONING OF VIRAL BACKBONE-PLASMIDS AND INSERTION OF
TRANSCRIPTION FACTOR
CODING SEQUENCES INTO VIRAL BACKBONES 126
6.2.9.16 QPCR ANALYSES OF THE SHH, WNT1 AND GAPDH EXPRESSION IN
DIFFERENTIATING
CELLS 129
6.2.10 HISTOLOGICAL AND CYTOCHEMICAL METHODS 129
VI
6.2.10.1 ISOLATION AND FIXATION OF TISSUE AND CELLS 129
6.2.10.2 PARAFFIN EMBEDDING OF TISSUE AND PREPARATION OF TISSUE SECTIONS
130
6.2.10.3 IMMUNOHISTOCHEMISTRY (IHC) 130
6.2.10.4 IMMUNOCYTOCHEMISTRY (ICC) 131
6.2.10.5 5-ETHYNYL-2 -DEOXYURIDINE (EDU) STAINING 131
6.2.10.6 HEMATOXYLIN-EOSIN STAINING 131
6.2.10.7 IN SITU HYBRIDIZATION (ISH) 131
6.2.11 CELL COUNTING AND AREA DETERMINATION 134
6.2.11.1 ANALYSES OF THE MDDA NEURONS GENERATED BY 5 STAGE
DIFFERENTIATION
PROTOCOL 134
6.2.11.2 ANALYSES OF THE MDDA NEURONS GENERATED BY THE NEWLY ESTABLISHED
MONOLAYER DIFFERENTIATION PROTOCOL 134
6.2.11.3 ANALYSES OF THE CEREBELLUM DEVELOPMENT IN FGFR2 MUTANT EMBRYOS
135
6.2.12 STATISTICAL ANALYSES (DONE BY THERESIA FAUS-KEBLER) 135
7 APPENDIX 137
7.1 SUPPLEMENTARY DATA 137
7.1.1 SUPPLEMENTARY DATA 1: FACS ANALYSIS GFP
+
MDDA DIFFERENTIATED FROM PITX3
GFP/
*
IPSC^S 137
7.1.2 SUPPLEMENTARY DATA 2: ACT VALUES OF QPCR FOR SHH EXPRESSION 138
7.1.3 SUPPLEMENTARY DATA 3: ACT VALUES OF QPCR FOR WNT1 EXPRESSION 139
7.1.4 SUPPLEMENTARY DATA 4: ACTIVATION OF A TOPFLASH REPORTER CONSTRUCT
BY THE MEDIA
COMPOSITION APPLIED AT DAY3 TO DAY5 OF THE MONOLAYER DIFFERENTIATION
PROTOCOL
INCLUDING NODAL AND BMP SIGNALING INHIBITORS SB AND LDN 140
7.1.5 SUPPLEMENTARY DATA 5: AVERAGE CLUSTER SIZE OF TH
+
/PITX3
+
CELL CLUSTERS FOR
DIFFERENT TREATMENT CONDITIONS 141
7.1.6 SUPPLEMENTARY DATA 6: VIRAL BACKBONES WHICH WERE USED OR GENERATED
IN THIS
THESIS 142
7.1.7 SUPPLEMENTARY DATA 7: AVERAGE NUMBER OF BG CELLS IN 50-PM-BINS 142
7.2 ABBREVIATIONS 143
8 REFERENCES 146
9 ACKNOWLEDGEMENTS / DANKSAGUNG
VII
161
|
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| genre_facet | Hochschulschrift |
| id | DE-604.BV041710699 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T01:03:28Z |
| institution | BVB |
| isbn | 9783000449925 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-027157896 |
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| owner_facet | DE-M49 DE-BY-TUM DE-91 DE-BY-TUM DE-12 |
| physical | VII, 161 Bl. Ill., graph. Darst. |
| publishDate | 2014 |
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| spelling | Meier, Florian Verfasser aut Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo Florian Meier 2014 VII, 161 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2014 Zsfassung in dt. Sprache (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027157896&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Meier, Florian Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |
| subject_GND | (DE-588)4113937-9 |
| title | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |
| title_auth | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |
| title_exact_search | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |
| title_full | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo Florian Meier |
| title_fullStr | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo Florian Meier |
| title_full_unstemmed | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo Florian Meier |
| title_short | Signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |
| title_sort | signaling pathways in mesodiencephalic dopaminergic and cerebellar neural development in vitro and in vivo |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027157896&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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