Title Page
Contents
Chapter 1. Bmp4 synexpression gene, sizzled, transcription is collectively modulated by Smad1 and Ventx1.1 and Ventx2.1 in early Xenopus embryos 15
ABSTRACT 16
1. INTRODUCTION 17
2. MATERIALS AND METHODS 21
2.1. Ethic declaration 21
2.2. Nucleic acids (DNA, RNA) preparation 22
2.3. Cloning of szl, bmp7.1 and bambi promoter constructs 22
2.4. Xenopus embryo microinjection 23
2.5. Embryos animal cap explants culture 24
2.6. RT-PCR experiment 24
2.7. Quantitative RT-PCR (qRT-PCR) analysis 24
2.8. Luciferase reporter gene assay 24
2.9. Site-directed mutagenesis of Smad1 and Ventx response elements (SRE and VRE) 25
2.10. Chromatin immunoprecipitation (ChIP) assay 25
2.11. Immunoprecipitation assay 26
2.12. Statistical analysis 26
3. RESULTS 31
3.1. Spatio-temporal expression of szl and Bmp-mediated modulation of bmp7.1 and bambi promoter activities 31
3.2. Isolated szl(-1566bp) promoter contains the Bmp4 regulatory elements required for Bmp4 synexpression 35
3.3. Direct Smad1 binding on Bmp4/Smad1 response cis-acting element (BRE) site of szl promoter is essential for Bmp4 synexpression 42
3.4. Two direct targets of Bmp4 signaling, Ventx1.1 and Ventx2.1 oppositely modulate but Ventx1.1/2.1 together jointly upregulate szl transcription 48
3.5. Site-directed mutagenesis of one VRE site on the szl promoter abolishes Ventx1.1 as well as Ventx2.1 mediated szl transcription regulation 54
3.6. The triad combination of Smad1 and Ventx1.1/Ventx2.1 collectively modulate szl transcription 59
4. DISCUSSION 66
4.1. Szl is a direct target as well as a secondary response gene of Bmp4 signaling 66
4.2. Ventx1.1 enhances Ventx2.1's stimulatory effect 68
4.3. Smad1 and Ventx1.1/Ventx2.1 collectively upregulates szl expression 70
5. REFERENCES 73
Chapter 2. Molecular cloning and transcription regulation of tbc1d24.2.L by Bmp signaling during early Xenopus laevis embryo 82
ABSTRACT 83
1. INTRODUCTION 84
2. MATERIALS AND METHODS 86
2.1. Ethic declaration 87
2.2. Xenopus laevis embryos manipulation 87
2.3. Cloning of tbc1d24.2.L promoter 87
2.4. Constructs and mRNA preparation 88
2.5. Embryos animal cap explants culture 88
2.6. RNA isolation and cDNA preparation 88
2.7. Luciferase reporter gene assay 89
2.8. eGFP fluorescence 89
2.9. Statistical analysis 89
3. RESULTS 94
3.1. Cloning of Xenopus laevis tbc1d24.2.L gene and analysis of protein homology with other vertebrates 94
3.2. Xenopus laevis tbc1d24.2.L is zygotically expressed in ventral region of the embryo during early development 100
3.3. Tbc1d24.2.L overexpression gives gastrulation defects and headless embryos 104
3.4. Bmp signaling modulates isolated promoter of tbc1d24.2.L(-2887 bps) during early gastrula stage 108
4. DISCUSSION 111
5. REFERENCES 115
Chapter 3. The hairy and enhancer of split 3 (hes3) disrupts Bmp signaling to promote neural fate in Xenopus embryos animal cap explants 121
ABSTRACT 122
1. INTRODUCTION 124
2. MATERIALS AND METHODS 126
2.1. Ethics statement 126
2.2. Xenopus laevis embryos manipulation 127
2.3. Constructs and mRNA preparation 127
2.4. Embryos animal cap explants culture 128
2.5. RNA isolation and cDNA preparation 128
2.6. Luciferase assay 128
2.7. Western blotting 128
2.8. Statistical analysis 129
3. RESULTS 131
3.1. Developmental expression pattern of Xenopus laevis hes3.s is zygotically expressed in dorsal region of embryo 131
3.2. Analysis of Xenopus laevis hes3.S protein sequence homology with other vertebrates 134
3.3. Xenopus laevis hes3.S overexpression induces neural genes expression in animal cap explants 138
3.4. Hes3.S overexpression inhibits Bmp4 signaling mediator, Smad1's C-terminal phosphorylation and its targets in animal cap explants 142
4. DISCUSSION 146
4.1. Hes3.S is a zygotic gene and expressed from mid blastula transition (MBT) stage 146
4.2. Hes3.S as a neural inducer factor 147
4.3. Hes3.S behaves like Bmp signaling antagonist 147
5. REFERENCES 150
Abstract 156
Chapter 1. Bmp4 synexpression gene, sizzled, transcription is collectively modulated by Smad1 and Ventx1.1/Ventx2.1 in early Xenopus embryos 11
Table 1-1. Primers for szl.L, bmp7.1.l and bambi.s promoters 27
Table 1-2. Marker genes primers used for RT-PCR amplification 29
Table 1-3. Primers for BRE and VRE site-directed mutagenesis 30
Table 1-4. ChIP-PCR primers 30
Chapter 2. Molecular cloning and transcription regulation of tbc1d24.2.L by Bmp signaling during early Xenopus laevis embryos 11
Table 2-1. List of marker genes primers used for RT-PCR amplification 90
Table 2-2. Primers used to clone tbc1d24.2.L promoters into pGL3.luc+ and pGL3.eGFP+ vectors 91
Table 2-3. Primers used to clone tbc1d24.2.L CDS region into pCS4.3Flag vector 92
Table 2-4. TBC domain containing genes list from DNBR transcriptome. 93
Chapter 3. Hairy and enhancer of split 3 (hes3) disrupts Bmp signaling to promote neural fate in Xenopus embryos animal cap explants 11
Table 3-1. List of marker genes primers used for RT-PCR amplification 130
Chapter 1. Bmp4 synexpression gene, sizzled, transcription is collectively modulated by Smad1 and Ventx1.1/Ventx2.1 in early Xenopus embryos 12
Figure 1. Developmental expression pattern of szl and Bmp4-mediated modulation of synexpression genes promoter activities. 34
Figure 2. Bmp4 signaling modulates the isolated promoter activity of szl(-1566bps) mimicking endogenous mRNA expression. 41
Figure 3. Bmp4 signaling upregulates szl reporter gene activity via Smad1 regulatory response elements within the szl promoter. 47
Figure 4. Ventx1.1 and Ventx2.1 mediated szl transcriptional modulation acts as a secondary Bmp4 signaling response. 53
Figure 5. One VRE (ventx cis-acting response element) site for Ventx1.1/2.1 binding in szl promoter is required for szl transcription. 58
Figure 6. Smad1 enhances Ventx2.1-mediated szl activation and rescues Ventx1.1 mediated szl suppression. 65
Figure 7. Proposed model 72
Chapter 2. Molecular cloning and transcription regulation of tbc1d24.2.L by Bmp signaling during early Xenopus laevis embryos 12
Figure 1. Tbc1d24.2.L gene sequence (promoter and CDS region) analysis and comparison with human and mouse Tbc1d24 proteins. 99
Figure 2. Tbc1d24.2.L spatio-temporal expression in early Xenopus embryos. 103
Figure 3. Morphological effect of cloned tbc1d24.2.L on Xenopus embryos during early developmental stages. 107
Figure 4. Tbc1d24.2.L cloned promoter was modulated by Bmp4 signaling and a neural transcription factor, Foxd4l1.1 during early embryogenesis. 110
Figure 5. Putative model 114
Chapter 3. Hairy and enhancer of split 3 (hes3) disrupts Bmp signaling to promote neural fate in Xenopus embryos animal cap explants 14
Figure 1. Spatio-temporal expression of hes3.S gene in early developmental stages of Xenopus embryos. 133
Figure 2. Xenopus laevis Hes3 protein and its domains comparison with human and mouse Hes3 protein. 137
Figure 3. Hes3 mRNA overexpression induces neural genes in ectodermal explants. 141
Figure 4. Hes3 inhibits Bmp4 signaling via blocking Smad1 C-terminal phosphorylation and its target genes. 145
Figure 5. Putative model of Hes3-mediated inhibition of Bmp signaling and its target genes at different levels of signal transduction. 149