MOLECULAR CHARACTERIZATION AND DEVELOPMENTAL ANALYSIS OF INTERFERON REGULATORY FACTOR (IRF6) GENE IN ZEBRAFISH BEN JIN NATIONAL UNIVERSITY OF SINGAPORE 2006 MOLECULAR CHARACTERIZATION AND DEVELOPMENTAL ANALYSIS OF INTERFERON REGULATORY FACTOR (IRF6) GENE IN ZEBRAFISH BEN JIN (M.Sc., National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PAEDIATRICS NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENT I wish to express my cordially gratitude to my supervisor, Associate Professor Samuel S Chong for the guidance in this Ph.D pursuing His direction, criticisms, encouragement, patience and incessant push powerfully support me to go through the whole process of this study I would like to acknowledge Drs Karuna Sampath, Korzh Vladmir, Yiwen Liu, Peng Jingrong, Wen Zilong and their group members for offering the reagents and technical assistance I would like to give my thanks to Drs Violet P.E Phang and Dong Liang for the suggestion and encouragement I would sincerely appreciate the colleagues, Siew Hong Cheah, Wen Wang, Yayun Yang, Arnold Tan for the material and technical support, collaboration and lab maintenance Finally, I would like to appreciate my mother and father Their love and their enthusiasms on scientific truth accompany me and initiate my career in science i Page No i ii v vii viii CONTENTS ACKNOWLEDGEMENT TABLE OF CONTENTS SUMMARY LIST OF TABLES LIST OF FIGURES CHAPTER 1 2.1 2.1.1 2.1.2 2.1.2.1 2.1.2.2 2.1.2.3 2.1.3 2.1.3.1 2.1.3.2 2.1.3.3 2.1.4 2.1.5 2.2 2.3 2.4 3.1 3.2 4.1 4.2 CHAPTER 2 GENERAL INTRODUCTION Human Genetic Diseases/Inherited disorders Animal models for interpretation of human genome and genetic diseases Zebrafish as a model for Human disorders Forward genetics - Mutagenesis Reverse genetics - Perturbation of gene of interest Gain of function Loss of function by knockdown Loss of function - dominant negative perturbation Developmental studies of digestive organs using zebrafish Gut Liver and pancreas Molecular pathways regulating the development of the digestive system Studies of craniofacial development using zebrafish Molecular markers for expression analysis of phenotypes Mouse Chick Xenopus Human Van der Woude (VWS) and popliteal pyterygium syndromes (PPS) Orocleft disorders VWS and PPS IRF6 IRFs IRF6 Objectives of characterization of zebrafish irf6 MATERIALS AND METHODS Zebrafish strains Isolation of total RNA and Genomic DNA Full-length cDNA cloning 1 9 10 13 17 19 22 22 23 23 23 24 25 25 27 29 32 32 32 33 ii 10 11 12 13 14 15 16 17 18 19 20 CHAPTER 3 CHAPTER 4 5.1 Phylogenetic analysis Promoter cloning Identification of Genomic structure Chromosome mapping The probe syntheses for in situ RNA hybridization analysis Whole mount RNA in situ hybridization Cryosection Paraffin section H and E staining Head cartilage detection with alcian blue staining Staining of red blood cells Image process Microinjection Knockdown analysis Dominant negative perturbation and ectopic expression Detection of putative promoter Western Blot to detect Irf6 protein 37 37 38 42 42 RESULTS Characterization of the full-length zebrafish irf6 cDNA Genomic organization and map location of zebrafish irf6 Developmental expression pattern of irf6 Effectiveness of morpholino knockdowns and dominant negative perturbation Phenotypes of irf6 morpholino knockdown and dominant negative perturbation The expression of molecular markers shows the organogenesis affected by irf6 Defective pharyngeal arches in loss of irf6 function larvae Expression of EGFP driven by the kb irf6 promoter 58 58 DISCUSSION AND CONCLUSION Zebrafish Irf6 and its cDNA Conserved genomic structures of IRF6 gene among organisms Comparison of IRF6 expression patterns The association of irf6 expression and human VWS and PPS disorders Loss of function analysis Phenotypes of morphants and dominant 106 106 106 44 47 48 48 49 50 51 51 52 54 55 56 63 69 75 82 89 96 99 107 109 110 110 iii 5.2 5.3 5.4 8.1 8.2 8.3 8.4 8.5 8.6 negative perturbation Loss of irf6 function causes digestive organs under-develop Loss of irf6 function leads to deforms in pharyngeal arches Loss of irf6 function does not produce the zebrafish that phenocopy Van der Woude syndrome or Popliteal Pterygium syndrome Two forms of irf6 Intron Anti- G124SVPTYETDGDEDDI138 polyclonal antibody can not work in Western Blot analysis Conclusion and Future studies Genomic and transcript information of irf6 and its expression pattern irf6 affects the digestive and pharyngeal arch organogenesis Clinical significance of perturbation of zebrafish irf6 Unknown interacting partners of Irf6 Is Irf6 an activator or repressor, and what are the genes regulated by Irf6 What are those transcription factors regulating irf6 expression REFERENCES 112 114 114 117 117 118 118 112 119 120 120 121 122 iv SUMMARY Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant clefting disorders recently discovered to be caused by mutations in the IRF6 (Interferon Regulatory Factor 6) gene The IRF gene family consists of nine members encoding transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain Most IRFs regulate the expression of interferon-α and -β after viral infection; however, the function of IRF6 remains unknown In this project, a full length zebrafish irf6 cDNA was isolated It encodes a 492 amino acid protein that contains a protein-IRF interaction motif and a DNA-binding domain The zebrafish irf6 gene was identified to consist of eight exons and maps to linkage group 22 closest to marker unp1375 The in situ hybridization analysis of whole mounts and cryosections demonstrates that irf6 was first expressed as a maternal transcript During gastrulation, irf6 expression was concentrated in the forerunner cells From the bud stage to the 3-somite stage, irf6 expression was observed in the Kupffer’s vesicle No expression could be detected at the 6-somite and 10-somite stages At the 14-somite stage, expression was detected in the otic placode At the 17somite stage, strong expression was also observed in the cloaca During the pharyngula, hatch and larva periods up to days post-fertilization, irf6 was expressed in the pharyngeal arches, olfactory and otic placodes, and in the epithelial cells of endoderm derived tissues The latter tissues include the mouth, pharynx, esophagus, endodermal lining of swim bladder, liver, exocrine pancreas, and associated ducts The zebrafish expression data are consistent with the observations of lip pits in VWS patients, as well v as more recent reports of alae nasi, otitis media and sensorineural hearing loss documented in some patients The translation-blocking and splice-modifying morpholino-mediated gene knockdown analyses were performed to observe the effect of reduction or loss of Irf6 function on organogenesis during zebrafish embryonic development Additionally, microinjection of capped mRNA carrying an Arg84 to Cys (R84C) dominant-negative point mutation was performed All loss of function treatments produced larvae with identical gross morphologies, including bloated abdomen, small eyes and head, and delayed yolk resorption Major pharyngeal arch deformities were observed, including misalignments, degenerations and absent structures Furthermore, the liver and pancreas were reduced in size compared to wildtype fish The intestinal lumen was constricted, with absent or reduced folding of the epithelial cell monolayer The epithelial cells of the intestine maintained their cuboidal shape, with the nuclei still situated in the center and not the base of the cells A milder phenotype showing reddish intestine was observed in larvae injected with splice-modifying morpholinos and dominant negative perturbation By the inspiration that both irf6 and sox17 have the expression in the forerunner cells and endoderm organs, we propose and test the hypothesis that Irf6 is an effector of Nodal signaling pathway, upstream or downstream of sox17 However, our results not support this hypothesis vi LIST OF TABLES Table No Page No 2.1 Primer pairs for PCR detection of introns 41-42 2.2 The syntheses of RNA probes and in situ hybridization conditions 47 3.1 Percentage amino acid similarity of zebrafish IRF6 compared with other species 61 3.2 Summary of rescue experiments for morpholino knockdown 3.3 Phenotypes of Loss of irf6 function 82-83 88 vii LIST OF FIGURES Fig No 1.1 Page No Flowchart of characterization of irf6 using zebrafish model 31 2.1 Amplification of irf6 cDNA from AB line zebrafish embryos at the age of 24 hpf old 37 2.2 Amplification of the putative irf6 promoter fragments using Universal GenomeWalker™ kit 38 2.3 Diagram of putative genomic structure and the PCR detection of exon-intron junction using primers 40-41 2.4 (A) PCR fragment amplified from genomic DNA using PaeIIRF-Pf4 and BamHI-5’E2r primers (B) Expression plasmid to detect the cis-elements upstream of irf6 coding region 57 3.1 Full-length nucleotide sequence of zebrafish irf6 cDNA and its deduced amino acid sequence 3.2 Phylogenetic analysis of the IRF gene family 60 3.3 Alignment of the predicted IRF6 proteins from five species 62 3.4 Genomic organization of the IRF6 orthologs in human, mouse, Fugu, and zebrafish 63-64 3.5 Assembled sequences of irf6 genomic fragments 64-66 3.6 The irf6 gene locus on the LG22 in T51 RH panel and in the Ensembl zebrafish version (ZV6) 68-69 3.7 Whole-mount (A-I) and sagittal section (J-L) analysis of irf6 expression in early zebrafish embryos 71-72 3.8 Expression of irf6 transcript in the developing zebrafish during pharyngula, hatching and larval stages 73-74 3.9 The antibody targeting to the Irf6 epitope G124SVPTYETDGDEDDI138 is not specific to detect the Irf6 protein in the Western Blot analysis 75 58-59 viii Du,X.Y., Tang,W., Tian,W.D., Li,X.Y., Liu,L., and Zheng,X.H (2006) [Identification of three novel mutations of 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