def (digestive-organ expansion factor) IS A CRUCIAL GENE FOR THE DEVELOPMENT OF ENDODERM-DERIVED ORGANS IN ZEBRAFISH (Danio rerio) RUAN HUA NATIONAL UNIVERSITY OF SINGAPORE 2008 def (digestive-organ expansion factor) IS A CRUCIAL GENE FOR THE DEVELOPMENT OF ENDODERM-DERIVED ORGANS IN ZEBRAFISH (Danio rerio) RUAN HUA (M.Sc., Wuhan University, P.R.China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCE NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgement I would like to express my most sincere gratitude to my supervisor, Dr. Peng Jinrong, for giving me a chance to pursue my Ph. D degree. In the past years, he not only provided insightful suggestions on my project but also trained me to become a genuine scientific researcher with the impersonal and honest attitude. Special thankfulness also goes to my PhD committee members, Dr. Cai Mingjie, Dr. Cao Xinmin and Dr. Wen Zilong for their invaluable comments and suggestions on my work and their encouragement throughout this study. I would like to extend my heartfelt thanks to my colleagues in the Functional Genomics Laboratory, Changqing, Chaoming, Chen Jun, Cheng Hui, Cheng Wei, Dongni, Evelyn, Gao Chuan, Guo Lin, Honghui, Husain, Jane, Linda, Mengyuan, Qian Feng, Shulan, Zhenhai and all other ex-members for creating a joyful and conductive working environment. In addition, I was also thankful to all other members in the exMolecular and Developmental Immunology laboratory for their valuable supports and advice on my experiments. Furthermore, I would like to show earnest appreciations of the professional supports from the fish facility, sequencing facility and histology facility in the Institute of Molecular and Cell Biology, and the financial support from the Institute of Molecular and Cell Biology. Finally, I would like to thank my parents and my family. My parents have been an incredible source of strength throughout my life, and I could not accomplish my academic without their encouragement and support. In addition, I would like to thank my husband, Honghui, for his spiritual support and invaluable suggestion on my project, and thank my son, Zhaoxi, for the joy he brings to us. i Table of Contents Acknowledgement i Table of Contents ii Summary vii List of Abbreviations . ix List of Tables . x List of Figures . xi List of Publications xiii Chapter Introduction . 1.1 Current knowledge of endoderm organ development in vertebrate 1.1.1 Morphogenesis of endoderm-derived organs . 1.1.2 Small intestine development 1.1.2.1 Structure and functions of small intestine . 1.1.2.2 Morphogenetic events of small intestine development in mouse . 1.1.2.3 Signals and factors controlling small intestine development in mouse 1.1.3 Liver development . 1.1.3.1 Structure and functions of liver . 1.1.3.2 Different stages of liver organogenesis . 1.1.3.3 Different signals and factors in controlling liver development 1.1.4 Pancreas development 1.1.4.1 Structure and functions of Pancreas 1.1.4.2 Overview of pancreas development 10 1.1.4.3 Regulation of pancreas development by signals and factors 11 1.2 Zebrafish as a good model organism 12 1.2.1 General advantages of zebrafish 13 1.2.2 Genetic analysis in zebrafish . 13 1.2.2.1 Forward genetics in zebrafish . 14 1.2.2.1.1 ENU mutagenesis screens 14 1.2.2.1.2 Insertional mutagenesis screens . 15 1.2.2.2 Reverse genetic study in zebrafish:TILLING . 16 1.2.3 Molecular techniques in zebrafish . 17 1.2.3.1 Microarray . 17 1.2.3.2 Morpholino and SiRNA 18 1.2.3.3 Transgenic fish 19 1.2.4 Genomics and zebrafish community 19 1.3 Development of endoderm-derived organs in zebrafish . 20 1.3.1 Endoderm formation in zebrafish 21 1.3.1.1 Endoderm formation and endoderm marker genes . 21 1.3.1.2 Regulators of zebrafish endoderm formation . 22 1.3.2 Intestine morphogenesis in zebrafish . 23 1.3.2.1 The anatomy of the zebrafish intestine . 23 ii 1.3.2.2 The morphogenesis of the zebrafish intestine . 24 1.3.2.3 Regulators of intestine development . 27 1.3.3 Liver morphogenesis in zebrafish 28 1.3.3.1 The morphogenetic events during liver development . 28 1.3.3.2 Regulators of liver development . 30 1.3.4 Pancreas morphogenesis in zebrafish 32 1.3.4.1 Exocrine and endocrine pancreas in zebrafish 32 1.3.4.2 The morphogenetic events occur during pancreas development 33 1.3.4.3 Regulators of pancreas development 34 1.3.4.3.1 Regulators of endocrine pancreas development 35 1.3.4.3.2 Regulators of exocrine pancreas development 37 1.4 Aim of this project 38 Chapter Material and method 47 2.1 Zebrafish . 47 2.1.1 Fish strains and maintenance . 47 2.1.2 Collection of fertilized eggs . 47 2.1.3 Collection of unfertilized eggs . 48 2.2 E. coli strains . 48 2.3 General DNA application . 48 2.3.1 Gene Cloning . 48 2.3.1.1 Polymerase Chain Reaction (PCR) . 48 2.3.1.2 Purification of PCR product/DNA fragments . 49 2.3.1.3 Plasmid DNA extraction . 49 2.3.1.4 Ligation of DNA inserts into plasmid vectors 49 2.3.1.5 Transformation of DH5α competent cells with plasmids or ligation products using a heat-shock method . 50 2.3.1.5.1 Preparation of DH5α competent cells for a long-term storage 50 2.3.1.5.2 Heat-shock transformation . 51 2.3.2 DNA sequencing 51 2.3.3 Site-directed mutagenesis 52 2.3.4 Southern Blot analysis . 52 2.3.4.1 Preparation of DIG-labeled DNA probes . 52 2.3.4.2 DNA gel electrophoresis . 53 2.3.4.3 Transfer of DNA from gel to Hybond-N membrane 53 2.3.4.4 Hybridization 53 2.4 Zebrafish genomic DNA extraction 54 2.4.1. Genomic DNA extraction from adult zebrafish 54 2.4.2 Isolation of genomic DNA from embryos or scales of adult zebrafish . 55 2.5 Genotyping defhi429+/- fish or defhi429-/- embryos . 55 iii 2.6 General RNA application 56 2.6.1 RNA extraction from embryos or adult zebrafish 56 3.6.2 Removal of genomic DNA 57 2.6.3 mRNA isolation . 57 2.6.4 5’-RACE and 3’-RACE . 57 2.6.5 Reverse Transcription PCR (RT-PCR) 57 2.6.5.1 One-step RT-PCR . 58 2.6.5.2 Two-step RT-PCR 58 2.6.6 mRNA synthesized by in vitro transcription . 58 2.6.7 Northern Blot analysis . 59 2.6.7.1 Probe preparation 59 2.6.7.2 RNA sample preparation . 59 2.6.7.3 RNA gel electrophoresis . 59 2.6.7.4 Hybridization analysis 60 2.7 General protein Application 60 2.7.1 Protein expression in E. Coli cells . 60 2.7.1.1 Heat-shock transformation of M15 or BL21 competent cells 60 2.7.2 Protein expression 61 2.7.3 Protein purification 61 2.7.3.1 Purification of soluble protein 62 2.7.3.2 Purification of insoluble protein . 62 2.7.4 Antibody generation . 63 2.7.5 Antibody affinity purification 63 2.7.6 Western Blot 64 2.7.6.1 Protein sample preparation . 64 2.7.6.2 SDS-PAGE gel electrophoresis and membrane transfer 65 2.7.6.3 Signal detection of target protein 66 2.7.7 Immunochemical whole mount staining 67 2.8 Co-immunoprecipitation (Co-IP) analysis 67 2.9 Yeast two-hybrid assay . 68 2.9.1 Bait constructs and cDNA expression library 68 2.9.2 Preparation of yeast competent cells 69 2.9.3 Transformation of yeast competent cells with plasmids 70 2.9.4 Extraction of plasmids from yeast cells . 71 2.9.5 Electroporation of XL1-Blue competent cells with plasmids 71 2.9.5.1 Preparation of XL1-Blue competent cells . 71 2.9.5.2 Electroporation 72 2.10 Microinjection . 72 2.10.1 Preparation of injected materials . 72 2.10.2 Preparation of accessory items, needles and supporter dishes . 73 2.10.3 Microinjection 73 iv 2.11 Phenol red injection 74 2.12 Sectioning of zebrafish embryo 74 2.12.1 Sectioning of paraffin-embedded embryos 74 2.12.2 Cryosectioning . 75 2.13 Whole Mount in situ Hybridization (WISH) 76 2.13.1 Preparation of DIG-labeled RNA probe 76 2.13.2 High-resolution WISH protocol . 77 2.13.3 High throughput WISH protocol . 78 2.13.4 Double WISH protocol 79 2.14 Alcine Blue staining 80 2.15 Alkaline phosphatase staining . 81 2.16 Microscope and Photograph . 81 Chapter Results 86 3.1 Characterization of defhi429 mutant 86 3.1.1 Major digestive organs in the defhi429 mutant are severely hypoplastic . 86 3.1.2 Detailed characterization of defhi429 mutant phenotype using molecular markers . 87 3.1.2.1 Def is not essential for the early development of digestive organs 88 3.1.2.2 Def is essential for the intestine expansion growth but not the endoderm– intestine transition . 89 3.1.2.3 Def is required for liver expansion growth . 90 3.1.2.4 Def is required for expansion growth of the exocrine but not the endocrine pancreas . 91 3.1.2.5 Def is also required for the growth of other endoderm organs . 92 3.1.3 Discussion 94 3.2 def gene . 95 3.2.1 5’ RACE and 3’ RACE of def gene . 95 3.2.2 Cloning and sequence analysis of def gene 95 3.2.3 def genomic DNA 96 3.2.4 Examination of def expression during embryo development 97 3.2.4.1 def expression during embryogenesis . 97 3.2.4.2 def is enriched in the digestive organs 98 3.2.5 Discussion 98 3.3 The retroviral insertion causes the defhi429 mutant phenotype 99 3.3.1 The retroviral insertion in the def gene is closely linked to the defhi429 mutant phenotype 99 v 3.3.2 Complementation Test . 100 3.3.2.1 def mRNA rescued the defhi429 mutant intestine . 100 3.3.2.2 def mRNA restored the defhi429 mutant liver to normal 101 3.3.2.3 Exocrine pancreas in the defhi429 mutant could be rescued by the def mRNA . 101 3.3.3 defhi429 mutant phenotype is mimicked in wild-type embryos by def splicing morpholino injection . 102 3.3.4 Discussion 102 3.4 Def protein 103 3.4.1 Generation of Def antibody . 103 3.4.1.1 Preparation of Def truncated proteins . 103 3.4.1.2 Def antibodies . 104 3.4.2 Def is a nuclear localized protein . 105 3.4.3 Discussion 106 3.5 Microarray . 107 3.5.1 Up-regulation of p53, mdm2 and cyclin G1 in the defhi429 mutant . 107 3.5.2 Discussion 108 3.6 Yeast two-hybrid to identify protein interacting with Def 109 3.6.1 Yeast two-hybrid assay 109 3.6.1.1 Construction of a cDNA expression library and def-baits for yeast twohybrid assay 109 3.6.1.2 Identification of proteins interacting with Def via yeast two-hybrid screen . 111 3.6.2 Preliminary analysis of genes obtained from yeast two-hybrid screen by WISH . 113 3.6.3 Discussion 114 3.7 Functional studies of five Def interacting proteins, Rybp, Appbp2, L159, L221 and L245 115 3.7.1 rybp gene 115 3.7.1.1 Rybp interacts with Def protein in vivo 117 3.7.1.2 rybp expression pattern in zebrafish embryogenesis 117 3.7.1.3 Rybp was antagonistic to Def protein during intestine organogenesis . 118 3.7.2 appbp2 119 3.7.3 L159 . 121 3.7.4 L221 . 122 3.7.5 L245 . 124 3.7.6 Discussion 125 Chapter Conclusions 162 Reference List 168 vi Summary Digestive organs are essential in the human body. The vertebrate digestive organs are all derived from the endoderm. Endoderm development is conserved among the vertebrates and is characterized by several basic morphogenesis processes, including endoderm formation, gut formation, organ budding, and cell differentiation and proliferation within the organ buds. Through studies in frog, chicken and mouse, several key regulators important for the development of digestive organs have been identified. However, due to the complexity of the endoderm organogenesis, little is known about the molecular mechanisms underlying these factors. Owing to its advantages for genetic and developmental studies, zebrafish has recently emerged as a good model organism to study the digestive organogenesis. The main aim of this study is to determine the functional role of the def (digestive-organ expansion factor) gene in the development of endoderm-derived organs through studying a loss-of-function mutation in the def gene in zebrafish. The defhi429 mutation is caused by a retroviral vector insertion in the second intron of the def gene. Characterization of defhi429 mutants using different organ specific markers showed that the def mutation affected cell proliferation, not cell differentiation, in the developing digestive organs except the endocrine pancreas at the later stage of endoderm organogenesis. The mutant phenotype coincides with the spatial expression pattern of def. The data from the complementation test and morpholino knockdown assay confirmed that the retroviral insertion in the def gene resulted in the compromised growth of the digestive organs in the defhi429 mutant. vii Immunostaining revealed that def encodes a novel nuclear-localized pan-endodermspecific factor. To identify Def interacting proteins functioning in regulation of the growth of the digestive organs, Def was used as a bait in yeast two-hybrid screening and 16 candidates showing strong interaction with Def were identified. Whole-mount in situ hybridization showed that 15 candidates are enriched in one or more digestive organs during embryogenesis. To gain further insight into the biological functions of these Def interacting proteins, we designed gene-specific morpholinos targeting appbp2, L159, L221, L245 and rybp, five genes and observed that knock-down of appbp2, L159, L221 and L245 in developing zebrafish embryos caused a phenotype mimicking the phenotype of defective digestive organs in the defhi429 mutant. In contrast, rybp MO did not cause obvious phenotype in the wild type embryos but could partially rescue ifabp expression in the intestine in the defhi429 mutant. Co-IP showed that Def and Rybp physically interact with each other in vivo. 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Molecular basis of vertebrate endoderm development. Int. Rev Cytol. 259, 49-111. 184 [...]... anemia Thirdly, the anatomy, function and cell composition of the digestive organs in zebrafish are very similar to those in the mammalian, the knowledge obtained from the study of the development of endoderm- derived organs in zebrafish will be directly applicable to other vertebrates As in the mammalian, the swim bladder and all digestive organs in zebrafish, including intestine, liver, pancreas and gall... organs, endoderm formation in zebrafish is briefly summarized before reviewing zebrafish intestine, liver and pancreas organogenesis 1.3.1 Endoderm formation in zebrafish 1.3.1.1 Endoderm formation and endoderm marker genes Fate mapping studies in zebrafish have shown that the endoderm progenitors are located within the four-cell-diameter region close to the blastoderm margin (Kimmel et al., 1990; Warga and... out in zebrafish, the literature review has also introduced the advantages of using zebrafish as a genetic model for studying vertebrate development and followed by a review on our current knowledge about endoderm organogenesis in zebrafish 1.1 Current knowledge of endoderm organ development in vertebrate 1.1.1 Morphogenesis of endoderm- derived organs In vertebrates, the development of endoderm and endoderm- derived. .. stock that contains mutations in all zebrafish genes, just as the T-DNA insertion line collection in Arabidopsis That means any desired mutant could be directly obtained from a library containing all zebrafish mutagenized genes Recently, Wang et al reported that they were establishing a retroviral insertion library made by cryopreserving sperm samples containing zebrafish gene disruptions (Wang et al.,... model organisms to bridge the gap between nematode/fly and mouse/human genetics for better understanding of life 1.2.1 General advantages of zebrafish Zebrafish was firstly described in the study of inheritance as early as in 1973 (Kosswig, 1973) However, it was until recently that this small animal became an excellent model for genetic and developmental studies in vertebrate Zebrafish has many advantages... differentiation timing of transplanted endoderm intrinsically occurs according to the correct anterior-posterior wave (Rubin et al., 1992; Duan et al., 1993; Falk et al., 1994) These data suggests that the intestinal anlage holds all necessary information for correct patterning of the small intestine along the A- P axis before E15 Expression patterns and results of gene targeting knock-out have revealed that... http://zfin.org, provides all information, including genomic sequences, mutants and gene expression pattern database This website provides not only a large amount of information about zebrafish but also serve as a unique platform to share and exchange experiences among researchers within or outside the zebrafish community 1.3 Development of endoderm- derived organs in zebrafish The advantages for embryological... of using zebrafish as the model organism 18 1.2.3.3 Transgenic fish Transgenic approach is another powerful tool for the analysis of gene function during development Injection of either plasmid DNA or BAC into the cytoplasm of one-cell stage embryos is a reliable method for making transgenic lines to express the gene of interest For example, the Tg(gut GFP)s854 transgenic line (gutGFP) was generated... The size of the zebrafish genome is about 1.5-1.63 x 109 base pairs and is predicted to contain around 23,500 genes The total number of genes in zebrafish is very similar to that in the mammalian With the high homology among zebrafish, human and mouse, studies from the zebrafish will provide a powerful means to elucidate the complexities of the development in vertebrates To date, about 75% of zebrafish. .. essential Angioblasts, precursors of endothelial cells, start to appear near the hepatoblasts at the stage of hepatic specification During the outgrowth of the liver bud, primitive endothelial cells invade in the STM as the hepatoblasts and eventually form the vascular structure in the nascent liver (Matsumoto et al., 2001) The hepatoblasts remain in a morphologically undifferentiated state until day 12 of . 1.2 Zebrafish as a good model organism 12 1.2.1 General advantages of zebrafish 13 1.2.2 Genetic analysis in zebrafish 13 1.2.2.1 Forward genetics in zebrafish 14 1.2.2.1.1 ENU mutagenesis. NATIONAL UNIVERSITY OF SINGAPORE 2008 def (digestive- organ expansion factor) IS A CRUCIAL GENE FOR THE DEVELOPMENT OF ENDODERM- DERIVED ORGANS IN ZEBRAFISH (Danio rerio) . These data suggests that the intestinal anlage holds all necessary information for correct patterning of the small intestine along the A- P axis before E15. Expression patterns and results of gene