xuehui thesis 2007

DIFFERENT FUNCTIONS OF NOTCH ACTIVATION ON FORMATION AND MAINTENANCE OF RHOMBOMERE BOUNDARIES QIU XUEHUI A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISRTY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements I would like to express my deepest gratitude to my supervisor, Dr Yun-Jin Jiang for his invaluable advice, guidance and forbearance over the past five years. Dr Jiang’s depth of scientific knowledge never ceased to amaze me and I have truly enjoyed working under him during my studies. I would like to thank Dr Karuna Sampath, Dr Peng Jinrong and Dr Alan Munn for guidance during my rotating period. Their passion in science has provided me a lot of inspiration and also gave me a chance to pursue my real interest. I would also like to thank Dr Wen Zilong for providing me this opportunity to my PhD. work in IMCB. I would like to extend my sincere thanks to my fellow colleagues in the laboratory, Chiaw-Hwee, Chengjin, Ma Ming, Kate, Ashok, Shangwei, Li Qing, Kenny, Steven and Kian Hong for creating a joyful and conductive working environment. I would also like to thank my ex-collegues, Haoying, Stephanie, Nick, Nguyet, Rida and our visiting scientist Hsiao Chung-Der for the helpful discussion during my project. Furthermore, I would like to specifically thank Chen Jun and Tang Lang for their encouragement during my studies. Most importantly, the support from Danio Unit has greatly facilitated my work, especially to Dr May-Su You. Finally, I would like to thank my family members. My parents have been taking care of my daughter for me without any complain. Moreover, they have given me their encouragements and made me feel confident. In addition, I would like to thank my husband Chen Chunhua. He gives me his moral support and without his love I could not complete my projects. Lastly, I would like to thank my lovely daughter, Chen Jiayi. She brings me a lot of happiness since she was born. i Table of contents Page Acknowledgements i Table of contents ii List of abbreviations vii List of tables ix List of figures x List of publications xii Summary xiv Chapter Introduction 1.1 General introduction 1.2 Hindbrain segmentation 1.2.1 Hox genes in hindbrain patterning 1.2.2 Meis and Pbx in hindbrain patterning 1.2.3 Val and Krox-20 in hindbrain patterning 1.2.4 Vhnf1 and Iro7 in hindbrain patterning 1.2.5 Cdx factors in hindbrain segmentation 1.2.6 Fgf signaling in hindbrain segmentation 10 1.2.7 RA signaling in hindbrain patterning 12 1.3 Ephs and ephrins mediate cell sorting in the hindbrain 13 1.4 Zebrafish as a research model 16 1.5 Sequence of appearance of zebrafish hindbrain boundaries 17 1.6 Boundaries act as signaling center 17 1.7 General introduction of Notch signaling pathway 20 1.8 Notch signaling in Boundary formation 23 ii 1.8.1 Notch signaling in somite boundary formation 24 1.8.2 Notch signaling in the D/V boundary formation in Drosophila wing disc 26 1.8.3 Notch signaling in Drosophila leg segment boundary formation 27 1.8.4 Notch signaling in rhombomere boundary formation 28 1.9 Wnt signaling pathway 30 1.10 Wnt signaling interplays with Notch signaling in the rhombomere boundary formation/maintenance 32 1.11 Glial boundaries 34 1.12 Notch signaling in gliogenesis 35 1.13 Glia-derived signals in gliogenesis 37 1.14 Aim and object of this study 38 Chapter Materials and Methods 2.1 Production and purification of GST fusion proteins 46 2.2 GST pull-down assays 46 2.3 Immunoprecipitation (IP) 47 2.4 in vitro ubiquitination assay 47 2.5 Antisense and sense probe synthesis 48 2.6 Whole-mount in situ hybridization 48 2.7 Detection of fluorescein-labelled probe 49 2.8 Crystat sectioning 49 2.9 Fish maintenance 50 2.10 Viewing anesthetized embryos 50 2.11 Inhibition of cell proliferation 50 2.12 Live embryo imaging 50 2.13 Quick lysis of adult fins 51 2.14 Total RNA extraction 51 iii 2.15 Reverse transcription PCR (RT-PCR) 51 2.16 Immunohistochemical staining 52 2.17 5’/3’ Rapid amplification of cDNA end (RACE) by kit (Roche) 53 2.18 Brdu staining 53 2.19 Cell death assay 54 2.20 Morpholino microinjections 55 2.21 Synthesis of 5’-capped mRNA 55 2.22 DAPT treatment 56 2.23 Heat shock induction 56 Chapter Notch activation is required for the rhombomere boundary maintenance 3.1 Summary 63 3.2 Results 3.2.1 rfng is expressed in the rhombomere boundaries 64 3.2.2 mibta52b mutants display more severe rhomobomere boundary defects than mibtfi91 mutants 3.2.3 Progressive cellular changes within rhomomere boundaries in mibta52b mutants 65 66 3.2.4 Cell adhesion and Wnt signaling are affected differentially in mibtfi91 and mibta52b mutants 66 3.2.5 Cell proliferation is reduced in mibta52b mutants, while inhibition of cell proliferation in wild-type embryos did not result in boundary defects 67 3.2.6 Influence of neurogenesis on rhombomere boundary maintenance 3.2.6.1 Nrarp-deficient embryos have more boundary cells 68 3.2.6.2 hdac1-MO can rescue the boundary defects in mibta52b mutants 69 3.2.7 Disruption of rhombomere boundaries is Su(H)-dependent and dosage-dependent 70 3.2.8 Overexpression of dn-xSu(H) by heat shock beginning at tail bud to 8s stage results in rhombomere boundary disruption 72 iv 3.3 Discussion 73 Chapter Notch1a and Notch3 play a distinct role from Notch1b in rhombomere boundary formation 4.1 Summary 86 4.2 Results 4.2.1 Effects on the expression of notch genes in mibta52b mutants 87 4.2.2 Clone of full-length notch1b 87 4.2.3 Knock-down of notch3 function in noctch1a mutants led to the loss of rhombomere boundaries and neuronal hyperplasia 88 4.2.4 Loss of notch1b function led to a transformation of non-boundary cells into boundary cells and neuronal hypoplasia 89 4.2.5 Two dachsous1 homologues are expressed in the hindbrain and are affected differentially to the change of Notch activation 91 4.2.6 Overexpression of activation of Notch1a leads to excessive gliogenesis at the expense of neurons 4.3 Discussion 92 93 Chapter Wnt signaling in the rhombomere boundary formation 5.1 Summary 117 5.2 Results 5.2.1 Expression of Wnt pathway genes in the rhombomere boundaries 117 5.2.2 Impairment of rhomomere boundaries in Frizzled7b-deficient embryos 118 5.2.3 Mib can interact and ubiquitinate Wnt1, Wnt4 and Wnt8b 119 5.3 Discussion 120 Chapter Loss of Cntfrα results in p53-dependent rhombomere boundary expansion 6.1 Summary 128 6.2 Results v 6.2.1 Clone of full-length cntfrα gene 128 6.2.2 Expression of cntfrα in wild-type zebrafish embryos 129 6.2.3 Mopholino-induced blocking of cntfrα in vivo 129 6.2.4 Loss of cntfrα results in rhombomere boundary expansion 130 6.2.5 Regulation of neurogenesis and gliogenesis in the hindbrain by Cntf signaling 131 6.2.6 Developmental defects observed in Cntfrα-deficient zebrafish embryos are caused by a massive cellular apoptosis and is p53-dependent 132 6.2.7 Cntf signaling interplays with Notch signaling in regulating neurogenesis and boundary formation 133 6.3 Discussion 134 Chapter General discussion 148 Chapter References 154 vi List of abbreviations aa amino acid ANK ankyrin repeats AP alkine phosphatase BCIP 5-Bromo-4-chloro-3-indolyl-phosphate bp base pair BrdU bromodeoxyuridine cntfr α ciliary neutrophic factor receptor α component DAB diaminobenzidine DAPT N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl ester DEAB 4-(diethylamino)benzaldehyde DIG digoxygenin DMSO dimethyl sulfoxide DNA deoxyribonucleotide triphosphate DOTAP N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammonium methylsulfate dpf days post fertilization EDTA Ethylenediaminetetracetic acid disodium Hdac1 histone deacetylase HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid HES Hairly/Enhancer of Split hpf hours post-fertilization IPTG isopropyl β-D thiogalactoside kb kilo base pair kD kilo dalton MAB maleic acid buffer minute vii MO morpholino mRNA messenger RNA NBT 4-Nitroblue tetrazolium chloride ng nanogram NTP 2'-nucleoside 5'-triphosphate PBS phosphate-buffered saline PBST phosphate-buffered saline plus 1% Tween-20 PFA paraformaldehyde PMSF phenylmethylsulfonyl fluoride POD peroxidase PTU 1-pheny-2-thiourea r rhombomere RNA ribonucleic acid RT-PCR reverse transcription polymerase chain reaction rTdT recombinant terminal deoxynucleotidyl transferase s somite SDS sodium dodecyl sulfate SSC sodium chloride-trisodium citrate solution SSCT sodium chloride-trisodium citrate solution plus 1% Tween-20 Su(H) Suppressor of Hairless TCA trichloroacetic acid TCF T cell factor Tricaine 3-amino benzoic acidethylester TSA tyramide signal amplification TUNEL terminal transferase mediated dUTP nick end-labeling UTR untranslated region viii List of tables Table 2.1 Primers used in CS2-myc or PGEX-wnt constructs. Table 2.2 List of molecular makers used in this study. 57 57-58 Table 2.3 Primers used in identifying hsp70:GAL4/+ and UAS:myc-notch1a-intra/+ transgenic line. Table 2.4 Primers used in RT-PCR for cloning partial or full-length genes. 58 58-59 Table 2.5 Primes used in functional studies of cntfrα morphants. 59 Table 2.6 Primers used in 5’-RACE to clone full-length notch1b sequence. 59 Table 2.7 Primers used in 3’-RACE to clone full-length cntfrα sequence. 60 Table 2.8 Morpholinos used in the thesis. 61 Table 2.9 Primers used for checking splicing products. 62 Table 2.10 Function of the markers and antibodies used in this thesis. 62 Table 3.1 Effects of foxb1.2 expression in su(h) morpholino injected embryos. 84 ix Leimeister C., Dale K., Fischer A., Klamt B., Hrabe dA, Radtke F., McGrew M.J., Pourquie O. and Jouve C., Palmeirim I., Henrique D., Beckers J., Gossler A., Ish-Horowicz D. and Pourquie O. 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Wilkinson, D.G (2004) Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain Dev Cell 6, 539-50 Zhang C., Li Q., Lim C.H., Qiu X and Jiang Y.J (2007) The characterization of zebrafish antimorphic mib alleles reveals that Mib and Mind bomb-2 (Mib2) function redundantly Dev Biol 305, 14-27 xiii Summary In vertebrates, the hindbrain is subdivided... (Kanki and Ho, 1997) Loss of cdx1a and cdx4 results in the failure of the development of spinal cord together with the posterior expansion of hindbrain territory (Shimizu et al., 2006; Skromne et al., 2007) Despite the expansion of r7/r8 region, the native anterior hindbrain appeared to be intact More interestingly, the expanded hindbrain is organized into segmental units and is arranged in a mirror-image... val expression is expanded All these results support a posteriorizing role for RA (Begemann et al., 2001; Grandel et al., 2002) However, neither the concentration of RA nor the localization of its synthesis is critical for the proper pattern of hindbrain (Maves and Kimmel, 2005) Moreover, the duration of RA exposure is also not critical for the generation of nested domains of RA-responsive gene expression... exogenous RA Therefore, it is believed that the RA responsiveness along the anterior-posterior axis of hindbrain is regulated primarily by the dynamic expression of RA-degrading enzymes (Hernandez et al., 2007) In addition to the Hox gene expression, RA signaling is also required for the expression of vHnf1 in the posterior hindbrain (Hernandez et al., 2004), which in turn acts as a repressor to regulate... homeodomain transcription factor Lmx1b is suggested to be essential for both the initiation of Fgf8 expression and the maintenance of several other genes, such as Wnt, En1, Pax2 and Gbx2 (Guo et al., 2007) Similar results are attained from zebrafish and chick, indicating that Lmx1b is indispensable for the initiation and maintenance of the induction activity of the isthmic organizer during MHB development