1 INTRODUCTION ................................................. 1
1.1 Cell Signaling (Biological Information Processing) ...... 2
1.1.1 The general logic of cell signaling .............. 2
1.1.2 WNT signaling .................................... 5
1.1.3 Developmental signaling .......................... 8
1.2 Endocytosis and the organization of the endocytic
system ................................................. 10
1.2.1 The multiple endocytic entry routes ............. 11
1.2.2 Compartment identity and regulation of
endocytic trafficking by Rab GTPases ............ 13
1.2.3 The early endosome-specific Rab GTPase Rab5 ..... 15
1.2.4 Endocytic cargo sorting ......................... 16
1.3 Regulation of signal transduction by endocytic
trafficking ............................................ 18
1.3.1 Mechanisms: signal attenuation, signal
propagation, signal amplification ............... 19
1.3.2 Endosomes function as intracellular signaling
platforms ....................................... 20
1.3.3 Regulation of developmental signaling by
endocytic trafficking ........................... 22
1.4 The Zebrafish as a model system to investigate
developmental signaling ................................ 25
1.4.1 Overview of early zebrafish development (1st
day of development) ............................. 26
1.4.2 The complex morphogenetic movements during
zebrafish gastrulation .......................... 28
1.4.3 Developmental signaling during zebrafish
gastrulation .................................... 32
1.5 The novel Rab5 effector P95 ............................ 37
2 PROJECT AIMS ................................................ 40
3 RESULTS ..................................................... 41
3.1 Full-length zebrafish P95 is expressed during early
zebrafish embryogenesis ................................ 41
3.1.1 Zebrafish full-length P95 is the homolog of
human full-length P95 ........................... 41
3.1.2 Full-length zebrafish P95 is ubiquitously
expressed during zebrafish gastrulation ......... 43
3.2 The novel Rab5 effector P95 is essential for
zebrafish embryogenesis ................................ 44
3.2.1 Systemic interference with zfP95 expression
results in complex morphogenetic defects
during the first day of zebrafish
embryogenesis ................................... 45
3.2.2 The P95 morphant phenotypes can be partially
rescued with human P95 mRNA ..................... 49
3.3 P95 is required for patterning and morphogenetic
movements during zebrafish gastrulation ................ 50
3.3.1 Systemic interference with zfP95 expression
causes an expansion of the dorsal organizer at
the onset of zebrafish gastrulation ............. 52
3.3.2 P95 MO KD results in moderate C/E defects,
while P95 OE disrupts patterning during
zebrafish gastrulation .......................... 55
3.4 P95 is required for WNT/β-catenin signaling during
zebrafish gastrulation ................................. 59
3.4.1 P95 knockdown, but not overexpression,
significantly reduces nuclear translocation of
β-catenin ....................................... 59
3.4.2 P95 knockdown induces downregulation of WNT/β-
catenin signaling during zebrafish
gastrulation .................................... 66
3.5 P95 is required to maintain activity of developmental
signaling .............................................. 68
3.6 P95 localizes to Rab5 endosomes and modulates the
endosomal recruitment of β-catenin in vivo ............. 71
3.6.1 P95 localizes to Rab5 early endosomes in
shield-stage zebrafish embryos .................. 71
3.6.2 Systemic interference with zfP95 expression
does not disrupt the early endocytic system in
vivo ............................................ 79
3.6.3 P95 MO KD amplifies the recruitment of
β-catenin to early endosomes in vivo ............ 81
3.6.4 P95 MO KD affects the sub-cellular
distribution of E-Cadherin ...................... 88
4 DISCUSSION .................................................. 92
4.1 P95 as novel endocytic protein in vertebrate
development ............................................ 92
4.2 The functional requirement of P95 for zebrafish
gastrulation ........................................... 94
4.3 The P95-dependent modulation of developmental
signaling .............................................. 97
4.4 The significance of endosomal transport of β-catenin ... 98
5 CONCLUSION & PERSPECTIVE ................................... 102
6 METHODS & MATERIALS ........................................ 105
6.1 Methods ............................................... 105
6.1.1 Zebrafish animal husbandry, embryo collection
and staging of embryos ......................... 105
6.1.2 Zebrafish lines used in this study ............. 105
6.1.3 Cloning of the zebrafish full-length P95
coding sequence ................................ 106
6.1.4 Synthesis of mRNA for microinjection ........... 109
6.1.5 Microinjections of Morpholinos and mRNA ........ 109
6.1.6 Heat-Shock of the transgenic line
Tg(Hsp70:Wnt3a+exon3-HA-EGFP) .................. 110
6.1.7 Transplantation experiments .................... 111
6.1.8 Dechorionation of embryos ...................... 111
6.1.9 Whole-mount in situ hybridization (WISH) ....... 112
6.1.10 Basic light microscopy of living and fixed
whole-mount embryos ............................ 114
6.1.11 Quantification of embryonic expression
domains and epiboly stages ..................... 114
6.1.12 Quantification of signaling activity in the
transgenic reporter lines ...................... 114
6.1.13 Whole-mount antibody staining for
immunofluorescence (IF) detection .............. 115
6.1.14 The "shield-stage imaging assay": Imaging of
fixed shield-stage zebrafish embryos across
several scales of biological organization ...... 116
6.1.15 Image acquisition by confocal laser scanning
microscopy ..................................... 117
6.1.16 Image analysis ................................. 118
6.1.17 Protein immunoblotting (Western Blot) and
quantitative detection of protein levels ....... 119
6.1.18 In vitro transcription/translation to confirm
P95 protein expression ......................... 120
6.1.19 Statistical analysis ........................... 121
6.2 Materials ............................................. 122
6.2.1 Antibodies used in this study .................. 122
6.2.2 DNA constructs (Plasmids) ...................... 123
6.2.3 Morpholino antisense oligos .................... 124
6.2.4 PCR Primer ..................................... 125
6.2.5 Buffer solutions ............................... 126
6.2.6 Chemicals, molecular biology kits and
reagents ....................................... 126
6.2.7 Technical equipment ............................ 128
7 REFERENCES ................................................. 129
7.1 Paper ................................................. 129
7.2 Textbooks ............................................. 137
7.3 Internet Platforms and Databases ...................... 138
8 DECLARATION ................................................ 139
9 ERKLÄRUNG .................................................. 140
10 ACKNOWLEDGEMENTS ........................................... 141
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