Gerstner N. Endocytic modulation of developmental signaling during zebrafish gastrulation (Dresden, 2014). - ОГЛАВЛЕНИЕ / CONTENTS

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ОбложкаGerstner N. Endocytic modulation of developmental signaling during zebrafish gastrulation: Diss. ... Dr. rer.nat. - Dresden: Technische Universitat Dresden, 2014. - vii, 142 p.: ill. - Bibliogr.: p.129-137.
 

Место хранения: 02 | Отделение ГПНТБ СО РАН | Новосибирск

Оглавление / Contents
 
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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