Mypt1-Mediated Spatial Positioning of Bmp2-Producing Cells Is Essential for Liver Organogenesis HUANG HONG HUI NATIONAL UNIVERSITY OF SINGAPORE 2008 Mypt1-Mediated Spatial Positioning of Bmp2-Producing Cells Is Essential for Liver Organogenesis HUANG HONG HUI (M.Sc., China Pharmaceutical University, P.R.China) (B.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 My Ph.D. study is really a long and tough journey and I am glad I am reaching the destination. I would like to thank my supervisor Dr. Peng Jinrong and my ex-supervisor Dr. Zhang Lianhui. Starting with a bacterial quorum sensing project in Dr. Zhang Lianhui’s lab, I learned the first molecular biology experiment, and when Dr. Peng Jinrong showed me the fantastic zebrafish model, I decided to initiate the liver development study in this organism under his supervision. During past nearly seven years, he has put extensive effort to systematically train me to be a good scientist, not only the correct way of doing science, but also the right attitude to thinking about science which definitely will benefit me in my career. At the same time, I want to thank my supervisory committee, Dr. Wen Zilong, Dr. Li Baojie, and Dr. Lim Seng Gee for their invaluable comments and advice throughout this study. I would like to express my sincere gratitude to my colleagues in the Functional Genomics Laboratory, Dr. Lee Sorcheng, Dr. Chen Jun, Dr. Cheng Wei, Dr. Alamgir Hussain, Aw Meng Yuan, Aw Siqi, Cao Dongni, Chang Changqing, Cheng Hui, Gao Chuan, Guo Lin, Lo Lijan, Low Swee Ling, Lu Peiying, Ma Weiping, Ng Sok Meng, Ruan Hua, Xu Min, Yang Shulan, Wen Chaoming, Wu Wei, Zhang Zhenhai and all other ex-members. Together with Guo Lin we carried out a fruitful genetic screening for liver defective mutant in zebrafish. My special thanks go to Ruan Hua and Aw Meng Yuan, they help me to perform huge amount of bench work. I would like to thank Dr. Alamgir Hussain for his great help of biochemical assay of the Mypt1-PP1c complex and stress fibres. My thanks also go to all the members of Molecular and Developmental Immunology Laboratory, especially Qian Feng, Jin Hao, Xu Jin, Liu Yanmei, Du Linsen. We shared the great experience of collaboration in the genetic screening and positional cloning. The constructive discussions and suggestions in the joint lab meeting indeed were of great help to my study, particularly the valuable comments from Dr. Chen Jun, Qian Feng, Jin Hao and Xu Jin. I would like also thank Zhang Zhenhai, my badminton partner and friend; I really enjoyed the friendship and joy in badminton game he brought to me. My special thanks go to Dr. Wen Zilong, his helpful suggestions and encouragements always are pushing me forward. I would like to say thanks to Prof. David Kimelman, Dr. Thomas Leung, and Dr. Song Haiwei for their precious help and suggestion to make my work publishable. I would like to thank for the financial support from the Institute of Molecular and Cell Biology and ex-Institute of Molecular Agrobiology for my Ph.D. study. My thanks also go to the fish facility, the sequencing facility, administration team, and technical supporting team of these two institutes for their great support. Finally, I would like to thank my parents and my family. My wife, Ruan Hua, and my lovely son, Huang Zhaoxi, always are my spiritual support and source of strength to move forward. i Table of Contents Acknowledgement . i Table of Contents ii Summary vi List of Abbreviations . viii List of Tables . ix List of Figures . x List of Publications . xii Chapter Introduction 1.1 The liver structure and functions 1.1.1 The liver structure 1.1.1.1 The hepatic vascular system . 1.1.1.2 The biliary system . 1.1.1.3 The three dimensional arrangements of the liver cells . 1.1.2 The liver functions . 1.2 Liver organogenesis 1.2.1 Liver is an endoderm derived organ 1.2.2 The liver morphogenesis 1.2.3 Molecular mechanism involved in the liver development . 11 1.2.3.1 Acquisition of competency . 11 1.2.3.2 Hepatic specification . 15 1.2.3.3 The liver bud formation and growth . 17 1.2.3.3.1 The liver bud formation . 17 1.2.3.3.2 Growth and apoptosis of hepatoblasts . 20 1.2.3.4 Hepatocyte differentiation and establishment of hepatic architecture 23 1.2.3.5 Cholangiocyte differentiation . 25 1.3 Zebrafish: an ideal model for studies of the liver development 29 1.3.1 Advantages of zebrafish . 29 1.3.2 Liver development study in zebrafish 34 1.4. Rationality and aim of the project 43 Chapter Material and method . 45 2.1 Zebrafish . 45 2.1.1 Fish strains and maintenance . 45 2.1.2 zebrafish embryos 46 2.1.3 Collection of unfertilized eggs . 46 2.2 General DNA application . 46 2.2.1 DNA fragment Cloning 46 2.2.1.1 Polymerase Chain Reaction (PCR) . 46 2.2.1.2 Purification of PCR product/DNA fragments . 47 2.2.1.3 Ligation of DNA inserts into vectors 47 2.2.1.4 Heat-shock transformation 48 2.2.1.4.1 Preparation of DH5α competent cells 48 2.2.1.4.2 Heat-shock transformation . 48 2.2.1.5 Colony PCR 49 2.2.1.6 Plasmid miniprep from bacteria 49 ii 2.2.2 DNA sequencing 49 2.2.3 Site directed mutagenesis . 50 2.3 Zebrafish genomic DNA extraction 50 2.3.1. Genomic DNA extraction from adult zebrafish 50 2.3.2 Isolation of genomic DNA from embryos or scales of adult zebrafish . 51 2.4 General RNA application 51 2.4.1 Total RNA extraction from embryos or adult zebrafish 51 2.4.2 Removal of genomic DNA from total RNA 52 2.4.3 mRNA isolation . 52 2.4.4 Reverse Transcription PCR (RT-PCR) 52 2.4.4.1 One-step RT-PCR . 52 2.4.4.2 Two-step RT-PCR 53 2.4.5 Capped mRNA synthesis by in vitro transcription 53 2.4.6 Northern Blot analysis . 53 2.4.6.1 Probe preparation 53 2.4.6.2 RNA sample preparation . 54 2.4.6.3 RNA denaturing gel electrophoresis . 54 2.4.6.4 Hybridization and autoradiography 55 2.5 Western Blot . 55 2.5.1 Protein sample preparation 55 2.5.2 SDS-PAGE and blot 56 2.6 Cryosectioning of zebrafish embryo . 57 2.7 Immunochemistry . 58 2.7.1 Whole mount antibody staining . 58 2.7.2 Antibody staining on sectioned samples 58 2.8 Microinjection . 59 2.8.1 Preparation of injected materials . 59 2.8.2 Preparation of accessory items, needles and supporter dishes . 59 2.8.3 Microinjection 60 2.9 Whole Mount in situ Hybridization (WISH) 60 2.9.1 Preparation of labeled RNA probe . 60 2.9.2 High-resolution WISH . 61 2.9.3 Two-color WISH . 62 2.9.4 High throughput WISH protocol . 63 2.10 SSLP and SNP marker detection 64 2.11 Assay of the Mypt1-PP1c Complex 64 2.11.1 Constructs 64 2.11.2 Co-immunoprecipitation (Co-IP) and Immunoblotting . 65 2.12 Stress Fiber Assay . 65 2.13 Mosaic Analysis via Cell Transplantation 66 2.13.1 Mutant donor cells to WT embryos for endoderm replacement 66 2.13.2 Wild-type mesoderm donor cells to mypt1 morphants for mesoderm replacement . 66 2.14 Mutant Rescue 67 2.15 Heatshock Treatment 67 2.16 p-Histone H3 Immunostaining and TUNEL Assay 68 iii Chapter Forward genetic screen for zebrafish liver defective mutants 74 3.1 Introduction . 74 3.2 Results . 74 3.2.1 Mutagenesis and generation of families for screen 74 3.2.2 Forward genetic screen 76 3.2.2.1 Setup of a high-throughput whole mount in situ hybridization (WISH) method for screen 76 3.3.2.2 The scheme of screen 76 3.2.2.3 First round screen (screen in F2 families) . 79 3.2.2.4 Second round screen (screen in F3 families) 81 3.2.2.5 Allelism test 84 3.2.2.6 Third round screen (screen in F4 families) . 84 3.3 Discussions . 91 Chapter Positional cloning reveals that a mutation alters a conserved motif in Mypt1 in sq181 . 95 4.1 Introduction . 95 4.2 Results . 99 4.2.1 Construction of the initial mapping panel 99 4.2.2 Positional cloning of sq181 100 4.2.2.1 Initial mapping of sq181 . 100 4.2.2.2 Fine mapping and chromosomal walking on BAC contig 103 4.2.2.3 The sq181 mutation alters a conserved motif in Mypt1 . 108 4.2.3 V36 to M36 substitution in Mypt1 causes the liverless phenotype in sq181 . 108 4.2.4 An insertion allele of sq181 also confers a liverless phenotype 111 4.2.5 Knockdown of mypt1 gene phenocopies the liverless phenotype in sq181 . 112 4.3 Discussions . 112 Charpter Mypt1-mediated spatial positioning of Bmp2-producing cells is essential for liver organogenesis . 115 5.1 Introductions . 115 5.2 Results . 118 5.2.1 The mypt1sq181 mutation confers a liverless phenotype . 118 5.2.2 The mypt1sq181 mutation does not block hepatic competency 118 5.2.3 The mypt1sq181 mutant hepatoblasts are not maintained . 120 5.2.4 The mutant Mypt1 binds PP1c poorly and is functionally attenuated . 123 5.2.5 Knockdown of PP1c phenocopy mypt1sq181 . 124 5.2.6 mypt1 is expressed in the liver primordium and surrounding lateral plate mesoderm 128 5.2.7 The mypt1sq181 mutation causes the liverless phenotype in a tissue nonautonomous manner 130 5.2.8 The mypt1sq181 mutation causes abnormal bundling of actomyosin filaments and disorganization of LPM cells . 130 5.2.9 Bmp2a rescues the liver development in mypt1sg181 135 5.2.10 Blocking Bmp signaling causes the liverless phenotype . 137 5.2.11 M36-Mypt1 disrupts the spatial coordination between the liver primordium and Bmp2a-producing cells 140 5.2.12 Mutant hepatoblasts are impaired in proliferation . 146 iv 5.2.13 Mutant hepatoblasts undergo apoptosis to cause the liverless phenotype . 146 5.3 Discussions . 149 5.3.1 V36 to M36 in Mypt1 confers the liverless phenotype 149 5.3.2 The myptsq181 mutation causes defective LPM displacement . 150 5.3.3 The defective LPM displacement leads to the failure of establishment a proper spatial positioning between Bmp2a producing cells and the liver primordium to support hepatoblasts proliferation . 150 5.3.4 Bmp signaling is essential for the hepatoblasts specification and proliferation . 152 5.3.5 A posterior shift of the liver primordium also appears in the mypt1sq181 mutant . 153 Chapter General conclusion and future prospects . 156 Appendix1………………………………………………………………………………160 Appendix2………………………………………………………………………………162 Reference list ………………………………………………………………………… 163 v Summary The liver is an essential organ and carries out many essential functions. Most studies in the liver development are carried out in mice and chick using reverse genetics and explants culture method, however, the whole picture of liver organogenesis is still mysterious due to limitations of such approaches and the early lethality of liver defect in mouse. Zebrafish emerges as an ideal model for forward genetics and liver organogenesis. To investigate molecular mechanism of the liver development without bias, we carried out a forward genetic screen for liver defective mutants in zebrafish assisted with a high throughput whole mount in-situ hybridization method using the liver specific marker prox1 as a probe. After screening 524 mutagenized genomes, we obtained 71 putative mutants which came from 51 F2 families. Of these mutants 19 lines showed liver defects with relatively normal morphology and were considered as interesting mutants for further study. To initiate positional cloning to reveal molecular lesion in a liverless mutant sq181 obtained in our screen, a total of 451 simple sequence length polymorphism (SSLP) markers from established panels were tested in our lines, and 226 markers that showed polymorphism were selected for construction of the initial mapping panel. Positional cloning identified a G to A substitution in the myosin phosphatase targeting subunit (mypt1) gene in sq181 mutants, which results in the V36 to M36 substitution in an RVxF motif in Mypt1. Genetic analyses unequivocally prove that the mypt1sq181 mutation is responsible for the liverless phenotype. Previous studies showed that mesodermal tissues produce various inductive signals essential for morphogenesis of endodermal organs. However, little is known about how vi the spatial relationship between the mesodermal signal-producing cells and their target endodermal organs is established during morphogenesis. The mypt1sq181 mutation attenuates the binding of Mypt1 to PP1c and leads to a compromised myosin phosphatase activity, and causes abnormal bundling of actin filaments and disorganization of lateral plate mesoderm (LPM) cells around the hepatic endoderm. As a result, the coordination between mesoderm and endoderm cell movements is disrupted. Consequently, the two stripes of Bmp2a-expressing cells in the LPM fail to align in a V-shaped pocket sandwiching the liver primordium. Mispositioning Bmp2a producing cells with respect to the liver primordium leads to a reduction of hepatoblast proliferation and final abortion of hepatoblasts by apoptosis that causes the liverless phenotype. Our results demonstrate that Mypt1 mediates coordination between mesoderm and endoderm cell movements in order to carefully position the liver primordium such that it receives a Bmp signal that is essential for liver formation in zebrafish. vii List of Abbreviations aa AP BCIP BMP BSA bp CIP DEPC DIG DMSO DNA dNTP dpf DTT EGFP ENU GFP hpf IPTG Kb LPM M MO mRNA NBT ng nl ORF PAGE PBS PCR PFA PTU RT-PCR SSC STM UV μl WISH amino acid alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate bone morphogenetic protein bovine serum albumin base pair calf intestinal alkaline phosphatase diethylpyrocarbonate digoxigenin dimethyl sulfoxide deoxyribonucleic acid deoxyribonucleotide triphosphate days post-fertilization dithiothreitol enhanced green fluorescence protein N-ethyl-N-nitrosourea green fluorescent protein hours post-fertilization isopropyl b-D-thiogalactopyranoside kilo base pair lateral plate mesoderm mole per liter morpholino messenger ribonucleic acid nitro blue tetrazolium nanogram nanoliter open reading frame polyacrylamide gel electrophoresis phosphate-buffered saline polymerase chain reaction paraformaldehyde 1-phenyl-2-thiourea reverse-transcription polymerase chain reaction sodium chloride-trisodium citrate solution septum transversum mesenchyme ultraviolet microliter whole mount in situ hybridization viii Davidson, A.E., Balciunas, D., Mohn, D., Shaffer, J., Hermanson, S., Sivasubbu, S., Cliff, M.P., Hackett, P.B., and Ekker, S.C. 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Nat Rev Drug Discov. 4, 35-44. 181 [...]... functions of the adult liver, the fetal liver serves as a site for haematopoiesis by mid-gestation Though the liver has diverse functions, only a small number of cell types is found in liver, which makes the liver as an ideal organ for studies of organogenesis Approximately 60% of cells in the adult liver are hepatocytes and the remaining cells are cholangiocytes (bile duct cells) , Kuppfer cells, stellate cells. ..List of Tables Table 2-1 List of methods used for genotyping 70 Table 2-2 List of primer pairs used in this project 71 Table 2-3 Preparation of denaturing agarose gel for northern blot analysis 72 Table 2-4 Preparation of SDS PAGE gel 72 Table 2-5 The sequences of gene specific morpholinos 72 Table 2-6 List of constructs for WISH RNA probes 73 Table 2-7 Duration of Proteinase K permeabilization for. .. Kimelman, Zilong Wen, and Jinrong Peng (2008); Mypt1- mediated spatial positioning of Bmp 2producing cells is essential for liver organogenesis, Development, 135 (19), 3209-3218 Participation at conference 1 Joint EMBO-IMA Workshop: Fish as model organisms in the genomic era, Singapore, 2001 2 3rd European Conference on Zebrafish and Medaka Genetics and Development, Paris, France, 2003 3 International Stem cell... cells and some endothelial cells Since liver plays such critic roles, it is of great importance to study liver development, not only for basic research, but also for clinic application 1.2 Liver organogenesis 1.2.1 Liver is an endoderm derived organ The endoderm is one of the three germ layers established during gastrulation In mouse embryo, gastrulation starts with the formation of the primitive streak... mutation disrupts LPM organization and causes posterior shift of the liver primordium 133 Figure 5-10 Bmp Signaling Is Essential for Liver Organogenesis 138 Figure 5-11 The mypt1sq181 mutation alters the spatial alignment between the liver primordium and the two stripes of LPM expressing Bmp2a 142 Figure 5-12 The has mutant gives rise to two smaller liver 145 Figure 5-13 Mutant hepatoblasts are impaired in... the failure of the formation of endothelial cells and blood vessels (Shalaby et al., 1995), 17 moreover, the formation of liver bud in flk1-/- embryo is blocked after the hepatic specification, indicating that endothelial cells are crucial for the early liver bud formation prior to vascular function, although the molecular mechanism underlying is unclear (Matsumoto et al., 2001) Besides the essential. .. invade STM to form a liver bud (Bort et al., 2004) Further investigation showed the failure of liver budding in hex-/- embryo is due to the disruption of Hex-dependent cell morphological change from columnar epithelia to pseudostratified epithelia which is necessary for hepatoblasts to undergo migration and 18 A Initiation of the liver bud B The liver bud formation Figure 1-6 The liver bud formation (A)... initiate the liver bud formation (B) The liver bud formation at 18-25 somite stage Liver budding morphogenesis is marked by the formation of the rostral diverticulum of the gut, remodelling of the extracellular matrix around the hepatoblasts and of E-cadherin-based connections between the cells, and proliferation and migration into the surrounding STM (beige) During this stage primitive endothelial cells. .. morphologically distinguishable structure of liver, an outgrowth named the primary liver bud, appears in the ventral floor of the foregut by E8.5 to E9.0 (10-12 somites) as a result of the proliferation of the hepatic endoderm, referred as hepatoblasts (Douarin, 1975; Gualdi et al., 1996) Cell linage tracing shows that two distinct populations of endoderm cells, lateral and medial, arising from three spatially... plates of hepatocytes, one or two cells thick, separated by capillaries, the liver sinusoids (Figure 1-3A, B) Sinusoids are vascular channels lined with highly fenestrated endothelial cells Between the sinusoids and the hepatocytes is a subendothelial space called the space of Disse Several types of cells are residents in the sinusoids or the space of Disse: Kupffer cells, stellate cells (Ito cells) . of mypt1 gene phenocopies the liverless phenotype in sq181 112 4.3 Discussions 112 Charpter 5 Mypt1- mediated spatial positioning of Bmp2- producing cells is essential for liver organogenesis. Mypt1- Mediated Spatial Positioning of Bmp2- Producing Cells Is Essential for Liver Organogenesis HUANG HONG HUI NATIONAL UNIVERSITY OF SINGAPORE. Peng (2008); Mypt1- mediated spatial positioning of Bmp2- producing cells is essential for liver organogenesis, Development, 135 (19), 3209-3218. 1 Chapter 1 Introduction The liver, the