Nanog in the Twin Fish Models Medaka and Zebrafish: Functional Divergence or Pleiotropy of Vertebrate Pluripotency Gene Li Zhendong (M.Sc, Nankai University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my deepest gratitude to my supervisor A/P Hong Yunhan for giving me the opportunity to carry out research under his guidance, for his great patience and encouragement, for imparting his invaluable knowledge and technical expertise to me during the last six years I would like to thank A/P Wang Shu, A/P Peng Jinrong and Dr Paul Robson, as my QE committee members, for their support and sharing the ideas Thanks go to A/P Ng Huck Hui for sharing mouse nanog construct I am also debt to Madam Deng Jiaorong and Mr Zeng Qinghua for medaka breeding; Mr Subhas Balan for zebrafish support; Ms Veronica Wong for the ordering matters I greatly thank my labmates: Chen Tiansheng, Xu Hongyan, Li Mingyou, Hong Ni, Tan Sze ley, Wang Li, Yovita Ida Purwanti, Yi Meisheng, Guan Guijun, Zhao Haobing, Lu Wenqing, Wang Weijia, Lim Meng Huat, Liu Rong, Liu Lixiu and Yan Yan for their kind help and the happy time they brought to the lab I also thank Liang Dong, Li Yan, Xiang Wei, Wang Xingang, Li Zhen, and Zhan Huiqin for their kind help in zebrafish I thank National University of Singapore and Department of Biological Sciences for providing me scholarship and opportunity to study here Finally, I would like to thank my parents for their support all these years i Table of contents Acknowledgements i Table of Contents -ii Summary -vi List of Figures and Tables -viii List of Abbreviations -x List of Publications -xii Chapter I: Introduction - 1.1 Embryogenesis and stem cells - 1.1.1 Embryonic stem cells - 1.1.2 Adult stem cells - 1.1.3 Germ stem cells Mechanisms modulating pluripotency in stem cells - 1.2.1 Intrinsic transcription factors 1.2.2 Signaling pathways in maintaining ES cell pluripotency: 10 1.2.2.1 LIF/Stat3 pathway -10 1.2.2.2 Transforming growth factor β (TGF-β) superfamily pathway 11 1.2.2.3 WNT-beta catenin pathyway -14 1.2.2.4 FGF pathway 16 1.3 The homeodomain transcription factor Nanog -20 1.3.1 General introduction -20 1.3.2 Expression pattern -20 1.3.3 Nanog target genes -22 1.3.4 Regulation of nanog 23 1.3.5 Cofactors of Nanog -26 1.3.6 iPS –induced pluripotent stem cells by defined factors -26 1.3.7 Domain structure of Nanog protein -28 1.4 Medaka as a vertebrate model -32 1.4.1 General introduction -32 1.4.2 Stem cell research in medaka 35 1.4.3 Medaka embryonic development 35 1.5 Zebrafish as a vertebrate model -36 1.5.1 Zebrafish as a popular vertebrate model 36 1.5.2 Early Stages of embryonic development of zebrafish -37 1.5.3 Molecular vertebrate axis formation in zebrafish -41 1.6 Objetives of this study -45 Chapter II: Materials and Methods -47 2.1 DNA manipulation 47 ii 2.1.1 Polymerase chain reaction (PCR) -47 2.1.2 Reverse-transcriptase PCR (RT-PCR) -47 2.1.3 Rapid amplification of cDNA ends (RACE) 48 2.1.4 Cloning of PCR products (T-A cloning) 49 2.1.5 DNA ligation -49 2.1.6 Preparation of competent cells -49 2.1.7 Transformation -50 2.1.8 Colony screening by restricion enzyme digestion 51 2.1.9 Automatic sequencing -51 2.1.10 Isolation of plasmid DNA 52 2.1.11 Isolation of genomic DNA -53 2.1.12 Purification of DNA fragments from agarose gel -54 2.1.13 Purification of DNA from enzyme reaction solution -54 2.1.14 Restriction endonuclease (RE) digestion of DNA 54 2.1.15 DNA gel electrophoresis -55 2.1.16 Quantification of DNA by spectrophotometry 55 2.1.17 Bioinformatic analysis -55 2.1.18 Vectors -56 2.2 RNA manipulation 56 2.2.1 Isolation of total RNA -56 2.2.2 Synthesis of 5’ capped mRNA 56 2.2.3 Quantification of RNA by spectrophotometry 57 2.2.4 In situ hybridization -57 2.2.4.1 Probe synthesis 57 2.2.4.2 Whole mount in situ hybridization (WISH) 58 2.2.4.3 Section in situ hybridization (SISH) 60 2.2.4.4 Fluorescent in situ hybridization 60 2.3 Protein manipulation -61 2.3.1 His-tagged fusion protein expression and purification -61 2.3.2 Antibody preparation 62 2.3.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) -62 2.3.4 Western Blot -63 2.3.5 Immunohistochemical staining 64 2.4 Culture of medaka ES cells -64 2.4.1 Preparation of medaka embryo extract 64 2.4.2 Preparation of fish serum -65 2.4.3 Preparation of tissue culture plate -65 2.4.4 Preparation of ES cell medium ESM4 -65 2.4.5 Subculture of ES cells 66 2.4.6 Counting Cells -66 2.4.7 Freezing of ES cells -66 2.4.8 Thawing of ES cells 66 2.4.9 Cell Transfection with GeneJuice -67 Microinjection into cytoplasm of medaka embryos -67 iii Chapter III: Characterization, expression and function of medaka nanog -69 3.1 Results -69 3.1.1 Isolation of a nanog homolog from medaka -69 3.1.2 Gene structure of Ong -70 3.1.3 Phylogenetic analysis 77 3.1.4 Synteny analysis of Ong -77 3.1.5 Expression analysis -79 3.1.5.1 Spatial and temporal expression analysis by RT-PCR -79 3.1.5.2 Expression analysis in ovary by ISH -81 3.1.5.3 Expression analysis in testis by ISH 81 3.1.5.4 Expression in early embryonic stages 84 3.1.5.5 Protein expression analysis by western blot 87 3.1.5.7 Ong protein localization in ovary -90 3.1.5.8 Ong protein expression in germ cells of fry fish -90 3.1.6 Functional analysis -93 3.1.6.1 Function analysis by overexpression in ES cells 93 3.1.6.2 ES cell identity is associated with Ong expression -94 3.1.6.3 Dominant Negative Mutant analysis -98 3.1.6.3 Functional analysis by Morpholino oligonucleotides (MO) knockdown - 102 3.1.7 Ong is required for cell fate decision - 111 3.2 Discussion - 114 3.2.1 Ong is specifically expressed in pluripotent cells - 114 3.2.2 Ong is maternally supplied - 115 3.2.3 Expression in spermatogonia 116 3.2.4 Expression in early cleavage stage 117 3.2.5 Expression in primordial germ cells (PGCs) - 118 3.2.6 Western blot analysis 118 3.2.7 Function analysis by DNM analysis 119 3.2.8 Ong function in medaka ES cells 120 Chapter IV: Expression and function of zebrafish nanog - 122 4.1 Results - 122 4.1.1 Isolation of zebrafish nanog (Zng) 122 4.1.2 Expression analysis of Zng 122 4.1.2.1 Expression of Zng by RT-PCR - 122 4.1.2.2 Expression of Zng in adult tissues by ISH. 122 4.1.2.3 Spatial expression of Zng in early embryos by WISH 123 4.1.3 Functional analysis by MO knockdown - 129 4.1.4 Rescue of MO knockdown phenotypes by Ong and mouse nanog - 133 4.1.5 Mechanisms of DV and neuroectoderm patterning defects 136 4.2 Discussion - 142 4.2.1 Zng is expressed in adult brain, kidney and gonad - 142 4.2.2 Zng is maternally expressed in all blastomeres - 142 4.2.3 Zng is weakly expressed in somitogensis stage - 142 4.2.4 Maternal Zng is required for neuroectoderm specification 143 iv 4.2.5 Zng is required for tail formation - 143 Chapter V: General Discussion - 145 5.1 Why choosing fish nanog 145 5.2 Existence of fish nanog - 146 5.3 Ong and Zng are homologues of mammalian nanog 147 5.4 Divergence of nanog among vertebrates - 147 5.5 Nanog regulates pluripotency or DV patterning - 148 5.6 Models of Nanog function in fish 148 Chapter VI: Conclusion and perspective - 151 Conclusion - 151 Perspective - 152 Reference 153 Appendix 175 v Summary Pluripotency maintenance and embryo patterning are key events of early embryogenesis Transcription factors Nanog, Oct4 and Sox2 constitute a core circuit for regulating pluripotency in mouse embryonic stem (ES) cells and early developing embryos However, zebrafish Pou2, homolog of mammalian Oct4, is a maternal determinant for dorsoventral patterning Nanog shows extensive sequence divergence, producing an as low as 26% identity between mammals and chicken Whether nanog exists and plays a conserved role in pluripotency or patterning in lower vertebrates, in particular in fish, the ancient vertebrate lineage, has been unclear This work was aimed at the identification, expression and function of nanog in the medaka and zebrafish as excellent models for analyzing pluripotency and patterning The medaka and zebrafish nanog termed Ong and Zng respectively, encode proteins of 420 amino acids (aa, Ong) and 384 aa (Zng), which exhibit a best but 16-18% low sequence identity to tetrapod Nanog and lacks chromosomal synteny to tetrapod vertebrates It has, however, the conserved 4-exon structure and a unique motif The homology between fish and mouse nanog genes was established by the experiments where the mouse nanog can produce gain-of-function phenotype and rescue the loss-of-function phenotype in both fish species In vivo, Ong is expressed throughout the pluripotency cycle, including the zygote and germline In vitro, Ong RNA and protein are high in ES cells and down-regulated upon differentiation Importantly, forced Ong expression supported ES cell proliferation under differentiation conditions Upon zygotic RNA injection, Ong overexpression affected blastomeres proliferation, whereas Ong interference by dominant-negative mutants or morpholino-based knockdown compromised cell divisions and lineage commitment, leading to gastrula arrest as well as to the loss of yolk vein and tail defects Strikingly, despite extensive sequence divergence and chromosome rearrangements, medaka nanog possesses the conserved role in pluripotency maintenance at early stages and previously unidentified roles in late stages Zng exhibits a similar expression pattern to Ong with a salient difference: in contrast vi to restricted Ong expression in the central blastomeres, Zng distributes in all blastomeres Zng knockdown led to strongly dorsalized embryos and other profound defects including eyeless phenotype, loss of yolk vein and tail defects Zng knockdown led to severe reduction in the early zygotic expression of ventralizing genes (vox, ved and vent), dorsoventral patterning gene pou2 and neuroectodermal genes (pax2.1 and pax6.1) and to the expanded expression of the mesendodermal gene ntl (no tail) Therefore, similar to zebrafish Pou2, Zng becomes another determinant for dorsoventral patterning in early embryogenesis In conclusion, nanog plays an essential role in pluripotency maintenance in medaka but in dorsoventral patternining in zebrafish This striking finding provides direct evidence for functional conservation and divergence or pleiotropism of a patterning or pluripotency gene vii List of Figures and Tables Fig 1-1 Fig 1-2 Fig 1-3 Schematic diagram showing embryonic cell lineage and derivation of stem cells Models of core transcription networks containing Oct4, Sox2 and Nanog Nanog mRNA is confined to pluripotent/multipotent cells 19 Table 1-1 Summary of nanog expression pattern Fig 1-4 Fig 1-5 Fig 1-6 Fig 1-7 Fig 1-8 Fig 1-9 Fig 3-1 Fig 3-2 Fig 3-3 Fig 3-4 Fig 3-5 Fig 3-6 Fig 3-7 Fig 3-8 Fig 3-9 Fig 3-10 Fig 3-11 Fig 3-12 Fig 3-13 Fig 3-14 Regulation of Nanog by other transcription factors Alignment analysis of homeodomains from various homeoproteins Nanog domain structure The phylogeny of vertebrates Zebrafish fate maps Fig 3-15 Fig 3-16 Fig 3-17 Fig 3-18 Fig 3-19 Fig 3-20 Fig 3-21 Fig 3-22 Table 3-1 Fig 3-23 Fig 3-24 Fig 3-25 20 25 30 31 34 40 44 Transcriptional interactions patterning the dorsal-ventral axis PCR cloning of the Ong cDNA 71 Ong cDNA sequence with deduced anmino acid sequence 72 Alignment of homeodomains of Nanog with related Nkx and Vent family 73 Genomic organizations of nanog, vent and nkx2.5 74 Protein domain structure of Nanog 75 Phylogenetic tree of Nanog 76 Chromosomal location and synteny analysis of Ong 78 RT-PCR analysis of Ong 80 Ong mRNA expression in oocytes and oogonia of adult ovary 82 Ong RNA expression in adult testis 83 Ong RNA expression during early embryogenesis until gastrulation 85 Ong RNA expression in primordial germ cells 86 Ong protein presents in gonads and ES cells 88 Nanog protein expression and nuclear localization in medaka ES cells and 89 embryos Ong protein localization in oocytes 91 Ong protein is localized to vasa positive germ cells from male fry 92 Ong protein colocalizes with vasa in female germ cells 92 Ong expression is sufficient to prevent ES cell differentiation 95 Ong expression is sufficient for clonal expansion of ES cells in the 96 absence of growth factor bFGF ES cell differentiation is associated with loss of Ong expression 97 DNM analysis of Ong in early development by injection of OngDN2 100 DNM analysis of Ong in early development by injection of OngDN3 101 Dose-dependent phenotypes by OngMO knockdown 104 Examination of OngMO1 specificity 105 Classification of phenotypes caused by OngMO1 knockdown 107 Phenotype class IV caused by OngMO 108 viii Fig 3-26 Fig 3-27 Fig 3-28 Fig 4-1 Fig 4-2 Fig 4-3 Fig 4-4 Fig 4-5 Table 4-1 Fig 4-6 Fig 4-7 Table 4-2 Fig 4-8 Fig 4-9 Fig 4-10 Fig 4-11 Fig 4-12 Fig 4-13 Fig 5-1 Fig 5-2 Rescue of OngMO phenotype by mouse nanog mRNA Ong knockdown affects blastula cell fate Ong knockdown led to 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2003 An SP1-like transcription factor Spr2 acts downstream of Fgf signaling to mediate mesoderm induction EMBO J 22, 6078-88 174 Appendix Alignment of Nanog homologues Appendix Identity value is given to the end of each sequence 175 Appendix Primer List name sequence ongF CTCCACATGTCCCCCCTTATC ongR AGGATAGAATAGTCACATCAC ongb1 CGCGGATCCATGGCGGAGTGGAAAACTCAGGTC ongx1 ACCGCTCGAGTCATTGGACAGCATTGTGCAAAATG oeomesF ATGCAGTTGGAGAACATCCTTC oeomesR TAGGCAAAGGCAGAGTCTG ovoxF ATGGTCAAATACTTTTCAGTAGACTG ovoxR TCAGAAGAAATGTTGGTAGTGGA osox32F ATGCGTTTCAGCCACGACC osox32R TTATGACACTGAATTTGTTTCTTTGTTG ontlF: CTGCCTACCAGAACGAAGAGA ontlR: TTCGATCAGTAGAAGGCACGT oactinF CCACACATTTCTCGCTCACTC oactinR TTAAGTATGCTCAACCACTGG ooct4F: GTAGGTCACCTGACAGGATGATG ooct4R CTGATTGCACTCTGACAGCAAGT zngstart2 GAGCGCTTCAATCAGCATCC zngexon2R GTTAAGTTCCGTTCTCCACTGTC zngB1 TGTCGGATCCATGGCGGACTGGAAGATGC zngsal1 AAAGTCGACTCACAGCAAAGTTATTCCTTTAGTTGC zactin-F GTTTTCCCCTCCATTGTTG zactin-R GGTGTTGAAGGTCTCGAACA mNg B1 CGCGGATCCATGAGTGTGGGTCTTCCTGGTC mNgx1 ACCGCTCGAGTCATATTTCACCTGGTGGAGTCAC zoct4F ATGCTTCAGGATCCAAGTCTGA zoct4R TAATTTGGGACAGTCCAGGC zpax6.1F GAGGGAGAGTCCAATATTCGAC zpax6.1R GCATAGTTCCAACAGCCTTTGTA zbrachyuryF TGTCGGATCCTCTTGGATGATAG zBrachyuryR CTATAAGGATGGTGGTGCCAC zntlF ATGTCTGCCTCAAGTCCCGAC zntlR GAGAGTCGTCCCTGCAACTG zpax2.1F ATGGATATTCACTGCAAAGC zpax2.1R CTAGTGGCGGTCATAGGCAG zgscF ATGCCCGCTGGGATGTTTAG zgscR TCAGCTGTCAGAATCCACGTC use RT-PCR Size (bp) 591 expression, probe, 1263 RT-PCR 1586 RT-PCR 735 RT-PCR 918 RT-PCR 985 RT-PCR 317 RT-PCR 660 RT-PCR 609 expression, probe, 1155 RT-PCR 300 expression, probe, 915 probe 418 probe 418 probe 1298 probe 1046 probe 1176 probe 723 176 Appendix Primer List zvedF ATGAAGGGTCAGTTCTCCATC zvedR TCAGTGTGTGCTGTAATACTG zvoxF ATGGTGAAGAACTTTTCCGTG zvoxR TCAGTAGTAATGATGTCTGGG zventF ATGATACCCAGCAAGTTCTC zventR TTAGAAGTAGCAGCGTGTGAAC probe 837 probe 729 probe 513 o, medaka; z, zebrafish; m, mouse Underline indicates restriction enzyme site 177 Appendix Medaka stage map, from Takashi IWAMATSU (2004) 178 Appendix Zebrafish stage map 179 Appendix Zebrafish stage map 180 Appendix Plasmid map From http://www.imagenes-bio.de/info/vectors/pCS2plus_pic.shtml HindIII (29) SP6 promoter Bam HI (80) CMV Eco RI (139) mouseNanog Sal I (4005) f1 origin T7 pCS2mNanog 4991 bp Xho I (1004) SV40 polyA Not I (1243) 181 Appendix Plasmid map SP6 Bam HI (80) CMV Ong Eco RI (1211) pCS2ong Xho I (1349) 5336 bp poly A Not I (1588) CMV promoter Xho I (1) Eco 255I (647) Nanog Eco RI (1164) Fse1 pCVongCVpf 7794 bp CMV promoter Kana/neo pf2 Bam HI (3831) Not I (3850) 182 Appendix Plasmid map Bam HI (80) OngDN1 Xho I (893) pCS2ongDN1 4880 bp poly A Not I (1132) Bam HI (80) OngDN2 pCS2ongDN2 5177 bp Xho I (1190) poly A Not I (1429) 183 Bam HI (80) Nanog partial Eco RI (1211) pCS2ongDN3 5924 bp GFP Xho I (1937) poly A Not I (2176) CMV Apa LI (4776) Nco I (514) HindIII (749) DraI (4335) Apa BI (796) DraI (4316) Pst I (831) Ava I (894) Xma I (894) pCVpf2 Sma I (896) 5106 bp Sal I (953) DraI (3624) Sac II (1059) Apa LI (3530) Apa BI (1417) PF Bam HI (2178) Apa BI (3033) Apa LI (3033) Not I (2197) DraI (2401) ClaI (2435) Bam HI (2442) 8888 Appendix Plasmid map 185 ... at the identification, expression and function of nanog in the medaka and zebrafish as excellent models for analyzing pluripotency and patterning The medaka and zebrafish nanog termed Ong and. .. patternining in zebrafish This striking finding provides direct evidence for functional conservation and divergence or pleiotropism of a patterning or pluripotency gene vii List of Figures and Tables... zebrafish Pou2, Zng becomes another determinant for dorsoventral patterning in early embryogenesis In conclusion, nanog plays an essential role in pluripotency maintenance in medaka but in dorsoventral