Molecular cloning and characterization of sq163, a zebrafish liver mutant

MOLECULAR CLONING AND CHARACTERIZATION OF sq163, A ZEBRAFISH LIVER MUTANT LO LI JAN (B.Sc., Melbourne University, Australia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgement The pursuit of my Ph.D. study, like the truth in science, did not take a straightforward path. Across two continents and shift of three projects, it is interesting that I have come so far, considerably. That does not come easy. For those people that have either directly or indirectly made this possible and even enjoyable at times, my deepest appreciation goes to them. My supervisor, Associate Prof. Hong Yunhan, he is not only a great scientist but has been an unbelievable mentor in the past years. My Ph.D. conversion committee members, Dr. Vladimir KORZH and Dr. Wangshu, and my pre-thesis committee members, Prof. Ding Jeakling and Associate Prof. Christoph Winkler, my special thanks go to them for giving my project invaluable comments and suggestions and eventually an unequivocal green light during the conversion and pre-thesis presentation respectively. To all my colleagues in the Functional Genomics Laboratory (IMCB), Changqing, Chaoming, Chen Jun, Cheng Hui, Cheng Wei, Dongni, Evelyn, Gao Chuan, Honghui, Hussain, Linda, Mengyuan, Peiying, Qian Feng, Sharon, Shulan and Zhenhai, and in the Developmental Laboratory (DBS), Mingyou, Tianshen, Veron and Zhendong, I extend my heartfelt thanks for their technical help and active discussion and most importantly, their invaluable friendship. Not forgetting the enormous supports from the zebrafish and sequencing facility in the Institute of Molecular and Cell Biology, I would like to acknowledge their contributions. To the scholarship offered by the National University of Singapore, my gratitude goes for the opportunity given. Finally, to my parents, my husband and three daughters, I cannot thank them enough for their boundless love. I think I can only repay them by loving them back, twice as much. ii Table of Contents Acknowledgement . ii Table of Contents . iii Summary . ixii List of Abbreviations . ix List of Tables . ixi List of Figures xii List of Publications and List of Conference Participation xiv Chapter Introduction . 1.1 Germ layers and organogenesis 1.2 Liver: Structure and Functions 1.2.1 The liver structure 1.2.1.1 The hepatic vascular system . 1.2.1.2 The biliary system . 1.2.1.3 The three dimensional architecture of the liver 1.2.2 The liver functions . 1.3 Liver organogenesis 10 1.3.1 Liver is an endodermal-derived organ . 11 1.3.2 Liver morphogenesis 12 1.3.3 Molecular mechanisms underlying liver development 13 1.3.3.1 Acquisition of competency . 14 1.3.3.2 Hepatic specification . 18 1.3.3.3 Liver bud formation and growth . 21 1.3.3.3.1 Liver bud formation . 21 1.3.4.3.2 Growth and apoptosis of hepatoblasts . 23 1.3.3.4 Hepatocyte differentiation and establishment of hepatic architecture 27 1.3.3.5 Cholangiocyte differentiation . 30 1.4 Zebrafish: A model for studies of liver development . 34 1.4.1 Advantages of zebrafish . 34 1.4.2 Liver development study in zebrafish 40 1.4.2.1 Morphological description of liver development 40 1.4.2.2 Molecular mechanisms of liver development . 42 1.4.2.3 Signaling molecules and transcription factors 43 1.5 Bms1l 50 1.6 Rationales and aims of the project 51 iii Chapter Material and method 52 2.1 Zebrafish . 53 2.1.1 Fish strains and maintenance . 52 2.1.2 Collection of fertilized eggs . 53 2.1.3 Collection of unfertilized eggs . 54 2.2 General DNA application . 55 2.2.1 Gene Cloning . 55 2.2.1.1 Polymerase Chain Reaction (PCR) . 55 2.2.1.2 Purification of PCR product/DNA fragments . 56 2.2.1.3 Plasmid DNA extraction . 56 2.2.1.4 Ligation of DNA inserts into plasmid vectors 57 2.2.1.5 Transformation of DH5α competent cells with plasmids or ligation products using a heat-shock method . 57 2.2.1.5.1 E.coli strain 56 2.2.1.5.2 Preparation of competent cells . 58 2.2.1.5.3 Heat-shock transformation . 58 2.2.2 DNA sequencing 59 2.2.3 Site-directed mutagenesis 59 2.2.4 Zebrafish genomic DNA extraction . 60 2.2.4.1 Genomic DNA extraction from adult zebrafish 60 2.2.4.2 Isolation of genomic DNA from embryos or scales of adult zebrafish 61 2.2.5 Preparation of ‘home-made’ Taq . 61 2.3 General RNA application 62 2.3.1 RNA extraction from embryos or adult zebrafish 63 2.3.2 Removal of genomic DNA 63 2.3.3 mRNA isolation . 63 2.3.4 Reverse Transcription PCR (RT-PCR) 64 2.3.4.1 One-step RT-PCR . 64 2.3.4.2 Two-step RT-PCR 64 2.3.5 mRNA synthesized by in vitro transcription . 65 2.3.6 Northern Blot analysis . 65 2.3.6.1 Probe preparation 66 2.3.6.2 RNA sample preparation . 66 2.3.6.3 RNA gel electrophoresis . 67 2.4 Mapping 68 2.4.1 Preparation of mapping pairs . 68 2.4.2 Genomic DNA preparation 68 2.4.3 Rough mapping 69 2.4.3.1 Mapping panel 70 2.4.3.2 BSA . 71 2.4.4 Intermediate mapping 75 iv 2.4.5 Fine mapping and candidate gene approach 75 2.4.6 Genotyping sq163+/- fish or sq163-/- embryos . 76 2.5 Whole Mount in situ Hybridization (WISH) 77 2.5.1 Preparation of DIG-labeled RNA probe 77 2.5.2 High resolution WISH . 79 2.5.3 High throughput WISH 81 2.6 Microinjection . 82 2.6.1 Preparation of injected materials . 82 2.6.2 Preparation of injection needles and embryos supporter . 83 2.6.3 Microinjection 83 2.7 Immunochemistry . 84 2.7.1 Cryosectioning of zebrafish embryos 84 2.7.2 phospho-Histone H3 Immunostaining and TUNEL Assay . 85 2.8 Microscopy and picture capturing 85 Chapter Isolation of sq163 . 86 3.1 Identification of sq163 by large-scale phenotypic screening 86 3.1.1 Forward genetic screen 86 3.1.2 sq163 confers a small liver phenotype 87 3.2 Positional cloning of sq163 . 89 3.2.1 Introduction 89 3.2.2 Generation of sq163 mapping families 94 3.2.3 Initial mapping of sq163 95 3.2.4 Intermediate mapping 97 3.2.5 Fine mapping and chromosomal walking on BAC contig . 101 3.3 Candidate gene approach of sq163 . 103 3.3.1 sq163 alters a conserved domain in Bms1l 103 3.3.2 L154 to Q154 substitution in Bms1l causes small liver phenotype in sq163 108 3.3.3 bms1l mRNA can rescue sq163 small liver phenotype . 108 3.3.4 Knockdown bms1l gene phenocopies the small liver phenotype in sq163 110 3.3.5 Expression patterns of bms1l . 112 3.4.5 Knockdown of rcl1 114 3.4 Discussion . 116 3.4.1 Positional cloning of sq163 116 3.4.2 Mutations in bms1l . 117 v 3.4.3 Ribosomal proteins, development and cancer . 118 Chapter Characterization of bms1lsq163 . 122 4.1 Introduction . 122 4.2 Results . 123 4.2.1 bms1lsq163 confers a small liver phenotype 123 4.2.2 bms1lsq163 and hepatic competency 127 4.2.3 bms1lsq163 and hepatoblasts proliferation . 129 4.2.4 Mutant hepatoblasts are impaired in proliferation . 130 4.3 Discussion . 132 Chapter Conclusions 136 Reference List 139 vi Summary The liver is one of the main organs of endodermal origin. Most knowledge of liver development is obtained from reverse genetics and explants culture approaches performed on mice and chick. However, various gaps still exist in the whole picture of liver organogenesis due to limitations of such methodologies and early lethality of liver defects. Zebrafish, a recently chosen model for the study of vertebrate development, is particularly suitable for studying liver organogenesis via forward genetics. To take advantage of the zebrafish system for the investigation of the molecular mechanisms of liver development, we carried out a middle-scale genetic screen for liver defective mutants and adopted the map-based cloning method for the identification of mutated genes. By exploiting the polymorphisms exhibited in 226 pairs of simple sequence length polymorphism (SSLP) markers and polymorphic mapping families, the bulk segregation analysis (BSA) protocol has mapped one of the small liver mutants, sq163 to linkage group 12. From ~6800 meiotic events, subsequent detailed mapping in combination with candidate gene approach have identified a T to A mutation in the ribosomal biogenesis protein (Bms1l) gene, which results in the L154 to Q154 substitution in a GTPase motif in Bms1l. Genetic evidence from co-segregation analysis, morpholino knockdown and phenotypic rescuing experiment unequivocally demonstrated that the bms1lsq163 mutation is responsible for the small liver phenotype. Bms1l is a key component in the 40S ribosomal biogenesis pathway that recruits many other ribosomal proteins onto the preribosome-rRNA complex. Its role in this universal mechanism of ribosomes production has been well studied and established in yeast. The positional cloning of sq163 is the first vii genetic indication of Bms1l possibly playing a specific function in vertebrate liver organogenesis. Preliminary phenotypic characterization of the mutant using digestive organ specific molecular markers suggested that liver budding and initial growth are affected in the homozygous mutant which continues to impinge on subsequent expansion of the liver, as well as other digestive organs such as the intestine and pancreas, resulting in their retardation after 3dpf. Whole mount in situ hybridization on wildtype embryos showed that bms1l is enriched in the entire digestive tract and its accessory organs, consistent with the bms1lsq163 mutant phenotypes. Proliferation assay suggests that impairment of hepatoblasts proliferation is one of the consequences of bms1lsq163 that give rise to the small liver phenotype. Excitingly, one of the main interacting partners in the ribosomal biogenesis pathway, rc1l, was shown to share highly similar expression patterns with bms1l in the digestive organs, further suggesting that the ribosomal pathway is necessary for zebrafish liver development. While both examination of earlier mutant embryos with more extensive markers and investigation at the cellular and biochemical level will be necessary to reveal further insights into the functional consequences of bms1lsq163, the work reported in this thesis has demonstrated the possible involvement of a seemingly housekeeping gene in specific development process such as liver formation, of which eventually may be instrumental in filling up some of the gaps in liver organogenesis. viii List of Abbreviations A aa AP BAC BCIP BMP BSA1 BSA2 bp adenine amino acid alkaline phosphatase Bacterial Artificial Chromosomes 5-bromo-4-chloro-3-indolyl phosphate bone morphogenetic protein bulk segregation analysis bovine serum albumin DEPC DIG DMSO DNA dNTP dpf DTT EF-Tu ENU Fgf GDP GEF GFP Gln GTP hnf hpf ifabp IPTG Kb Leu (L) lfabp M base pair carboxyl domain calf intestinal alkaline phosphatase centimorgan diamond-blackfan anemia digestive-organ expansion factor diethylpyrocarbonate digoxigenin dimethyl sulfoxide deoxyribonucleic acid deoxyribonucleotide triphosphate days post-fertilization dithiothreitol eubacterial protein elongation factor N-ethyl-N-nitrosourea fibroblast growth factor guanosine diphosphate guanine exchange factor green fluorescent protein glutamine Guanosine-5'-triphosphate hepatocyte nuclear factor hours post-fertilization intestine fatty acid binding protein isopropyl b-D-thiogalactopyranoside kilo base pair leucine liver fatty acid binding protein mole per liter MGH Massachussets General Hospital MPNST MO mRNA N-domain ng malignant peripheral nerve sheath tumors morpholino messenger ribonucleic acid amino domain nanogram C-domain CIP CM DBA def ix nl npo ORF PBS PCR PFA PTU Q rp RPS19 rRNA RT-PCR SSC STM T UTR UV μl WISH nanoliter nil per os open reading frame phosphate-buffered saline polymerase chain reaction paraformaldehyde 1-phenyl-2-thiourea glutamine ribosomal protein ribosomal protein S19 ribosomal RNA reverse-transcription polymerase chain reaction sodium chloride-trisodium citrate solution septum transversum mesenchyme Thymine untranslated region ultraviolet microliter whole mount in situ hybridization x Fukuda-Taira, S. 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Nat Rev Drug Discov. 4, 35-44. 159 [...]... gastrulation at E7.5 The mRNA of Foxa1 can be first detected at E7 in the late primitive streak and then takes similar pattern as Foxa2 Unlike Foxa1 and Foxa2, the expression of Foxa3 extends from hindgut to the foregut/ midgut boundary from E8.5 onwards (Lai et al., 1991; Ang et al., 1993; Kaestner et al., 1993; Monaghan et al., 1993; Altaba et al., 1993; Sasaki and Hogan, 1993) The embryonic liver histology... tympanic cavity, the trachea, bronchi, and alveoli of the lungs, the urinary bladder and part of the urethra, and the follicles of the thyroid gland and thymus (Stainier, 2002) However although well defined by fate maps, due to the close proximity and the nature of the mechanistic movements among germ layers, especially between mesoderm and endoderm, an internal organ may be constituted by more than... primary liver bud, appearing at the ventral floor of the foregut by E8.5 to E9.0 (Douarin, 1975; Gualdi et al., 1996) Cell linage tracing experiments showed that two distinct populations of endodermal cells, lateral and medial, arising from three spatially separated embryonic domains, converge to generate the epithelial cells of the liver bud (Tremblay and Zaret, 2005) At this early stage the primary liver. .. terminal branches of the hepatic portal vein and hepatic artery coalesces into sinusoids in the liver and drains into the central vein in each lobule (Figure 1- 2A) The hepatic vein collects the blood from the central vein and leaves the liver and links to the inferior vena cava 4 Figure 1-1 Hepatic structure of the human liver showing the vascular and biliary system Adapted from http://www.moondragon.org/health/disorders/gallbladder.html... found that hepatic specification is normal in both chimeric embryos but the liver bud failed to expand, suggesting that although not needed for the specification step, these factors are essential for hepatoblast proliferation and differentiation (Zhao and Duncan, 2005; Watt et al., 2007) Interestingly but not surprisingly, like Foxa1 and Foxa2, Gata4 and Gata6 may have redundant functions during hepatic... physical regions based on a fate map generated by single endoderm cell labeling experiments at E7.5 (Lawson et al., 1986) Region I, the ventral foregut, gives rise to the thyroid, lung, liver and ventral pancreas Albumin, a characteristic marker of hepatic specification, can be detected at the ventral foregut at the stage E8.5 (Cascio and Zaret, 1991; Gualdi et al., 1996) Regions II and III, the dorsal... merges with the main 5 pancreatic duct in the hepatopancreatic ampulla that enters the duodenum at the major duodenal papilla (Figure 1-1) Figure 1-2 (A) Microscopic anatomy of the human liver highlighting (B) the lobule and portal triad Adapted from http://www.sacs.ucsf.edu/ 1.2.1.2 The three dimensional architecture of the liver A basic unit of the liver is a polygonal column called liver lobule The... (Bossard and Zaret, 2000) These data offered two possible explanations: the binding of Foxas and Gatas to the otherwise silent albumin enhancer will either facilitate the initiation of hepatic cell fate in the presence of the inductive signals in the ventral foregut, or remove repressive interaction in the dorsal endoderm The functional implication of the 17 occupancy of Foxa2 and Gata4 on the albumin... genetics and tissue explantation assay, majority of the mature knowledge of the molecular mechanisms governing liver development is obtained from mouse and chick, with data dated as far back as more than 30 years ago The findings so far can be summarized by a five-step model: (i) endoderm cells gaining competency to become hepatogenic cells, (ii) hepatoblast specification, (iii) liver bud 13 formation,... Specification (Foxa+,Gata+) 14 Figure 1-3 Hepatic competence and specification in the mouse liver (A) Acquisition of competence at 2-6 somite stage: The ventral foregut endoderm gains hepatic competence with the action of transcription factors Foxas and Gatas, and bone morphogenetic proteins (Bmps) that emanate from the adjacent cells of septum transversum mesenchyme (STM) (B) Hepatic specification at 7-8 . tube and tympanic cavity, the trachea, bronchi, and alveoli of the lungs, the urinary bladder and part of the urethra, and the follicles of the thyroid gland and thymus (Stain However although. formation, of which eventually may be instrumental in filling up some of the gaps in liver organogenesis. viii List of Abbreviations A adenine aa amino acid AP alkaline phosphatase. vertebrate liver organogenesis. Preliminary phenotypic characterization of the mutant using digestive organ specific molecular markers suggested that liver budding and initial growth are affected