Identification of factors that establish asymmetry and cell-death fate in the NSM lineage in Caenorhabditis elegans [Elektronische Ressource] / Julia Hatzold

Julia Hatzold

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Identification of Factors That Establish
Asymmetry and Cell-death Fate
in the NSM lineage
in Caenorhabditis elegans







Julia Hatzold


München 2006


Identification of Factors That Establish
Asymmetry and Cell-death Fate
in the NSM lineage
in Caenorhabditis elegans



Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München



vorgelegt von
Julia Hatzold
aus München



München, den 21. April 2006




















Erstgutachter: Prof. Dr. Charles N. David
Zweitgutachter: Prof. Dr. Stefan Jentsch
Tag der mündlichen Prüfung: 30. Mai 2006 Table of Contents
Table of Contents
Table of Contents...........................................................................................................1
1 Abstract..................................................................................................................5
2 Introduction............................................................................................................6
2.1 C. elegans represents an excellent model organism for studying
development and programmed cell death ..................................................................7
2.2 Cell death in C.elegans ..................................................................................7
2.3 The specification of cell death in the NSM sister cells..................................9
2.4 Thesis Aims.................................................................................................12
3 Material and Methods ..........................................................................................13
3.1 Materials......................................................................................................13
3.1.1 Chemicals.............................................................................................13
3.1.2 Devices14
3.1.3 Enzymes, antibodies, and standards.....................................................15
3.1.4 Kits.......................................................................................................16
3.1.5 Bacteria strains.....................................................................................16
3.1.6 Plasmids and Vectors...........................................................................17
3.1.6.1 Provided vectors and plasmids.........................................................17
3.1.6.2 Constructed plasmids.......................................................................17
3.1.7 Oligonucleotides..................................................................................19
3.1.8 C. elegans strains .................................................................................21
3.1.8.1 Strains used in this study..................................................................21
3.1.8.2 Strains constructed in this study ......................................................23
3.2 Methods........................................................................................................26
3.2.1 Molecular biological methods..............................................................26
3.2.1.1 Protein purification..........................................................................26
3.2.1.2 Electrophoretic mobility shift assays...............................................27
3.2.1.3 Immunohistochemistry....................................................................27
3.2.1.4 RT-PCR analysis of hlh-3 transcripts ..............................................28
3.2.2 Biological examinations of C. elegans ................................................28
3.2.2.1 Generation of transgenic animals.....................................................28
3.2.2.2 RNAi experiments...........................................................................29
1Table of Contents
3.2.2.2.1 RNAi by feeding as the method of delivering dsRNA ..............29
3.2.2.2.2 RNAi by injection as the me ............29
3.2.2.3 PCR-Screen for deletion mutants.....................................................30
3.2.2.4 EMS mutagenesis and hlh-2(bx108) enhancer screen .........................30
3.2.2.5 SNP mapping...................................................................................31
3.3.3.6 Estimation of the cell size of NSM and NSM sister cells....................31
4 Results..................................................................................................................33
4.1 The NSM sister cell death is specified by asymmetric expression of the cell-
death activator gene egl-1 ........................................................................................33
4.2 Region B of the egl-1 locus is required for egl-1 expression in the NSM
sister cells.................................................................................................................35
4.3 Identification of factors that contribute to the killing of the NSM sister cells
37
4.3.1 Candidate gene approach .....................................................................37
4.3.1.1 The bHLH gene hlh-2 is required for the NSM sister cell death .....39
4.3.1.2 hlh-2 acts synergistically with hlh-3 to kill the NSM sister cells ....40
4.3.1.2.1 RNAi of achaete-scute-like bHLH genes ..................................40
4.3.1.2.2 Isolation and characterization of hlh-3 deletions.......................41
4.3.1.2.3 hlh-2 and hlh-3 are specifically required for the NSM sister cell
death 43
4.3.1.2.4 Are HLH-2 and HLH-3 present in the NSM sister cell at the time
it is dying? 44
4.3.1.2.4.1 Co-localization of EGl-1 and HLH-2 using antibody staining
44
4.3.1.2.4.2 The NSM and NSM sister cell can be identified due to their
position within the embryo ......................................................................46
4.3.1.2.4.3 hlh-2::gfp is expressed in the NSM and the NSM sister cell
but not in the NSM mother cell................................................................49
4.3.1.2.4.4 hlh-3::gfpo49
4.3.1.2.5 HLH-2 and HLH-3 bind to Region B of the egl-1 locus in vitro
50
4.3.1.2.6 HLH-2 and HLH-3 can induce ectopic egl-1 expression...........51
4.3.2 hlh-2 enhancer screen ..........................................................................53
2Table of Contents
4.3.2.1 Summary of candidates....................................................................55
4.3.2.2 Analysis of the Ces phenotype of the identified mutants ................56
4.3.2.3 Linkage analysis and characterization of the candidates .................59
4.3.2.3.1 bc97............................................................................................59
4.3.2.3.2 bc211..........................................................................................62
4.3.2.3.3 bc213 and bc214 represent new alleles of ces-2........................63
4.3.2.3.3.1 bc213 results in an early stop codon in the ces-2 coding
region 64
4.3.2.3.4 bc240..........................................................................................65
4.3.2.3.5 bc24168
4.3.2.3.6 bc24269
4.3.2.3.7 bc25274
4.3.2.3.8 bc260..........................................................................................76
4.4 dnj-11 is required for the NSM sister cell death..........................................76
4.4.1 Cloning of bc212: bc212 is a mutation in dnj-11 ................................77
4.4.2 dnj-11(bc212) animals have a pleiotropic phenotype and bc212 is most
likely a missense mutation...................................................................................83
4.4.2.1 dnj-11(bc212) results in a cold-sensitive NSM sister cell survival
phenotype which is suppressed by a loss-of-function mutation in ces-1.........84
4.4.2.2 dnj-11 is not required for cell death in general................................85
4.4.2.3 Reduction of dnj-11 function results in morphological defects.......87
4.4.2.4 dnj-11(bc212) causes slow growth and embryonic lethality ...........89
4.4.2.5 The broodsize of dnj-11(bc212) animals is strongly reduced..........90
4.4.2.6 P dnj-11::gfp is Expressed Ubiquitously, and DNJ-11::GFP dnj-11
Localizes to the Cytosol...................................................................................90
4.4.2.7 dnj-11 encodes a protein with a DnaJ domain and two Myb-like
DNA binding domains and is a member of the MIDA1-like protein family...92
4.4.2.8 dnj-11 might be involved in establishing asymmetry during the
division of the NSM mother cell......................................................................95
4.4.2.9 Analysis of the domains of DNJ-11 for their functional relevance in
the NSM sister cell death .................................................................................99
4.4.2.10 Are Hsp70 proteins involved in establishing the cell death fate of
the NSM sister cell?.......................................................................................101
4.5 Identification of factors that repress egl-1 expr