Hedgehog signaling in the zebrafish embryo role of kif7 and DZIP1 1

ACKNOWLEDGEMENTS I would like to thank my PhD supervisor Dr. Sudipto Roy for the opportunity to work under his wing and for being a great mentor in science. The journey to success is never a bed of roses. With his guidance and support, I was able to overcome many obstacles that came along my way. I am also grateful to the members of my PhD Advisory Committee, Prof David Lane, Dr. Li Bao Jie and Dr. Jiang Yun-Jin for their praises and criticisms, and helpful suggestions over the last five years. I would also like to thank the past and present members of the SR lab for their support and friendships. I would like to thank Mr. Noel Wong Kangli for his technical assistance and tolerance of my work demands over the last three years. I would like to thank IMCB and A*STAR for funding this research and provided the scholarship for me to pursue PhD, the Zebrafish Facility for their excellent support in fish husbandry, and the DNA Sequencing Facility for their sequencing service. A million thanks to others in IMCB who had helped me in one way or another. Finally, I would like to thank my family members for their love and support for me. I would like to apologize to my parents who had to endure my long irregular working hours and hence, neglected their needs. Lastly, I would like to thank Ms Janice Oh Hsueh Ling, Ms Nur Khairiah Mohd Ismail, Dr Kua Hui Yi and Dr Liew Hoe Peng for proofreading the drafts of this manuscript. i TABLE OF CONTENTS Page Acknowledgements Table of Contents Summary List of Figures and Table List of Abbreviations List of Publications i ii v vii viii x Chapter Introduction 1.1. 1.2. 1.3. Origin of the Hedgehog (Hh) pathway Drosophila Hedgehog Signaling Pathway Vertebrate Hedgehog Signaling Pathway 1.3.1. Genetics in Vertebrate Models 1.3.2. The Signaling Cascade 1.3.3. Role of Sonic Hedgehog 1.3.4. Regulation of Smoothened Activity 1.4. Activation of Hedgehog protein 1.5. Comparing Hedgehog Orthologs 1.5.1. Patched 1.5.2. Smoothened 1.5.3. Hedgehog Signaling Complex (HSC) 1.5.3.1. HSC-Fused 1.5.3.2. HSC-Suppressor of Fused 1.5.3.3. HSC- Costal2 1.6. Cilia 1.6.1. Introduction to Cilia 1.6.2. Structure of the cilium 1.6.2.1. Axoneme 1.6.2.2. Basal Body 1.7. Primary Cilia 1.7.1. Primary cilium and Hedgehog 1.7.2. Basal Body and Hedgehog 1.8. Ancestral role of Hedgehog components 1.9. Cilia and other pathway(s) 1.9.1. Cilia and Wnt Pathway 1.9.2. Cilia and Fgf pathway 1.9.3. Cilia and Phosphoinositide Pathway 1.10. Aim of Thesis 6 7 9 10 11 12 13 14 16 16 19 19 19 21 21 26 26 28 28 30 31 32 ii Chapter Materials and Methods 2.1. 2.2. 2.3. 2.4. 2.5. 2.6. 2.7. 2.8. 2.9. 2.10. 2.11. 2.12. 2.13. 2.14. 2.15. 2.16. 2.17. 2.18. Zebrafish strains and husbandry Microinjections Generation of transgenic zebrafish line Whole mount immunohistochemistry on zebrafish embryos Whole mount in situ hybridization 2.5.1. In situ Hybridization 2.5.2. Fluorescent in situ hybridization Restriction Enzyme digests and cloning Synthesis and purification of antisense probes Synthesis of sense mRNA Polymerase Chain Reaction (PCR) 2.9.1. General PCR 2.9.2. Site Directed Mutagenesis (SDM) Plasmid DNA Purification Quantification of nucleic acid cDNA synthesis 2.12.1. Extraction of total RNA from zebrafish embryos 2.12.2. Zebrafish cDNA preparation DNA Sequencing Mammalian Tissue Culture 2.14.1. Maintenance of cell lines 2.14.2. Thawing and Freezing of cells 2.14.3. Transient transfection of mammalian cells 2.14.4. SDS PolyAcrylamide Gel Electrophoresis (PAGE) and western blot 2.14.5. ImmunoFluorescence (IF) 2.14.6. Protein Staining 2.14.7. Co-ImmunoPrecipitation (Co-IP) Iguana Antibody Production 2.15.1. Synthesis of Recombinant Protein for antibody production 2.15.2. Overexpression and Induction of GST-Igu (Small scale) 2.15.3. Overexpression and Induction of GST-Igu (Large scale) 2.15.4. Raising of antibodies (Rat) Yeast-2-Hybrid (Y2H) Microscopy and figure preparation Cryo Sectioning 33 33 34 34 35 35 36 37 37 37 38 38 38 39 39 39 39 39 40 40 40 41 41 41 43 43 44 44 44 44 45 46 46 47 47 Chapter An Elusive Member of The Vertebrate Hedgehog Signaling Pathway 3.1. 3.2. Introduction Results 3.2.1. Cloning of a vertebrate orthologue of cos2 49 51 51 iii 3.2.2. 3.3. Expression of the zebrafish cos2 gene and subcellular distribution of its protein 3.2.3. Cos2 as a repressor in Hh signaling pathway affecting myotome of zebrafish embryo 3.2.4 Cos2 inactivation affects the developing neural tube 3.2.5 The activity of zebrafish Cos2 is epistatic to Shh and Smo 3.2.6 Cos2 and Su(fu)- the two repressors of Hh signal transduction 3.2.7 Cos2 interacts with transcription factor Gli1 Discussion and Conclusion 52 58 61 61 64 67 69 Chapter The Iguana/DZIP1 Protein Is Important For Ciliogenic Pathway 4.1. 4.2. 4.3. Introduction Results 4.2.1. The role of Igu in primary cilia formation 4.2.2. Igu is not required for docking of basal bodies but essential for axoneme outgrowth 4.2.3. The role of Igu in motile cilia formation 4.2.4. Detection of endogenous Igu protein 4.2.5. Production of Igu Antibody 4.2.6. Igu proteins localize to the bases of primary and motile cilia 4.2.7. Smo proteins localize to the primary cilia in the zebrafish embryo in response to Hh signaling Discussion and Conclusion 73 76 76 82 85 89 90 93 96 99 Chapter Conclusion and Future Work 5.1. 5.2. Conclusion Future Work 103 108 References 112 Appendix Appendix – Buffer/Reagent Makeup 131 iv SUMMARY An elusive member of the vertebrate Hedgehog signaling pathway Hedgehog (Hh) signaling was first discovered in Drosophila melanogaster and misregulation of this pathway has been linked to a wide spectrum of congenital abnormalities and malignancies in human. Orthologs of nearly all the core components of the Hh pathway have now been discovered in vertebrates. Subsequent studies have shown that key components of the Hh pathway are conserved through to the vertebrates. However, amongst them, the kinesin-like protein Costal2 (Cos2), which plays an important role in controlling the transcriptional activity of the Hh pathway, remained elusive. I report the cloning of a kinesin-related gene from Danio rerio zebrafish and propose that it is the first vertebrate Cos2 and is annotated as kinesin family member (Kif7) in the mouse and human genome databases. Genetic and molecular analysis revealed that, similar to its function in Drosophila, zebrafish Cos2/Kif7 acts as an intracellular repressor of the Hh pathway. Cos2 has additive inhibitory effect on Hh signaling when it works together with Suppressor of Fused (Su(fu)), which is another protein that negatively regulates signaling in Hh-responsive cells. Co-immunoprecipitation shows that both the zebrafish Cos2 and Su(fu) bind to the vertebrate Hh transcription factor, Gli1. This collaborative effect of Cos2 and Su(fu) in regulating Gli activity is a conserved event in the Hh signaling cascade. v The Iguana/Dzip1 protein is important in Ciliogenic pathway Cilia are tiny hair-like organelles, which are present in two varieties: motile and nonmotile. Non-motile primary cilia are present on nearly all vertebrate cells but their function was unknown. Recently, it was discovered that these primary cilia play important roles in many developmental and physiological processes. We report that the zebrafish iguana (igu) gene is critical for the differentiation of primary cilia. Igu encodes DAZ-interacting protein (Dzip1) with zinc finger and coiled-coil domains and was first thought to play a role in the Hh pathway. Further molecular analyses show that the aberrant Hh activity in zebrafish igu mutants can now be explained as a result of primary cilia loss. By contrast, motile cilia formation appears less affected by the loss of Igu. In the absence of igu, basal bodies can still migrate and dock properly to the apical membrane. However, axonemal outgrowth and ciliary pits are completely absent. Igu proteins localize to the base of all cilia, and in close proximity to the basal bodies. These findings identify Igu as a novel and critical component of ciliogenesis. The importance of primary cilium in Hh signal transduction is absolute in mammals but not in Drosophila. Here, we showed that this requirement is also essential in zebrafish by demonstrating the localization of a key Hh component, Smoothened (Smo) to the primary cilia in response to Hh activation. This localization can be abolished by mutating a conserved cilia localizing domain. Mis-localization of Smo results in mis-regulation of the Hh pathway. Therefore, the primary cilium is an important organelle that serves as a platform for Hh components to aggregate, resulting in proper transduction of signals. vi LIST OF FIGURES AND TABLE Figure/ Table Title Page 1.1 The Hedgehog pathway in Drosophila and vertebrates 1.2 Makeup of a cilium consists of the axoneme and basal body 17 3.1 Alignment of Cos2 sequences from insects and vertebrates 53 3.2 Expression pattern of the zebrafish cos2 gene and sub-cellular distribution of the Cos2 protein Localisation of Cos2-GFP with microtubules in mammalian cells 55 3.3 3.4 57 Loss of Cos2 function results in de-repression of the Hh pathway in the myotome of the zebrafish embryo Inactivation of Cos2 function induces ectopic Hh signalling in the ventral neural tube Cos2 is epistatic to shh and smo 59 66 3.8 Loss of Su(fu) activity enhances de-repression of Hh signalling in Cos2 morphants Zebrafish Cos2 physically associates with Gli1 4.1 Staining of early stage embryos with acetylated tubulin 77 4.2 Igu is essential for primary cilia formation 78 Table Quantification of the phenotypes exhibited by embryos/morphants 80 4.3 84 4.4 Igu is required for axonemal biogenesis, but not for the docking of basal bodies to apical membranes Motile ciliogenesis is less strongly perturbed in igu mutants 4.5 Use of Igu antibody in zebrafish embryos and cell line 92 4.6 The Igu protein localizes to the bases of primary and motile cilia 95 4.7 Smo localizes to primary cilia in the zebrafish embryo in response to Hh signaling Updated vertebrate Hh pathway 98 3.5 3.6 3.7 5.1 62 63 68 87 106 vii LIST OF ABBREVIATIONS aa AP Amino acids Alkaline Phosphatase Ab antibody bp base pairs BSA Bovine Serum Albumin Cos2 Costal2 cDNA Complementary DNA CLD-Smo Ciliary Localizing Defective Smoothened CLM Ciliary Localizing Motif DAPI 4’6-diamidino-2-phenylindole DEPC Diethyl pyrocarbonate DNA Deoxyribonucleic Acid DIC Differential Interference Contrast DIG Digoxigenin dNTP Deoxynucleoside triphosphate dpf days post fertilization DZIP1 Daz-interacting protein DZIP1-L Daz-interacting protein 1-like EDTA Ethylene diamine tetraacetic acid EGTA ethylene glycol tetraacetic acid EtOH Ethanol FISH Fluorescence in situ hybridization Fu Fused GFP Green Fluorescent Protein HA Hemagglutinin Hh Hedgehog hpf hours post fertilization hph hours post heatshock viii HSC Hedgehog Signaling Complex IFT IntraFlagellar transport igu iguana IP Immuno precipitation ISH In situ hybridization kb kilobases KV Kupffer’s Vesicle MeOH methanol MFF Medial fast fibers MO Morpholino oligonucleotides MP Muscle pioneers NBT/BCIP Nitro Blue Tetrazolium/5-Bromo 4-chloro 3-indolyl phosphate mRNA messenger Ribonucleic Acid ORF Open reading frame PBS Phosphate Buffered Saline PCR Polymerase chain reaction POD Peroxidase ptc1 patched1 PTU 1-phenyl-2-thiourea SDS sodium dodecyl sulfate SDM Site directed mutagenesis Shh Sonic Hedgehog smo smoothened SSC saline-sodium citrate SSF Surface slow fibers Su(fu) Suppressor of fused TSA Tyramide Signal Amplification w/v Weight per volume v/v Volume per volume ix LIST OF PUBLICATIONS 1) Tay SY, Ingham PW, Roy S. A homologue of the Drosophila kinesin-like protein Costal2 regulates Hedgehog signal transduction in the vertebrate embryo. Development. 2005 Feb;132(4):625-34. 2) Xu J, Srinivas BP, Tay SY, Mak A, Yu X, Lee SG, Yang H, Govindarajan KR, Leong B, Bourque G, Mathavan S, Roy S. Genomewide expression profiling in the zebrafish embryo identifies target genes regulated by Hedgehog signaling during vertebrate development. Genetics. 2006 Oct;174(2):735-52. 3) Tay SY, Yu X, Wong KN, Panse P, Ng CP, Roy S. The iguana/DZIP1 protein is a novel component of the ciliogenic pathway essential for axonemal biogenesis. Dev Dyn. 2010 Feb;239(2):527-34. x . 1. 5 .1. Patched 9 1. 5.2. Smoothened 10 1. 5.3. Hedgehog Signaling Complex (HSC) 11 1. 5.3 .1. HSC-Fused 12 1. 5.3.2. HSC-Suppressor of Fused 13 1. 5.3.3. HSC- Costal2 14 1. 6. Cilia 16 1. 6 .1. . Chapter 1 Introduction 1. 1. Origin of the Hedgehog (Hh) pathway 1 1. 2. Drosophila Hedgehog Signaling Pathway 4 1. 3. Vertebrate Hedgehog Signaling Pathway 6 1. 3 .1. Genetics in Vertebrate. 16 1. 6 .1. Introduction to Cilia 16 1. 6.2. Structure of the cilium 19 1. 6.2 .1. Axoneme 19 1. 6.2.2. Basal Body 19 1. 7. Primary Cilia 21 1. 7 .1. Primary cilium and Hedgehog 21 1. 7.2. Basal