FUNCTIONAL STUDY OF ZEBRAFISH UDU AND ITS RELATIONSHIP TO THE NOTCH SIGNALING PATHWAY LIM CHIAW HWEE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2008 Acknowledgements I sincerely thank my supervisior, Dr Yun-Jin Jiang for acting as my mentor, providing guidance, and imparting knowledge throughout my PhD program I also thank Dr Liou Yih-Cheng, Dr Alan Porter and Dr Yang Xiaohang for serving on my PhD graduate committee I owe special thanks to all members of Danio unit: the aunties, the guys and Dr You May-Su for their help in rearing and maintaining zebrafish in this study I extend my sincere appreciation to all the members of JYJ laboratory: Steven and Qi Mei for their laboratory support, especially to Li Qing for imparting valuable techniques, Shang Wei for cryostat sectioning, William and Xuehui for valuable discussion; ex-members of JYJ laboratory: Haoying, Ma Ming, Chengjin, Nguyet, Kate, Stephanine, Nick, Kenny, Kian Hong, Ashok and Rida for their assistance that helped make my experience in JYJ laboratory a memorable one Special thank to visiting scientist Dr Hsaio Chung-Der for sharing imaging technique I also thank Dr Soojin Ryu from Prof Wolfgang Driever’s laboratory for sharing expertise on flow cytometry I thank Siew Chin from BSF facility for flow cytometry analysis I thank the IMCB community of researchers and graduate students for their technical advice and friendship Funding for this project was provided by A-STAR and graduate studies was funded by IMCB Finally, I thank my family for providing the encouragement, support and their understanding, patience that made my pursuit of a graduate degree possible i Tables of Contents Page Acknowledgements i Table of Contents ii Summary viii List of Tables xi List of Figures xii List of Abbreviations xvi List of Publications xix CHAPTER Introduction 1.1 Identification of udu gene 1.2 Udu and primitive erythropoiesis 1.3 p53, a tumor suppressor 1.4 p53 isoforms 1.5 DNA damage pathway and p53 response ii 1.6 Apoptosis-the elimination of tumorigenic cells 1.7 DNA repair 10 1.8 SANT and chromatin remodeling 12 1.9 Function of SIN3/PAH domain 13 1.10 Organization of eukaryotic DNA 14 1.11 DNA replication 17 1.12 Udu counterparts in human and mouse 20 1.13 Notch signaling and its core components 22 1.14 Regulation of Notch-ligand activity 23 1.15 Notch and lateral inhibition: neurogenesis 25 1.16 Notch and midline cell fate specification 26 1.17 Notch and boundaries formation: somitogenesis 27 1.18 Notch and diseases 29 1.19 Notch and p53 30 1.20 Regulation of Notch signaling by p53 32 1.21 Zebrafish: The model system 33 1.22 Advantages of using zebrafish 34 1.23 Zebrafish mutagenic screen 35 1.24 Formation of morphologically distinct somites 36 1.25 Somite patterning 37 iii 1.26 Aims of this study 39 Materials and Methods 44 2.1 Zebrafish maintenance and embryos 44 2.2 Irradiation of zebrafish embryos with ultraviolet light 44 2.3 Total RNA extraction 44 2.4 Reverse transcription PCR 45 2.5 Quantative Real-time PCR 45 2.6 Embryos preparation 46 2.7 Antisense probe synthesis 46 2.8 Whole-mount in situ hybridization 47 2.9 Injection experiments 48 2.10 Synthesis of 5’capped mRNA 48 2.11 Brdu incorporation and staining 49 2.12 Detection of apoptotic cells in whole-mount embryos 49 2.13 Cryostat sectioning 50 2.14 FACS and cell cycle analysis 50 2.15 Comet assay 51 2.16 Immunohistochemical staining 52 CHAPTER iv 2.17 Plasmids 53 2.18 Cell culture, transfection and Immunoprecipitation 53 2.19 Cells synchronization 54 2.20 Immunofluorescence 54 2.21 Microscopy 55 udutu24 mutants were characterized by defects in somites, myotome boundaries, muscle pioneers and midline structures 59 3.1 Background 59 3.2 Summary 60 3.3 Results 61 3.31 udutu24 mutants display somite defects 61 3.32 Segmentation clock is functioning in udutu24 mutants 63 3.33 Loss of udu function affects somite boundaries and muscle pioneers 64 3.34 udutu24 mutants show defects in midline structures 66 Discussion 67 Activation of ATM-Chk2-p53 signaling pathway in udutu24 mutants 77 Background 77 CHAPTER 3.4 CHAPTER 4.1 v 4.2 Summary 80 4.3 Results 81 4.31 Dramatic increase of apoptotic cell in udutu24 mutants 81 4.32 Elevated level of p53 mRNA transcript in udutu24 mutants 82 4.33 p53 and its downstream target genes are upregulated in udutu24 mutants 83 4.34 Loss of udu function in cell cycle defects 84 4.35 Activation of ATM-Chk2 pathway in udutu24 mutants 85 4.36 udu mutation causes DNA double stranded breaks 86 4.37 The induction of γ-H2AX after DNA damage 87 Discussion 88 PAH-L and SANT-L domains are essential for DNA replication 108 5.1 Background 108 5.2 Summary 109 5.3 Results 110 5.31 Udu counterparts in human and mouse 110 5.32 Udu is predominantly localized in the nucleus 111 5.33 PAH-L and SANT-L domains may be essential for DNA replication 112 4.4 CHAPTER vi 5.34 Interaction of PAH-L+SANT-L domains with Mcm3 and Mcm4 114 Discussion 114 Loss of functional udu up-regulates p53 and down-regulates Notch signaling during zebrafish development 130 6.1 Background 130 6.2 Summary 132 6.3 Results 133 6.31 Notch activity is down-regulated in udutu24 mutants 133 6.32 Elevated level of p53 could regulate Notch 133 6.33 Udu could regulate Notch 134 6.34 Differential level of Notch acts to regulate the elevated level of p53 in udu deficient background during zebrafish development 135 6.4 Discussion 136 CHAPTER Conclusions 145 CHAPTER References 153 5.4 CHAPTER vii Summary The zebrafish mutant, ugly duckling (udutu24) was isolated from the 1996 Tübingen Nethyl-N-nitrosourea (ENU) screen as a mutant affecting morphogenesis during gastrulation and tail formation (Hammerschmidt et al., 1996), while udusq1 mutant was isolated in a genetic screen aiming at mutants with defects in hematopoiesis (Liu et al., 2007) Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored Both udutu24 and udusq1 embryos share similar phenotypes and the earliest observable developmental defects in udutu24 and udusq1 mutants are the somite phenotypes In this study, I have used udutu24 mutants to carry out studies to further characterize their somite phenotype The expression pattern of somite morphological markers indicated that the anterior-posterior somite identity of udutu24 embryos were affected, while the segmentation clock regulating zebrafish somitogenesis was functioning in udutu24 mutants Overall, my results showed that udutu24 mutants have defects in somite boundaries, muscle pioneers and midline structures Zebrafish embryos with mutations for Notch components have deficits of floorplate and hypochord cells, thus indicating Notch signaling is an important regulator of midline cell fate specification (Appel et al., 1999; Latimer et al., 2002; Jülich et al., 2005; Latimer and Appel, 2006; Zhang et al., 2007) Whole mount in situ hybridization (WISH) of col2a1 and her4 have provided clues to suggest that the Notch signaling pathway may be impaired in udutu24 mutants Hence, further experiments were carried out to investigate the relationship of Udu, p53 and Notch Results indicated that elevated level of p53 in viii udutu24 mutants could regulate Notch activity negatively and experimental results from mib and udu double mutants showed that differential level of Notch can function to rescue the cell death phenotype that is mediated via the p53-dependent pathway in udu embryos Finally, current data suggested that Udu may function to activate Notch only in the absence of p53, possibly due to the interference of Notch activation by p53 The main aim of this study is to find out the causes for the up-regulation of p53 Terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling (TUNEL) assay showed that increased activation of cellular p53-apoptotic pathway correlated with regions of developmental abnormalities Experimental results showed that udutu24 mutants’ extensive p53-dependent apoptosis requires activation of the ATM-Chk2 pathway The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits an appropriate response to the damage The fluorescence-activated cell sorting (FACS) analysis showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udutu24 mutants Positional cloning revealed that Udu protein encodes a novel nuclear factor consisting of two PAH-L (Paired Amphipathic α-Helix like) repeats and a putative SANT-L (SW13, ADA2, N-Cor and TFIIIB like) domain and yeast-two hybrid (Y2H) screen has identified Mcm3 and Mcm4 as interacting partners of these domains Co-immunoprecipitation data indicated that PAH-L repeats and SANT-L domain of Udu interacted with Mcm3 and 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Histopathology 40:547-555 192 ... between the Notch and p53 pathway During mammalian neural development, activation of the Notch signaling pathway is known to be involved in the maintenance of neural progenitor identity and the suppression... Hence, the aim of this study is to investigate what causes the up-regulation of p53 and to further characterize other developmental defects that are associated with the loss of udu function in the. .. 2005) The intrinsic apoptotic pathway can also be activated by p53 involving the pro-apoptotic Bcl-2 family members The translocation of p53 to the mitochondria provoke the release of cytochrome