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  Endocrine regulation of development and aging in Caenorhabditis elegans

Rottiers, V. (2004). Endocrine regulation of development and aging in Caenorhabditis elegans. PhD Thesis, Universiteit, Gent.

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 Creators:
Rottiers, Veerle1, Author
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1Max Planck Society, ou_persistent13              

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Language(s): eng - English
 Dates: 2004
 Publication Status: Accepted / In Press
 Pages: -
 Publishing info: Gent : Universiteit
 Table of Contents: Acknowledgements ii
Aims of the present study 1

Part I: Introduction 4
1. Endocrine regulation of dauer formation and aging in Caenorhabditis elegans 5
1.1. The nematode C. elegans 5
1.2. The dauer larvae 5
1.3. Regulation of dauer formation 7
1.3.1. Insulin/IGF-I signaling 7
1.3.2. TGF-β signaling 11
1.3.3. cGMP signaling 13
1.3.4. Cross talk between the pathways 14
1.3.5. Cellular regulation of dauer formation 14
1.3.6. The nuclear hormone receptor daf-12 14
1.3.7. Other mediators of dauer formation 16
1.4. Regulation of fat storage 17
1.5. Regulation of aging 17
1.5.1. Theories of aging 17
1.5.2. Model organisms in aging research 18
1.5.2.1. Vertebrate models 18
1.5.2.2. Drosophila melanogaster 19
1.5.2.3. Caenorhabditis elegans 19
1.5.2.4. Saccharomyces cerevisae 20
1.5.3. Regulation of aging in C. elegans 20
1.5.3.1. Regulation of aging by insulin/IGF-I 20
1.5.3.2. Regulation of aging by“clk” and mitochondrial
genes 21
1.5.3.3. Regulation of aging by gonad and germ line signaling 22
1.5.3.4. Regulation of aging by caloric restriction 23
1.6. Sex specific differences in dauer formation and life span 23
1.7. Cholesterol, Steroid hormones and C. elegans 24
1.7.1. C. elegans requires exogenous cholesterol 24
1.7.2. The C. elegans genome: predictions on function of cholesterol and sterols 25
1.7.2.1. Cytochrome P450s 25
1.7.2.2. Nuclear hormone receptors 26
1.7.2.3. The sterol sensing domain 27

Part II: Results 28
2. DAF-9 CYP450 regulates C. elegans life history 29
2.1. Abstract 29
2.2. Introduction 29
2.3. Experimental Procedures 31
2.3.1. Culture Conditions 31
2.3.2. Mutant Isolation 31
2.3.3. Mutant Rescue 31
2.3.4. Molecular Biology 32
2.3.5. Life Span Assays 32
2.3.6. Cell Ablations 32
2.4. Results 33
2.4.1. daf-9 regulates reproductive growth and dauer formation 33
2.4.2. Cholesterol deprivation phenocopies daf-9 34
2.4.3. Genetic epistasis 36
2.4.4. daf-9 regulates fat storage 36
2.4.5. daf-9 encodes a cytochrome P450 37
2.4.6. Molecular lesions 39
2.4.7. Expression pattern 40
2.4.8. Life span phenotypes 41
2.5. Discussion 44
2.5.1. The hormone hypothesis 44
2.5.2. daf-9 forms a phenotypic map onto the daf-12 LBD 44
2.5.3. daf-9 couples TGF-β and insulin/IGF-I signals to daf-12 45
2.5.4. daf-9 expression suggests endocrine regulation 45
2.5.5. daf-9/daf-12 interaction 46
2.5.6. Regulation of diapause 46
2.5.7. Life span regulation 46
2.5.8. Conserved endocrine networks 48
2.6. Comments on later publications 48
3. DAF-36, a putative dioxygenase subunit regulating C. elegans dauer formation and life span 50
3.1. Abstract 50
3.2. Introduction 50
3.3. Experimental procedures 52
3.3.1. Culture Conditions 52
3.3.2. Mutant Isolation 52
3.3.3. Mapping daf-36 52
3.3.4. Molecular Biology 52
3.3.5. RNAi experiment 53
3.3.6. Life Span Assays 53
3.3.7. Germ line and Gonad Ablations 53
3.4. Results 54
3.4.1. daf-36 mutants are dauer constitutive and show gonadal migration defects 54
3.4.2. daf-36 is hypersensitive to cholesterol
deprivation 56
3.4.3. Life span phenotypes 56
3.4.4. Genetic epistasis experiments 57
3.4.5. daf-36/daf-9 interaction 58
3.4.6. daf-36/ncr-1 interactions 59
3.4.7. daf-36 is predicted gene C12D8.5 59
3.4.8. daf-36 encodes a terminal dioxygenase 60
3.4.9. Molecular lesions 61
3.4.10. Expression pattern 63
3.4.11. Rieske-like complexes 64
3.4.12. Cross species complementation experiments 65
3.5. Discussion 65
3.5.1. The hormone hypothesis 65
3.5.2. daf-36 is a novel metazoan Rieske oxygenase 66
3.5.3. The DAF-36 complex 69
3.5.4. The nonheme iron binding motive 70
3.5.5. The intestine as endocrine tissue 70
3.5.6. daf-36/daf-9 interaction 71
3.5.7. daf-36 acts in the gonad signaling pathway 72
4. Gonad signaling in hormone pathway mutants 74
4.1. Abstract 74
4.2. Introduction 74
4.3. Methods 77
4.3.1. Germ line and Gonad Ablations 77
4.3.2. Life span Assay 77
4.3.2.1. Liquid culture Life span 77
4.3.2.2. Life span assay on plates 78
4.4. Results 78
4.4.1. Effect of germ line and gonad ablation on wild type worms 78
4.4.2. Sex specific differences on wild type life span 80
4.4.3. Effect of germ line and gonad ablation on daf-12 mutants 81
4.4.4. Effect of germ line and gonad ablation on daf-9 and daf-36 mutants 83
4.5. Discussion 84
4.5.1. Gonad signaling is altered under different environmental conditions 84
4.5.2. Dependence for gonad signaling on daf-12 is dependent on the environmental conditions 85
4.5.3. Extreme long live in daf-12 daf-2 ablated animals 86
4.5.4. Sex differences in gonad signaling 87
4.5.5. Hormone pathway genes are necessary for gonad signaling 89

Part III: General Discussion and Perspectives 91
5. General Discussion and Perspectives 92
5.1. The daf-12 ligand instructs the choice between reproductive growth and dauer formation 92
5.2. Life span regulation by hormone signaling 93

Part IV: Supplemental Materials and Methods 95
6. Supplementary Materials and Methods 96
6.1. Culture Conditions 96
6.2. C. elegans nomenclature 96
6.3. Microscopy 97
6.4. Molecular biology 97
6.4.1. Isolation of genomic DNA of C. elegans 97
6.4.2. Cosmid DNA isolation 97
6.4.3. cDNA clone - DNA isolation and sequencing 98
6.4.4. Primer 98
6.4.5. Sequencing 98
6.4.6. Cloning 98
6.4.6.1. daf-36::gfp construct 99
6.4.6.2. Cloning KshA construct 99
6.4.7. Injection of cosmids and plasmids 99
6.4.8. Database analyses 100
6.5. Mapping daf-36 100
6.5.1. Three factor mapping 100
6.5.2. Deletion mapping 101
6.5.3. SNP mapping 103
6.5.3.1. Bulked segregate analysis 103
6.5.3.2. Using single recombinants 105
6.5.4. Injection of cosmids 106
6.5.5. Sequencing of genes on F21F2 107
6.6. Fat storage measurement 107
6.7. Laser ablation of cells 108
6.7.1. Ablation of germ line and gonad precursors 108
6.7.2. Ablation of neurons 109
6.8. Life span experiments 109
6.8.1. Life span experiments on agar plates 109
6.8.2. Life span experiments in liquid culture 110
6.9. Genetics 110
6.9.1. Epistasis analysis 110
6.9.2. Synergy analysis 111
6.9.3. EMS mutagenesis 111
6.9.4. gfp constructs crossed into mutants 112
6.10. RNAi 114
Summary/Samenvatting 116
Summary 117
Samenvatting 119
Annex 121
Appendices 124
References 132
 Rev. Type: -
 Identifiers: eDoc: 226539
 Degree: PhD

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