Key experiments in practical developmental biology /
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作 者：edited by Manuel Mari-Beffa, Jennifer Knight.
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ISBN：9780521833158
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Summary:
Publisher Summary 1
Including many described by the scientists who carried out the original pioneering research, 27 classic experiments in developmental biology provide key insights into developmental questions. They establish a bridge between state-of-the-art experimental work and the laboratory classes taken at the undergraduate and post-graduate levels. All chapters follow the same logical format, taking the students from materials and methods, through results and discussion, so that they learn the underlying rationale of the research.
Publisher Summary 2
This manual presents 27 laboratory exercises for student practical classes in developmental biology.
目录
Cover 1
Half-title 3
Title 5
Copyright 6
Dedication 7
CONTENTS 9
Preface 13
REFERENCES 14
CONTRIBUTORS 15
Introduction 19
KEEPING A LABORATORY NOTEBOOK 19
WRITING A LABORATORY REPORT 20
SECTION I. GRAFTINGS 22
1 Two developmental gradients control head formation in hydra 22
INTRODUCTION 22
MATERIALS AND METHODS 24
EQUIPMENT AND MATERIALS 24
REAGENTS 25
PREVIOUS TASKS FOR STAFF 25
TRANSPLANTATION PROCEDURE 25
OUTLINE OF THE EXPERIMENTS 28
A.A HEAD ACTIVATION GRADIENT IN THE BODY COLUMN 28
B.A HEAD INHIBITION GRADIENT IN THE BODY COLUMN 30
EXPECTED RESULTS AND DISCUSSION 31
DEMONSTRATION OF THE HEAD ACTIVATION PROPERTY 32
DEMONSTRATION OF THE DISTRIBUTION OF HEAD ACTIVATION 33
DEMONSTRATION OF THE HEAD INHIBITION PROPERTY 33
DEMONSTRATION OF THE DISTRIBUTION OF HEAD INHIBITION 33
TIME REQUIRED FOR THE EXPERIMENTS 33
POTENTIAL SOURCES OF FAILURE 34
TEACHING CONCEPTS 34
ALTERNATIVE EXERCISES 34
PROPOSED EXPERIMENTS 35
QUESTIONS FOR FURTHER ANALYSIS 36
REFERENCES 37
APPENDIX: MAINTENANCE OF A HYDRA CULTURE 37
MATERIALS AND EQUIPMENT (PER CLASS OF STUDENTS) 38
RAISING AND HANDLING HYDRA 38
FEEDING AND WASHING HYDRA 39
2 Embryonic regulation and induction in sea urchin development 41
INTRODUCTION 41
MATERIALS AND METHODS 43
BIOLOGICAL MATERIAL 43
LABORATORY EQUIPMENT AND SUPPLIES 43
CHEMICALS AND SOLUTIONS 45
OUTLINE OF THE EXPERIMENTS 46
EXPERIMENT 1. REGULATIVE DEVELOPMENT OF ISOLATED BLASTOMERES 46
EXPERIMENT 2. INDUCTION OF ENDODERM BY MICROMERES 47
EXPERIMENT 3. IMMUNOSTAINING OF EMBRYO WHOLE MOUNTS 48
EXPECTED RESULTS 50
EXPERIMENT 1 50
EXPERIMENT 2 50
EXPERIMENT 3 51
DISCUSSION 51
POTENTIAL SOURCES OF FAILURE 51
TIME REQUIRED FOR THE EXPERIMENTS 52
ADDITIONAL EXPERIMENTS 53
TEACHING CONCEPTS 53
REFERENCES 54
3 The isthmic organizer and brain regionalization in chick embryos 55
INTRODUCTION 55
MATERIALS AND METHODS 56
EQUIPMENT AND MATERIALS 56
PREVIOUS TASKS FOR STAFF 57
OUTLINE OF THE EXPERIMENT 58
SETTING EGGS INTO THE INCUBATOR 59
PROTOCOL FOR PREPARATION OF EGGS AND EMBRYOS FOR TRANSPLANTATION 60
PROTOCOL FOR TRANSPLANTING THE ISO REGION TISSUE INTO THE DIENCEPHALON 60
COLLECTION OF SURGICALLY MANIPULATED CHICK EMBRYOS 61
IDENTIFICATION OF THE QUAIL TISSUE BY IMMUNOHISTOCHEMISTRY 61
EXPECTED RESULTS 63
DISCUSSION 63
TIME REQUIRED FOR THE EXPERIMENTS 64
POTENTIAL SOURCES OF FAILURE 64
ALTERNATIVE EXERCISES 64
HISTOLOGICAL ANALYSIS OF THE ECTOPIC STRUCTURES FROM THE GRAFT EXPERIMENTS 64
IMPLANTATION OF FGF8-SOAKED HEPARIN ACRYLIC BEADS 65
IN SITU HYBRIDIZATION IN TOTO 65
QUESTIONS FOR FURTHER ANALYSIS 65
ACKNOWLEDGEMENTS 65
REFERENCES 66
APPENDIX 66
SECTION II. SPECIFIC CHEMICAL REAGENTS 68
4 Chemotaxis of aggregating Dictyostelium cells 68
INTRODUCTION 68
TIME REQUIRED FOR THE EXPERIMENT 72
MATERIALS AND METHODS 72
EQUIPMENT AND MATERIALS 72
REAGENTS 73
PREVIOUS TASKS FOR STAFF 73
PROCEDURES 74
OUTLINE OF THE EXPERIMENTS 74
CHEMOTACTIC STIMULATION 74
POTENTIAL SOURCES OF FAILURE 76
EXPECTED RESULTS 77
DISCUSSION 78
ALTERNATIVE EXERCISES 80
STIMULATION OF OTHER CELLS THROUGH A MICROPIPETTE 80
TEACHING CONCEPTS 81
ADDITIONAL INFORMATION AND TEACHING AIDS 82
APPENDIX 82
HISTORICAL OUTLOOK 82
REFERENCES 83
5 Inhibition of signal transduction pathways prevents head regeneration in hydra 85
INTRODUCTION 85
MATERIALS AND METHODS 86
OUTLINE OF THE EXPERIMENTS 90
EXPERIMENT: EFFECT OF THE INHIBITOR OF HEAD REGENERATION 90
EXPERIMENT: EFFECT OF THE INHIBITOR ON GENE EXPRESSION 91
EXPECTED RESULTS 92
DISCUSSION 92
TIME REQUIRED FOR THE EXPERIMENTS 93
TEACHING CONCEPTS 93
ALTERNATIVE EXERCISES 93
QUESTIONS FOR FURTHER ANALYSIS 93
REFERENCES 93
6 Retinoic acid during limb regeneration 95
INTRODUCTION 95
MATERIALS AND METHODS 98
EQUIPMENT AND MATERIALS 98
REAGENTS 98
PREVIOUS TASKS FOR STAFF 98
PROCEDURES 99
OUTLINE OF THE EXPERIMENT 99
EXPECTED RESULTS 100
DISCUSSION 100
TIME REQUIRED FOR THE EXPERIMENTS 100
POTENTIAL SOURCES OF FAILURE 101
TEACHING CONCEPTS 101
ALTERNATIVE EXERCISES 101
PROPOSED EXPERIMENT 101
ACKNOWLEDGEMENTS 101
REFERENCES 102
SECTION III. BEAD IMPLANTATION 103
7 Experimental manipulations during limb development in avian embryos 103
INTRODUCTION 103
MATERIALS AND METHODS 106
EQUIPMENT AND MATERIALS 106
PREVIOUS TASKS FOR STAFF 106
SOLUTIONS 106
BIOLOGICAL MATERIAL 107
OUTLINE OF THE EXPERIMENTS 107
INCUBATION OF EGGS 107
MANIPULATION OF EMBRYOS 107
PREPARATION OF EMBRYOS 110
NEUTRAL RED PROTOCOL FOR DETECTION OF CELL DEATH 110
CARTILAGE STAINING 110
EXPECTED RESULTS 111
REMOVAL OF AER 111
CHONDROGENESIS 111
DISCUSSION 111
CHONDROGENESIS 112
APOPTOSIS 113
TIME REQUIRED FOR EXPERIMENTS 113
POTENTIAL SOURCES OF FAILURE 113
TEACHING CONCEPTS 113
ALTERNATIVE EXERCISES 114
PROPOSED EXPERIMENTS 114
QUESTIONS FOR FURTHER ANALYSIS 114
ADDITIONAL INFORMATION 114
REFERENCES 114
8 Induction of ectopic limb outgrowth in chick with FGF-8 117
INTRODUCTION 117
MATERIALS AND METHODS 119
PROCEDURES 119
OUTLINE OF THE EXPERIMENT 121
EMBRYO MANIPULATION 121
EXPECTED RESULTS 121
DISCUSSION 122
ACKNOWLEDGEMENTS 122
REFERENCES 123
SECTION IV. NUCLEIC ACID INJECTIONS 124
9 RNAi techniques applied to freshwater planarians (Platyhelminthes) during regeneration 124
INTRODUCTION 124
MATERIALS AND METHODS 127
EQUIPMENT AND MATERIALS 127
PREVIOUS TASKS FOR STAFF 127
PROCEDURES 127
OUTLINE OF THE EXPERIMENTS 130
PLANARIANS GROUP 1\u2013CONTROL GROUP 130
PLANARIANS GROUP 2\u2013Gtsix-1 AND Tcen49 MINUS GROUP 130
EXPECTED RESULTS 131
Gtsix-1 RNAi EXPERIMENT 131
Tcen-49 RNAi EXPERIMENT 131
DISCUSSION 132
Gtsix-1 RNAi EXPERIMENT 132
Tcen-49 RNAi EXPERIMENT 132
TIME REQUIRED FOR THE EXPERIMENTS 133
POTENTIAL SOURCES OF FAILURE 133
TEACHING CONCEPTS 133
ALTERNATIVE EXERCISES 133
QUESTIONS FOR FURTHER ANALYSIS 133
REFERENCES 134
10 Microinjection of Xenopus embryos 135
INTRODUCTION 135
EQUIPMENT AND MATERIALS 135
INJECTION EQUIPMENT 135
INJECTION MATERIALS 137
PROCEDURES 138
ALTERNATIVE EXERCISE 143
TEACHING CONCEPTS 144
REFERENCES 144
SECTION V. GENETIC ANALYSIS 145
11 Segmental specification in Drosophila melanogaster 145
INTRODUCTION 145
MATERIALS AND METHODS 147
EQUIPMENT AND MATERIALS 147
REAGENTS 150
PREVIOUS TASKS FOR STAFF 150
PROCEDURES 150
OUTLINE OF THE EXPERIMENTS 152
EXPERIMENT 1A. STUDY OF THE PHENOTYPE OF DIFFERENT MUTATIONS AND COMPLEMENTATION ANALYSIS 152
EXPERIMENT 1B. PROTOCOL FOR THE COMPLEMENTATION ANALYSIS 153
EXPERIMENT 2. STUDY OF THE PHENOTYPE OF LETHAL MUTATIONS IN THE EMBRYONIC CUTICLE 155
EXPECTED RESULTS 156
PHENOTYPE OF DIFFERENT MUTATIONS AND COMPLEMENTATION ANALYSIS (EXPERIMENT 1) 156
STUDY OF THE PHENOTYPE OF LETHAL MUTATIONS IN THE EMBRYONIC CUTICLE (EXPERIMENT 2) 156
DISCUSSION 156
TIME REQUIRED FOR THE EXPERIMENTS 157
POTENTIAL SOURCES OF FAILURE 157
TEACHING CONCEPTS 157
ALTERNATIVE EXERCISES 158
QUESTIONS FOR FURTHER ANALYSIS 158
ADDITIONAL INFORMATION 158
THE HOX COMPLEX IN OTHER ORGANISMS 158
ACKNOWLEDGEMENTS 159
REFERENCES 159
APPENDIX 159
ADDITIONAL PROTOCOLS 159
12 Genetic analysis of flower development in Arabidopsis thaliana The ABC model of floral organ identity determination 161
INTRODUCTION 161
MATERIALS AND METHODS 163
EQUIPMENT AND MATERIALS 163
PROCEDURES 164
PREVIOUS TASKS FOR STAFF 166
OUTLINE OF THE EXPERIMENTS 166
EXPERIMENT: STUDY OF THE PHENOTYPE OF DIFFERENT MUTATIONS 166
EXPECTED RESULTS 166
PHENOTYPE OF DIFFERENT MUTATIONS 166
DISCUSSION 168
TIME REQUIRED FOR THE EXPERIMENTS 168
POTENTIAL SOURCES OF FAILURE 168
TEACHING CONCEPTS 168
ALTERNATIVE EXERCISES 168
PROPOSED EXPERIMENTS 168
ACKNOWLEDGEMENTS 170
REFERENCES 170
13 Genetic analysis of vulva development in C. elegans 171
INTRODUCTION 171
MATERIALS AND METHODS 174
EQUIPMENT AND MATERIALS 174
REAGENTS 175
PREVIOUS TASKS FOR STAFF 175
PROCEDURES 177
OUTLINE OF THE EXPERIMENTS 178
EXPERIMENT 1A. VULVAL PHENOTYPES IN MUTANTS OF THE RAS PATHWAY 178
EXPERIMENT 1B. CHARACTERISATION OF THE GENETIC INTERACTIONS BETWEEN FACTORS OF THE RAS PATHWAY DURING VULVAL DEVELOPMENT 179
EXPERIMENT 2. MAPPING OF A MUTATION OF THE RAS PATHWAY 179
EXPECTED RESULTS 182
EXPERIMENT 1 182
EXPERIMENT 2 182
DISCUSSION 182
EXPERIMENT 1A 182
EXPERIMENT 1B 182
EXPERIMENT 2 183
TIME REQUIRED FOR THE EXPERIMENTS 183
POTENTIAL SOURCES OF FAILURE 183
TEACHING CONCEPTS 183
ALTERNATIVE EXERCISES 184
QUESTIONS FOR FURTHER ANALYSIS 184
REFERENCES 184
SECTION VI. CLONAL ANALYSIS 185
14 The role of the gene apterous in the development of the Drosophila wing 185
INTRODUCTION 185
MATERIALS AND METHODS 188
EQUIPMENT AND MATERIALS 188
REAGENTS 188
PREVIOUS TASKS FOR STAFF 189
PROCEDURES 189
OUTLINE OF THE EXPERIMENTS 190
PROPOSED EXPERIMENT 190
EXPECTED RESULTS 191
DISCUSSION 191
TIME REQUIRED FOR EXPERIMENTS 192
POTENTIAL SOURCES OF FAILURE 192
TEACHING CONCEPTS 193
ALTERNATIVE EXERCISES 193
PROPOSED EXPERIMENT 193
QUESTIONS FOR FURTHER ANALYSIS 194
REFERENCES 194
15 Extramacrochaetae, an example of a gene required for control of limb size and cell differentiation during wing\u2026 196
INTRODUCTION 196
MITOTIC RECOMBINATION CLONES 198
MATERIALS AND METHODS 198
EQUIPMENT AND MATERIALS 198
REAGENTS 201
PREVIOUS TASKS FOR STAFF 201
PROCEDURES 201
OUTLINE OF THE EXPERIMENTS 201
EXPERIMENT 1. TWIN ANALYSIS 201
EXPERIMENT 2. MINUTE ANALYSIS 202
EXPECTED RESULTS 203
TWIN ANALYSIS (EXPERIMENT 1) 203
MINUTE ANALYSIS (emc 1 M + CLONES) (EXPERIMENT 2) 204
DISCUSSION 204
TIME REQUIRED FOR THE EXPERIMENTS 205
POTENTIAL SOURCE OF FAILURE 205
TEACHING CONCEPTS 205
ALTERNATIVE EXERCISES 206
PROPOSED EXPERIMENT 206
QUESTIONS FOR FURTHER ANALYSIS 206
ADDITIONAL INFORMATION 206
REFERENCES 207
GENERAL REFERENCES 207
16 Hedgehog transduction pathway is involved in pattern formation of Drosophila melanogaster tergites 208
INTRODUCTION 208
MATERIALS AND METHODS 212
EQUIPMENT AND MATERIALS 212
REAGENTS 212
PREVIOUS TASKS FOR STAFF 212
PROCEDURES 212
OUTLINE OF THE EXPERIMENT 213
EXPERIMENT 1. ANALYSIS OF A VIABLE HETEROALLELIC COMBINATION OF Patched 213
EXPERIMENT 2. CLONAL ANALYSIS 214
EXPECTED RESULTS 216
EXPERIMENT 1 216
EXPERIMENT 2 216
DISCUSSION 216
TIME REQUIRED FOR THE EXPERIMENTS 219
POTENTIAL SOURCES OF FAILURE 219
TEACHING CONCEPTS 219
ALTERNATIVE EXERCISES 220
PROPOSED EXPERIMENTS 220
QUESTIONS FOR FURTHER ANALYSIS 221
ACKNOWLEDGEMENTS 221
REFERENCES 221
SECTION VII. IN SITU HYBRIDIZATION 223
17 Retinoic acid signalling controls anteroposterior patterning of the zebrafish hindbrain 223
INTRODUCTION 223
MATERIALS AND METHODS 224
EQUIPMENT AND MATERIALS 224
PREVIOUS TASKS FOR STAFF 226
PROCEDURES 227
OUTLINE OF THE EXPERIMENTS 228
EXPERIMENT 1A. THE EFFECTS OF ALTERED RA SIGNALLING ON ZEBRAFISH HINDBRAIN PATTERNING 228
EXPERIMENT 1B. DETERMINING THE DEVELOPMENTAL STAGES DURING WHICH RA SIGNALLING PATTERNS THE HINDBRAIN 229
EXPERIMENT 2. THE EXPRESSION PATTERN OF RETINALDEHYDE DEHYDROGENASE 2 (raldh2)INTHE ZEBRAFISH EMBRYO 229
EXPECTED RESULTS 230
THE EFFECTS OF ALTERED RA SIGNALLING ON ZEBRAFISH HINDBRAIN PATTERNING (EXPERIMENTS 1A, B) 230
THE EXPRESSION OF RETINALDEHYDE DEHYDROGENASE 2 (raldh2) IN THE ZEBRAFISH EMBRYO (EXPERIMENT 2) 230
DISCUSSION 231
TIME REQUIRED FOR THE EXPERIMENTS 232
POTENTIAL SOURCES OF FAILURE 232
TEACHING CONCEPTS 233
ALTERNATIVE EXERCISES 233
QUESTIONS FOR FURTHER ANALYSIS 233
ACKNOWLEDGEMENTS 233
REFERENCES 233
18 Left\u2013right asymmetry in the mouse 235
INTRODUCTION 235
MATERIALS AND METHODS 237
EQUIPMENT AND MATERIALS 237
REAGENTS AND KITS 238
PREVIOUS TASKS FOR STAFF 238
OUTLINE OF THE EXPERIMENTS 241
EXPERIMENT 1: ANALYSIS OF ASYMMETRIC GENE TRANSCRIPTION BY ISH 241
EXPERIMENT 2: ASYMMETRIC MORPHOGENESIS 244
EXPECTED RESULTS 245
ASYMMETRIC GENE EXPRESSION 245
ASYMMETRIC ORGAN MORPHOGENESIS 245
DISCUSSION 246
TIME REQUIRED FOR THE EXPERIMENTS 246
POTENTIAL SOURCES OF FAILURE 246
TEACHING CONCEPTS 246
ALTERNATIVE EXERCISES 247
QUESTIONS FOR FURTHER ANALYSIS 247
ADDITIONAL INFORMATION 247
SYMMETRY BREAKAGE 247
EVOLUTIONARY CONSERVATION 247
ACKNOWLEDGEMENTS 247
REFERENCES 248
SECTION VIII. TRANSGENIC ORGANISMS 249
19 Bicoid and Dorsal: Two transcription factor gradients which specify cell fates in the early Drosophila embryo 249
INTRODUCTION 250
MATERIALS AND METHODS 252
EQUIPMENT AND MATERIALS 252
PROCEDURES 253
OUTLINE OF THE EXPERIMENTS 255
EXPERIMENT 1. CHANGING THE DOSE OF bicoid CAUSES SHIFTS IN THE ANLAGEN OF THE AP AXIS 255
EXPERIMENT 2. A GRADIENT OF DORSAL DETERMINES THE CELL FATES ALONG THE DV AXIS 259
EXPECTED RESULTS AND DISCUSSION 264
EXPERIMENT 1. CHANGING THE DOSE OF bicoid CAUSES SHIFTS IN THE ANLAGEN OF THE AP AXIS 264
EXPERIMENT 2. A GRADIENT OF DORSAL DETERMINES THE CELL FATES ALONG THE DV AXIS 265
TIME REQUIRED FOR THE EXPERIMENTS 266
TEACHING CONCEPTS 266
ADDITIONAL INFORMATION 267
EXPERIMENT 1. CHANGING THE DOSE OF bicoid CAUSES SHIFTS IN THE ANLAGEN OF THE AP AXIS 267
EXPERIMENT 2. A GRADIENT OF DORSAL DETERMINES THE CELL FATES ALONG THE DV AXIS 268
ACKNOWLEDGEMENTS 269
REFERENCES 269
20 Significance of the temporal modulation of Hox gene expression on segment morphology 273
INTRODUCTION 273
MATERIALS AND METHODS 275
REAGENTS 276
PREVIOUS TASKS FOR STAFF 276
PROCEDURES 277
OUTLINE OF THE EXPERIMENTS 279
EXPERIMENT 1. ANALYSIS OF THE EXPRESSION OF THREE HOX GENES 279
EXPERIMENT 2. ANALYSIS OF THE EFFECTS OF TEMPORAL REGULATION OF UBX AND ABD-B ON CUTICLE DEVELOPMENT 280
EXPERIMENT 3. ANALYSIS OF THE EXPRESSION OF UBX AND ONE OF ITS DOWNSTREAM TARGETS 281
EXPECTED RESULTS 282
EXPERIMENT 1. ANALYSIS OF THE EXPRESSION OF THREE HOX GENES 282
EXPERIMENT 2. ANALYSIS OF THE EFFECTS OF TEMPORAL REGULATION OF UBX AND ABD-B ON CUTICLE MORPHOLOGY 283
EXPERIMENT 3. ANALYSIS OF THE EXPRESSION OF UBX AND ONE OF ITS DOWNSTREAM TARGETS 283
DISCUSSION 283
TIME REQUIRED FOR THE EXPERIMENTS 284
POTENTIAL SOURCES OF FAILURE 285
TEACHING CONCEPTS 285
REFERENCES 285
21 The UAS/GAL4 system for tissue-specific analysis of EGFR gene function in Drosophila melanogaster 287
INTRODUCTION 289
MATERIALS AND METHODS 290
EQUIPMENT AND MATERIALS 290
PROCEDURES 291
OUTLINE OF THE EXPERIMENTS 291
EXPERIMENT 1. DEFINING THE TISSUE-SPECIFIC EFFECTS OF ALTERED EGFR SIGNALING DURING EYE DEVELOPMENT 291
EXPERIMENT 2. DEFINING THE TISSUE-SPECIFIC EFFECTS OF ALTERED EGFR SIGNALING DURING OOGENESIS 292
EXPERIMENT 3. REVEALING TISSUE-SPECIFIC EFFECTS OF VITAL LOCI USING DIRECTED MOSAICS 294
EXPECTED RESULTS 296
DISRUPTION OF EGFR SIGNALING IN THE EYE (EXPERIMENT 1) 296
DISRUPTION OF EGFR SIGNALING DURING OOGENESIS (EXPERIMENT 2) 296
CLONAL ANALYSIS OF VITAL LOCI REVEALS THEIR ROLE IN THE FOLLICLE CELLS (EXPERIMENT 3) 297
DISCUSSION 297
TIME REQUIRED FOR THE EXPERIMENT 297
POTENTIAL SOURCES OF FAILURE 297
TEACHING CONCEPTS 297
ALTERNATIVE EXERCISES 298
UNDERSTANDING THE TECHNOLOGY \u2013 LINKING THE METHODS TO THE OBSERVED RESULTS 298
QUESTIONS FOR FURTHER ANALYSIS 298
REFERENCES 298
22 Neurogenesis in Drosophila: A genetic approach 300
INTRODUCTION 300
THE PROCESS 300
THE GENES 302
MATERIALS AND METHODS 303
EQUIPMENT AND MATERIALS 303
PREVIOUS TASKS FOR STAFF 305
PROCEDURES 305
OUTLINE OF THE EXPERIMENTS 306
EXPERIMENT 1. OBSERVATION OF EMBRYONIC PHENOTYPES 306
EXPERIMENT 2. EPISTATIC RELATIONSHIPS 307
EXPERIMENT 3. ADULT PNS DEVELOPMENT 308
DISCUSSION 310
TIME REQUIRED FOR THE EXPERIMENTS 311
TEACHING CONCEPTS 312
ALTERNATIVE EXERCISES 312
PROPOSED EXPERIMENTS 312
QUESTIONS FOR FURTHER ANALYSIS 312
REFERENCES FOR FURTHER READING 312
23 Role of the achaete-scute complex genes in the development of the adult peripheral nervous system of Drosophila melanogaster 314
INTRODUCTION 314
MATERIALS AND METHODS 317
EQUIPMENT AND MATERIALS 317
REAGENTS AND KITS 318
PREVIOUS TASKS FOR STAFF 319
PROCEDURES 319
OUTLINE OF THE EXPERIMENTS 320
EXPERIMENT 1. ADULT PHENOTYPE OF achaete-scute MUTANTS 320
EXPERIMENT 2. DEVELOPMENT OF SMCS IN achaete-scute MUTANTS 321
EXPERIMENT 3. PRONEURAL ROLE OF sc 322
EXPERIMENT 4. CIS-REGULATION OF ac/sc EXPRESSION: THE DC ENHANCER 323
EXPERIMENT 5. TRANS-REGULATION OF ac/sc EXPRESSION: ACTIVATION OF THE DC ENHANCER BY PANNIER 323
EXPERIMENT 5A. EFFECT OF pnr LOSS-OF-FUNCTION ON BOTH THE ADULT PATTERN OF SOS AND THE DC ENHANCER 323
EXPERIMENT 5B. EFFECT OF THE OVER-EXPRESSION OF pnr ON BOTH THE ADULT PATTERN OF SO AND THE DC ENHANCER 323
GENERAL EXPECTED RESULTS 324
DISCUSSION 324
TIME REQUIRED FOR THE EXPERIMENTS 325
TEACHING CONCEPTS 325
ALTERNATIVE EXERCISES 325
QUESTIONS FOR FURTHER ANALYSIS 325
ADDITIONAL INFORMATION 326
REFERENCES 326
SECTION IX. VERTEBRATE CLONING 328
24 The conservation of the genome and nuclear reprogramming in Xenopus 328
INTRODUCTION 328
MATERIALS AND METHODS 329
EQUIPMENT AND MATERIALS 329
PREVIOUS TASKS FOR STAFF 329
OUTLINE OF THE EXPERIMENTS 330
PREPARATION OF DONOR CELLS 330
PREPARATION OF RECIPIENT EGGS 330
NUCLEAR TRANSPLANTATION 331
EXPECTED RESULTS 331
DISCUSSION 332
TEACHING CONCEPTS 332
ALTERNATIVE EXERCISES 333
REFERENCES 333
SECTION X. CELL CULTURE 334
25 In vitro culture and differentiation of mouse embryonic stem cells 334
INTRODUCTION 334
MATERIALS AND METHODS 336
EQUIPMENT AND MATERIAL 336
PREVIOUS TASKS FOR STAFF 336
OUTLINE OF THE EXPERIMENTS 338
EXPERIMENT 1: FEEDER LAYER (FL) CULTURE 338
EXPERIMENT 2: ESTABLISHMENT OF ES CELL LINES 339
EXPERIMENT 3: CULTURE AND CHARACTERIZATION OF UNDIFFERENTIATED ES CELLS 340
EXPERIMENT 4: DIFFERENTIATION OF ES CELLS AND CHARACTERIZATION OF DIFFERENTIATED CELL TYPES 341
EXPECTED RESULTS 343
CHARACTERIZATION OF UNDIFFERENTIATED ES CELLS 343
CARDIAC DIFFERENTIATION 343
NEURONAL DIFFERENTIATION 344
DISCUSSION 345
APPLICATIONS OF ES CELL TECHNOLOGY 345
TIME REQUIRED FOR THE EXPERIMENTS 345
TEACHING CONCEPTS 345
REFERENCES 346
SECTION XI. EVO\u2013DEVO STUDIES 348
26 Microevolution between Drosophila species 348
INTRODUCTION 349
MATERIALS AND METHODS 350
EQUIPMENT AND MATERIALS 350
PROCEDURES FOR ESTABLISHING CROSSES AND ANALYSIS OF THEIR RESULTS 351
OUTLINE OF THE EXPERIMENTS 354
EXPERIMENT 1. THE EFFECT ON HYBRID BRISTLE LOSS OF INTRODUCING DELETION CONSTRUCTS OF THE Achaete-Scute LOCUS\u2026 354
EXPERIMENT 2. THE EFFECT OF SUPPLYING HYBRIDS WITH AN EXTRA COPY OF THE AS-C VERSUS SUPPLYING AN EXOGENOUS SOURCE OF SCUTE\u2026 354
EXPERIMENT 3. THE EFFECT OF HYBRID SEX ON BRISTLE LOSS 355
EXPECTED RESULTS 356
EXPERIMENT 1. THE EFFECT ON HYBRID BRISTLE LOSS OF INTRODUCING DELETION CONSTRUCTS OF THE Achaete-Scute LOCUS\u2026 356
EXPERIMENT 2. THE EFFECT OF SUPPLYING HYBRIDS WITH AN EXTRA COPY OF THE AS-C VERSUS SUPPLYING AN EXOGENOUS SOURCE OF SCUTE\u2026 356
EXPERIMENT 3. THE EFFECT OF HYBRID SEX ON BRISTLE LOSS 359
DISCUSSION 359
TIME REQUIRED FOR THE EXPERIMENTS 361
POTENTIAL SOURCES OF FAILURE 361
TEACHING CONCEPTS 361
ALTERNATIVE EXERCISES 362
QUESTIONS FOR FURTHER ANALYSIS 362
REFERENCES 362
SECTION XII. COMPUTATIONAL MODELLING 364
27 Theories as a tool for understanding the complex network of molecular interactions 364
INTRODUCTION 364
HOWTO GENERATE AN ORGANIZING REGION 364
FORMULATION OF A PATTERN-FORMING INTERACTION 366
FORMATION OF PERIODIC STRUCTURES: BRISTLE FORMATION AS AN EXAMPLE 368
CONCLUSION 370
ACKNOWLEDGEMENTS 371
REFERENCES 371
APPENDIX 1. ABBREVIATIONS 373
APPENDIX 2. SUPPLIERS 381
INDEX 389
Half-title 3
Title 5
Copyright 6
Dedication 7
CONTENTS 9
Preface 13
REFERENCES 14
CONTRIBUTORS 15
Introduction 19
KEEPING A LABORATORY NOTEBOOK 19
WRITING A LABORATORY REPORT 20
SECTION I. GRAFTINGS 22
1 Two developmental gradients control head formation in hydra 22
INTRODUCTION 22
MATERIALS AND METHODS 24
EQUIPMENT AND MATERIALS 24
REAGENTS 25
PREVIOUS TASKS FOR STAFF 25
TRANSPLANTATION PROCEDURE 25
OUTLINE OF THE EXPERIMENTS 28
A.A HEAD ACTIVATION GRADIENT IN THE BODY COLUMN 28
B.A HEAD INHIBITION GRADIENT IN THE BODY COLUMN 30
EXPECTED RESULTS AND DISCUSSION 31
DEMONSTRATION OF THE HEAD ACTIVATION PROPERTY 32
DEMONSTRATION OF THE DISTRIBUTION OF HEAD ACTIVATION 33
DEMONSTRATION OF THE HEAD INHIBITION PROPERTY 33
DEMONSTRATION OF THE DISTRIBUTION OF HEAD INHIBITION 33
TIME REQUIRED FOR THE EXPERIMENTS 33
POTENTIAL SOURCES OF FAILURE 34
TEACHING CONCEPTS 34
ALTERNATIVE EXERCISES 34
PROPOSED EXPERIMENTS 35
QUESTIONS FOR FURTHER ANALYSIS 36
REFERENCES 37
APPENDIX: MAINTENANCE OF A HYDRA CULTURE 37
MATERIALS AND EQUIPMENT (PER CLASS OF STUDENTS) 38
RAISING AND HANDLING HYDRA 38
FEEDING AND WASHING HYDRA 39
2 Embryonic regulation and induction in sea urchin development 41
INTRODUCTION 41
MATERIALS AND METHODS 43
BIOLOGICAL MATERIAL 43
LABORATORY EQUIPMENT AND SUPPLIES 43
CHEMICALS AND SOLUTIONS 45
OUTLINE OF THE EXPERIMENTS 46
EXPERIMENT 1. REGULATIVE DEVELOPMENT OF ISOLATED BLASTOMERES 46
EXPERIMENT 2. INDUCTION OF ENDODERM BY MICROMERES 47
EXPERIMENT 3. IMMUNOSTAINING OF EMBRYO WHOLE MOUNTS 48
EXPECTED RESULTS 50
EXPERIMENT 1 50
EXPERIMENT 2 50
EXPERIMENT 3 51
DISCUSSION 51
POTENTIAL SOURCES OF FAILURE 51
TIME REQUIRED FOR THE EXPERIMENTS 52
ADDITIONAL EXPERIMENTS 53
TEACHING CONCEPTS 53
REFERENCES 54
3 The isthmic organizer and brain regionalization in chick embryos 55
INTRODUCTION 55
MATERIALS AND METHODS 56
EQUIPMENT AND MATERIALS 56
PREVIOUS TASKS FOR STAFF 57
OUTLINE OF THE EXPERIMENT 58
SETTING EGGS INTO THE INCUBATOR 59
PROTOCOL FOR PREPARATION OF EGGS AND EMBRYOS FOR TRANSPLANTATION 60
PROTOCOL FOR TRANSPLANTING THE ISO REGION TISSUE INTO THE DIENCEPHALON 60
COLLECTION OF SURGICALLY MANIPULATED CHICK EMBRYOS 61
IDENTIFICATION OF THE QUAIL TISSUE BY IMMUNOHISTOCHEMISTRY 61
EXPECTED RESULTS 63
DISCUSSION 63
TIME REQUIRED FOR THE EXPERIMENTS 64
POTENTIAL SOURCES OF FAILURE 64
ALTERNATIVE EXERCISES 64
HISTOLOGICAL ANALYSIS OF THE ECTOPIC STRUCTURES FROM THE GRAFT EXPERIMENTS 64
IMPLANTATION OF FGF8-SOAKED HEPARIN ACRYLIC BEADS 65
IN SITU HYBRIDIZATION IN TOTO 65
QUESTIONS FOR FURTHER ANALYSIS 65
ACKNOWLEDGEMENTS 65
REFERENCES 66
APPENDIX 66
SECTION II. SPECIFIC CHEMICAL REAGENTS 68
4 Chemotaxis of aggregating Dictyostelium cells 68
INTRODUCTION 68
TIME REQUIRED FOR THE EXPERIMENT 72
MATERIALS AND METHODS 72
EQUIPMENT AND MATERIALS 72
REAGENTS 73
PREVIOUS TASKS FOR STAFF 73
PROCEDURES 74
OUTLINE OF THE EXPERIMENTS 74
CHEMOTACTIC STIMULATION 74
POTENTIAL SOURCES OF FAILURE 76
EXPECTED RESULTS 77
DISCUSSION 78
ALTERNATIVE EXERCISES 80
STIMULATION OF OTHER CELLS THROUGH A MICROPIPETTE 80
TEACHING CONCEPTS 81
ADDITIONAL INFORMATION AND TEACHING AIDS 82
APPENDIX 82
HISTORICAL OUTLOOK 82
REFERENCES 83
5 Inhibition of signal transduction pathways prevents head regeneration in hydra 85
INTRODUCTION 85
MATERIALS AND METHODS 86
OUTLINE OF THE EXPERIMENTS 90
EXPERIMENT: EFFECT OF THE INHIBITOR OF HEAD REGENERATION 90
EXPERIMENT: EFFECT OF THE INHIBITOR ON GENE EXPRESSION 91
EXPECTED RESULTS 92
DISCUSSION 92
TIME REQUIRED FOR THE EXPERIMENTS 93
TEACHING CONCEPTS 93
ALTERNATIVE EXERCISES 93
QUESTIONS FOR FURTHER ANALYSIS 93
REFERENCES 93
6 Retinoic acid during limb regeneration 95
INTRODUCTION 95
MATERIALS AND METHODS 98
EQUIPMENT AND MATERIALS 98
REAGENTS 98
PREVIOUS TASKS FOR STAFF 98
PROCEDURES 99
OUTLINE OF THE EXPERIMENT 99
EXPECTED RESULTS 100
DISCUSSION 100
TIME REQUIRED FOR THE EXPERIMENTS 100
POTENTIAL SOURCES OF FAILURE 101
TEACHING CONCEPTS 101
ALTERNATIVE EXERCISES 101
PROPOSED EXPERIMENT 101
ACKNOWLEDGEMENTS 101
REFERENCES 102
SECTION III. BEAD IMPLANTATION 103
7 Experimental manipulations during limb development in avian embryos 103
INTRODUCTION 103
MATERIALS AND METHODS 106
EQUIPMENT AND MATERIALS 106
PREVIOUS TASKS FOR STAFF 106
SOLUTIONS 106
BIOLOGICAL MATERIAL 107
OUTLINE OF THE EXPERIMENTS 107
INCUBATION OF EGGS 107
MANIPULATION OF EMBRYOS 107
PREPARATION OF EMBRYOS 110
NEUTRAL RED PROTOCOL FOR DETECTION OF CELL DEATH 110
CARTILAGE STAINING 110
EXPECTED RESULTS 111
REMOVAL OF AER 111
CHONDROGENESIS 111
DISCUSSION 111
CHONDROGENESIS 112
APOPTOSIS 113
TIME REQUIRED FOR EXPERIMENTS 113
POTENTIAL SOURCES OF FAILURE 113
TEACHING CONCEPTS 113
ALTERNATIVE EXERCISES 114
PROPOSED EXPERIMENTS 114
QUESTIONS FOR FURTHER ANALYSIS 114
ADDITIONAL INFORMATION 114
REFERENCES 114
8 Induction of ectopic limb outgrowth in chick with FGF-8 117
INTRODUCTION 117
MATERIALS AND METHODS 119
PROCEDURES 119
OUTLINE OF THE EXPERIMENT 121
EMBRYO MANIPULATION 121
EXPECTED RESULTS 121
DISCUSSION 122
ACKNOWLEDGEMENTS 122
REFERENCES 123
SECTION IV. NUCLEIC ACID INJECTIONS 124
9 RNAi techniques applied to freshwater planarians (Platyhelminthes) during regeneration 124
INTRODUCTION 124
MATERIALS AND METHODS 127
EQUIPMENT AND MATERIALS 127
PREVIOUS TASKS FOR STAFF 127
PROCEDURES 127
OUTLINE OF THE EXPERIMENTS 130
PLANARIANS GROUP 1\u2013CONTROL GROUP 130
PLANARIANS GROUP 2\u2013Gtsix-1 AND Tcen49 MINUS GROUP 130
EXPECTED RESULTS 131
Gtsix-1 RNAi EXPERIMENT 131
Tcen-49 RNAi EXPERIMENT 131
DISCUSSION 132
Gtsix-1 RNAi EXPERIMENT 132
Tcen-49 RNAi EXPERIMENT 132
TIME REQUIRED FOR THE EXPERIMENTS 133
POTENTIAL SOURCES OF FAILURE 133
TEACHING CONCEPTS 133
ALTERNATIVE EXERCISES 133
QUESTIONS FOR FURTHER ANALYSIS 133
REFERENCES 134
10 Microinjection of Xenopus embryos 135
INTRODUCTION 135
EQUIPMENT AND MATERIALS 135
INJECTION EQUIPMENT 135
INJECTION MATERIALS 137
PROCEDURES 138
ALTERNATIVE EXERCISE 143
TEACHING CONCEPTS 144
REFERENCES 144
SECTION V. GENETIC ANALYSIS 145
11 Segmental specification in Drosophila melanogaster 145
INTRODUCTION 145
MATERIALS AND METHODS 147
EQUIPMENT AND MATERIALS 147
REAGENTS 150
PREVIOUS TASKS FOR STAFF 150
PROCEDURES 150
OUTLINE OF THE EXPERIMENTS 152
EXPERIMENT 1A. STUDY OF THE PHENOTYPE OF DIFFERENT MUTATIONS AND COMPLEMENTATION ANALYSIS 152
EXPERIMENT 1B. PROTOCOL FOR THE COMPLEMENTATION ANALYSIS 153
EXPERIMENT 2. STUDY OF THE PHENOTYPE OF LETHAL MUTATIONS IN THE EMBRYONIC CUTICLE 155
EXPECTED RESULTS 156
PHENOTYPE OF DIFFERENT MUTATIONS AND COMPLEMENTATION ANALYSIS (EXPERIMENT 1) 156
STUDY OF THE PHENOTYPE OF LETHAL MUTATIONS IN THE EMBRYONIC CUTICLE (EXPERIMENT 2) 156
DISCUSSION 156
TIME REQUIRED FOR THE EXPERIMENTS 157
POTENTIAL SOURCES OF FAILURE 157
TEACHING CONCEPTS 157
ALTERNATIVE EXERCISES 158
QUESTIONS FOR FURTHER ANALYSIS 158
ADDITIONAL INFORMATION 158
THE HOX COMPLEX IN OTHER ORGANISMS 158
ACKNOWLEDGEMENTS 159
REFERENCES 159
APPENDIX 159
ADDITIONAL PROTOCOLS 159
12 Genetic analysis of flower development in Arabidopsis thaliana The ABC model of floral organ identity determination 161
INTRODUCTION 161
MATERIALS AND METHODS 163
EQUIPMENT AND MATERIALS 163
PROCEDURES 164
PREVIOUS TASKS FOR STAFF 166
OUTLINE OF THE EXPERIMENTS 166
EXPERIMENT: STUDY OF THE PHENOTYPE OF DIFFERENT MUTATIONS 166
EXPECTED RESULTS 166
PHENOTYPE OF DIFFERENT MUTATIONS 166
DISCUSSION 168
TIME REQUIRED FOR THE EXPERIMENTS 168
POTENTIAL SOURCES OF FAILURE 168
TEACHING CONCEPTS 168
ALTERNATIVE EXERCISES 168
PROPOSED EXPERIMENTS 168
ACKNOWLEDGEMENTS 170
REFERENCES 170
13 Genetic analysis of vulva development in C. elegans 171
INTRODUCTION 171
MATERIALS AND METHODS 174
EQUIPMENT AND MATERIALS 174
REAGENTS 175
PREVIOUS TASKS FOR STAFF 175
PROCEDURES 177
OUTLINE OF THE EXPERIMENTS 178
EXPERIMENT 1A. VULVAL PHENOTYPES IN MUTANTS OF THE RAS PATHWAY 178
EXPERIMENT 1B. CHARACTERISATION OF THE GENETIC INTERACTIONS BETWEEN FACTORS OF THE RAS PATHWAY DURING VULVAL DEVELOPMENT 179
EXPERIMENT 2. MAPPING OF A MUTATION OF THE RAS PATHWAY 179
EXPECTED RESULTS 182
EXPERIMENT 1 182
EXPERIMENT 2 182
DISCUSSION 182
EXPERIMENT 1A 182
EXPERIMENT 1B 182
EXPERIMENT 2 183
TIME REQUIRED FOR THE EXPERIMENTS 183
POTENTIAL SOURCES OF FAILURE 183
TEACHING CONCEPTS 183
ALTERNATIVE EXERCISES 184
QUESTIONS FOR FURTHER ANALYSIS 184
REFERENCES 184
SECTION VI. CLONAL ANALYSIS 185
14 The role of the gene apterous in the development of the Drosophila wing 185
INTRODUCTION 185
MATERIALS AND METHODS 188
EQUIPMENT AND MATERIALS 188
REAGENTS 188
PREVIOUS TASKS FOR STAFF 189
PROCEDURES 189
OUTLINE OF THE EXPERIMENTS 190
PROPOSED EXPERIMENT 190
EXPECTED RESULTS 191
DISCUSSION 191
TIME REQUIRED FOR EXPERIMENTS 192
POTENTIAL SOURCES OF FAILURE 192
TEACHING CONCEPTS 193
ALTERNATIVE EXERCISES 193
PROPOSED EXPERIMENT 193
QUESTIONS FOR FURTHER ANALYSIS 194
REFERENCES 194
15 Extramacrochaetae, an example of a gene required for control of limb size and cell differentiation during wing\u2026 196
INTRODUCTION 196
MITOTIC RECOMBINATION CLONES 198
MATERIALS AND METHODS 198
EQUIPMENT AND MATERIALS 198
REAGENTS 201
PREVIOUS TASKS FOR STAFF 201
PROCEDURES 201
OUTLINE OF THE EXPERIMENTS 201
EXPERIMENT 1. TWIN ANALYSIS 201
EXPERIMENT 2. MINUTE ANALYSIS 202
EXPECTED RESULTS 203
TWIN ANALYSIS (EXPERIMENT 1) 203
MINUTE ANALYSIS (emc 1 M + CLONES) (EXPERIMENT 2) 204
DISCUSSION 204
TIME REQUIRED FOR THE EXPERIMENTS 205
POTENTIAL SOURCE OF FAILURE 205
TEACHING CONCEPTS 205
ALTERNATIVE EXERCISES 206
PROPOSED EXPERIMENT 206
QUESTIONS FOR FURTHER ANALYSIS 206
ADDITIONAL INFORMATION 206
REFERENCES 207
GENERAL REFERENCES 207
16 Hedgehog transduction pathway is involved in pattern formation of Drosophila melanogaster tergites 208
INTRODUCTION 208
MATERIALS AND METHODS 212
EQUIPMENT AND MATERIALS 212
REAGENTS 212
PREVIOUS TASKS FOR STAFF 212
PROCEDURES 212
OUTLINE OF THE EXPERIMENT 213
EXPERIMENT 1. ANALYSIS OF A VIABLE HETEROALLELIC COMBINATION OF Patched 213
EXPERIMENT 2. CLONAL ANALYSIS 214
EXPECTED RESULTS 216
EXPERIMENT 1 216
EXPERIMENT 2 216
DISCUSSION 216
TIME REQUIRED FOR THE EXPERIMENTS 219
POTENTIAL SOURCES OF FAILURE 219
TEACHING CONCEPTS 219
ALTERNATIVE EXERCISES 220
PROPOSED EXPERIMENTS 220
QUESTIONS FOR FURTHER ANALYSIS 221
ACKNOWLEDGEMENTS 221
REFERENCES 221
SECTION VII. IN SITU HYBRIDIZATION 223
17 Retinoic acid signalling controls anteroposterior patterning of the zebrafish hindbrain 223
INTRODUCTION 223
MATERIALS AND METHODS 224
EQUIPMENT AND MATERIALS 224
PREVIOUS TASKS FOR STAFF 226
PROCEDURES 227
OUTLINE OF THE EXPERIMENTS 228
EXPERIMENT 1A. THE EFFECTS OF ALTERED RA SIGNALLING ON ZEBRAFISH HINDBRAIN PATTERNING 228
EXPERIMENT 1B. DETERMINING THE DEVELOPMENTAL STAGES DURING WHICH RA SIGNALLING PATTERNS THE HINDBRAIN 229
EXPERIMENT 2. THE EXPRESSION PATTERN OF RETINALDEHYDE DEHYDROGENASE 2 (raldh2)INTHE ZEBRAFISH EMBRYO 229
EXPECTED RESULTS 230
THE EFFECTS OF ALTERED RA SIGNALLING ON ZEBRAFISH HINDBRAIN PATTERNING (EXPERIMENTS 1A, B) 230
THE EXPRESSION OF RETINALDEHYDE DEHYDROGENASE 2 (raldh2) IN THE ZEBRAFISH EMBRYO (EXPERIMENT 2) 230
DISCUSSION 231
TIME REQUIRED FOR THE EXPERIMENTS 232
POTENTIAL SOURCES OF FAILURE 232
TEACHING CONCEPTS 233
ALTERNATIVE EXERCISES 233
QUESTIONS FOR FURTHER ANALYSIS 233
ACKNOWLEDGEMENTS 233
REFERENCES 233
18 Left\u2013right asymmetry in the mouse 235
INTRODUCTION 235
MATERIALS AND METHODS 237
EQUIPMENT AND MATERIALS 237
REAGENTS AND KITS 238
PREVIOUS TASKS FOR STAFF 238
OUTLINE OF THE EXPERIMENTS 241
EXPERIMENT 1: ANALYSIS OF ASYMMETRIC GENE TRANSCRIPTION BY ISH 241
EXPERIMENT 2: ASYMMETRIC MORPHOGENESIS 244
EXPECTED RESULTS 245
ASYMMETRIC GENE EXPRESSION 245
ASYMMETRIC ORGAN MORPHOGENESIS 245
DISCUSSION 246
TIME REQUIRED FOR THE EXPERIMENTS 246
POTENTIAL SOURCES OF FAILURE 246
TEACHING CONCEPTS 246
ALTERNATIVE EXERCISES 247
QUESTIONS FOR FURTHER ANALYSIS 247
ADDITIONAL INFORMATION 247
SYMMETRY BREAKAGE 247
EVOLUTIONARY CONSERVATION 247
ACKNOWLEDGEMENTS 247
REFERENCES 248
SECTION VIII. TRANSGENIC ORGANISMS 249
19 Bicoid and Dorsal: Two transcription factor gradients which specify cell fates in the early Drosophila embryo 249
INTRODUCTION 250
MATERIALS AND METHODS 252
EQUIPMENT AND MATERIALS 252
PROCEDURES 253
OUTLINE OF THE EXPERIMENTS 255
EXPERIMENT 1. CHANGING THE DOSE OF bicoid CAUSES SHIFTS IN THE ANLAGEN OF THE AP AXIS 255
EXPERIMENT 2. A GRADIENT OF DORSAL DETERMINES THE CELL FATES ALONG THE DV AXIS 259
EXPECTED RESULTS AND DISCUSSION 264
EXPERIMENT 1. CHANGING THE DOSE OF bicoid CAUSES SHIFTS IN THE ANLAGEN OF THE AP AXIS 264
EXPERIMENT 2. A GRADIENT OF DORSAL DETERMINES THE CELL FATES ALONG THE DV AXIS 265
TIME REQUIRED FOR THE EXPERIMENTS 266
TEACHING CONCEPTS 266
ADDITIONAL INFORMATION 267
EXPERIMENT 1. CHANGING THE DOSE OF bicoid CAUSES SHIFTS IN THE ANLAGEN OF THE AP AXIS 267
EXPERIMENT 2. A GRADIENT OF DORSAL DETERMINES THE CELL FATES ALONG THE DV AXIS 268
ACKNOWLEDGEMENTS 269
REFERENCES 269
20 Significance of the temporal modulation of Hox gene expression on segment morphology 273
INTRODUCTION 273
MATERIALS AND METHODS 275
REAGENTS 276
PREVIOUS TASKS FOR STAFF 276
PROCEDURES 277
OUTLINE OF THE EXPERIMENTS 279
EXPERIMENT 1. ANALYSIS OF THE EXPRESSION OF THREE HOX GENES 279
EXPERIMENT 2. ANALYSIS OF THE EFFECTS OF TEMPORAL REGULATION OF UBX AND ABD-B ON CUTICLE DEVELOPMENT 280
EXPERIMENT 3. ANALYSIS OF THE EXPRESSION OF UBX AND ONE OF ITS DOWNSTREAM TARGETS 281
EXPECTED RESULTS 282
EXPERIMENT 1. ANALYSIS OF THE EXPRESSION OF THREE HOX GENES 282
EXPERIMENT 2. ANALYSIS OF THE EFFECTS OF TEMPORAL REGULATION OF UBX AND ABD-B ON CUTICLE MORPHOLOGY 283
EXPERIMENT 3. ANALYSIS OF THE EXPRESSION OF UBX AND ONE OF ITS DOWNSTREAM TARGETS 283
DISCUSSION 283
TIME REQUIRED FOR THE EXPERIMENTS 284
POTENTIAL SOURCES OF FAILURE 285
TEACHING CONCEPTS 285
REFERENCES 285
21 The UAS/GAL4 system for tissue-specific analysis of EGFR gene function in Drosophila melanogaster 287
INTRODUCTION 289
MATERIALS AND METHODS 290
EQUIPMENT AND MATERIALS 290
PROCEDURES 291
OUTLINE OF THE EXPERIMENTS 291
EXPERIMENT 1. DEFINING THE TISSUE-SPECIFIC EFFECTS OF ALTERED EGFR SIGNALING DURING EYE DEVELOPMENT 291
EXPERIMENT 2. DEFINING THE TISSUE-SPECIFIC EFFECTS OF ALTERED EGFR SIGNALING DURING OOGENESIS 292
EXPERIMENT 3. REVEALING TISSUE-SPECIFIC EFFECTS OF VITAL LOCI USING DIRECTED MOSAICS 294
EXPECTED RESULTS 296
DISRUPTION OF EGFR SIGNALING IN THE EYE (EXPERIMENT 1) 296
DISRUPTION OF EGFR SIGNALING DURING OOGENESIS (EXPERIMENT 2) 296
CLONAL ANALYSIS OF VITAL LOCI REVEALS THEIR ROLE IN THE FOLLICLE CELLS (EXPERIMENT 3) 297
DISCUSSION 297
TIME REQUIRED FOR THE EXPERIMENT 297
POTENTIAL SOURCES OF FAILURE 297
TEACHING CONCEPTS 297
ALTERNATIVE EXERCISES 298
UNDERSTANDING THE TECHNOLOGY \u2013 LINKING THE METHODS TO THE OBSERVED RESULTS 298
QUESTIONS FOR FURTHER ANALYSIS 298
REFERENCES 298
22 Neurogenesis in Drosophila: A genetic approach 300
INTRODUCTION 300
THE PROCESS 300
THE GENES 302
MATERIALS AND METHODS 303
EQUIPMENT AND MATERIALS 303
PREVIOUS TASKS FOR STAFF 305
PROCEDURES 305
OUTLINE OF THE EXPERIMENTS 306
EXPERIMENT 1. OBSERVATION OF EMBRYONIC PHENOTYPES 306
EXPERIMENT 2. EPISTATIC RELATIONSHIPS 307
EXPERIMENT 3. ADULT PNS DEVELOPMENT 308
DISCUSSION 310
TIME REQUIRED FOR THE EXPERIMENTS 311
TEACHING CONCEPTS 312
ALTERNATIVE EXERCISES 312
PROPOSED EXPERIMENTS 312
QUESTIONS FOR FURTHER ANALYSIS 312
REFERENCES FOR FURTHER READING 312
23 Role of the achaete-scute complex genes in the development of the adult peripheral nervous system of Drosophila melanogaster 314
INTRODUCTION 314
MATERIALS AND METHODS 317
EQUIPMENT AND MATERIALS 317
REAGENTS AND KITS 318
PREVIOUS TASKS FOR STAFF 319
PROCEDURES 319
OUTLINE OF THE EXPERIMENTS 320
EXPERIMENT 1. ADULT PHENOTYPE OF achaete-scute MUTANTS 320
EXPERIMENT 2. DEVELOPMENT OF SMCS IN achaete-scute MUTANTS 321
EXPERIMENT 3. PRONEURAL ROLE OF sc 322
EXPERIMENT 4. CIS-REGULATION OF ac/sc EXPRESSION: THE DC ENHANCER 323
EXPERIMENT 5. TRANS-REGULATION OF ac/sc EXPRESSION: ACTIVATION OF THE DC ENHANCER BY PANNIER 323
EXPERIMENT 5A. EFFECT OF pnr LOSS-OF-FUNCTION ON BOTH THE ADULT PATTERN OF SOS AND THE DC ENHANCER 323
EXPERIMENT 5B. EFFECT OF THE OVER-EXPRESSION OF pnr ON BOTH THE ADULT PATTERN OF SO AND THE DC ENHANCER 323
GENERAL EXPECTED RESULTS 324
DISCUSSION 324
TIME REQUIRED FOR THE EXPERIMENTS 325
TEACHING CONCEPTS 325
ALTERNATIVE EXERCISES 325
QUESTIONS FOR FURTHER ANALYSIS 325
ADDITIONAL INFORMATION 326
REFERENCES 326
SECTION IX. VERTEBRATE CLONING 328
24 The conservation of the genome and nuclear reprogramming in Xenopus 328
INTRODUCTION 328
MATERIALS AND METHODS 329
EQUIPMENT AND MATERIALS 329
PREVIOUS TASKS FOR STAFF 329
OUTLINE OF THE EXPERIMENTS 330
PREPARATION OF DONOR CELLS 330
PREPARATION OF RECIPIENT EGGS 330
NUCLEAR TRANSPLANTATION 331
EXPECTED RESULTS 331
DISCUSSION 332
TEACHING CONCEPTS 332
ALTERNATIVE EXERCISES 333
REFERENCES 333
SECTION X. CELL CULTURE 334
25 In vitro culture and differentiation of mouse embryonic stem cells 334
INTRODUCTION 334
MATERIALS AND METHODS 336
EQUIPMENT AND MATERIAL 336
PREVIOUS TASKS FOR STAFF 336
OUTLINE OF THE EXPERIMENTS 338
EXPERIMENT 1: FEEDER LAYER (FL) CULTURE 338
EXPERIMENT 2: ESTABLISHMENT OF ES CELL LINES 339
EXPERIMENT 3: CULTURE AND CHARACTERIZATION OF UNDIFFERENTIATED ES CELLS 340
EXPERIMENT 4: DIFFERENTIATION OF ES CELLS AND CHARACTERIZATION OF DIFFERENTIATED CELL TYPES 341
EXPECTED RESULTS 343
CHARACTERIZATION OF UNDIFFERENTIATED ES CELLS 343
CARDIAC DIFFERENTIATION 343
NEURONAL DIFFERENTIATION 344
DISCUSSION 345
APPLICATIONS OF ES CELL TECHNOLOGY 345
TIME REQUIRED FOR THE EXPERIMENTS 345
TEACHING CONCEPTS 345
REFERENCES 346
SECTION XI. EVO\u2013DEVO STUDIES 348
26 Microevolution between Drosophila species 348
INTRODUCTION 349
MATERIALS AND METHODS 350
EQUIPMENT AND MATERIALS 350
PROCEDURES FOR ESTABLISHING CROSSES AND ANALYSIS OF THEIR RESULTS 351
OUTLINE OF THE EXPERIMENTS 354
EXPERIMENT 1. THE EFFECT ON HYBRID BRISTLE LOSS OF INTRODUCING DELETION CONSTRUCTS OF THE Achaete-Scute LOCUS\u2026 354
EXPERIMENT 2. THE EFFECT OF SUPPLYING HYBRIDS WITH AN EXTRA COPY OF THE AS-C VERSUS SUPPLYING AN EXOGENOUS SOURCE OF SCUTE\u2026 354
EXPERIMENT 3. THE EFFECT OF HYBRID SEX ON BRISTLE LOSS 355
EXPECTED RESULTS 356
EXPERIMENT 1. THE EFFECT ON HYBRID BRISTLE LOSS OF INTRODUCING DELETION CONSTRUCTS OF THE Achaete-Scute LOCUS\u2026 356
EXPERIMENT 2. THE EFFECT OF SUPPLYING HYBRIDS WITH AN EXTRA COPY OF THE AS-C VERSUS SUPPLYING AN EXOGENOUS SOURCE OF SCUTE\u2026 356
EXPERIMENT 3. THE EFFECT OF HYBRID SEX ON BRISTLE LOSS 359
DISCUSSION 359
TIME REQUIRED FOR THE EXPERIMENTS 361
POTENTIAL SOURCES OF FAILURE 361
TEACHING CONCEPTS 361
ALTERNATIVE EXERCISES 362
QUESTIONS FOR FURTHER ANALYSIS 362
REFERENCES 362
SECTION XII. COMPUTATIONAL MODELLING 364
27 Theories as a tool for understanding the complex network of molecular interactions 364
INTRODUCTION 364
HOWTO GENERATE AN ORGANIZING REGION 364
FORMULATION OF A PATTERN-FORMING INTERACTION 366
FORMATION OF PERIODIC STRUCTURES: BRISTLE FORMATION AS AN EXAMPLE 368
CONCLUSION 370
ACKNOWLEDGEMENTS 371
REFERENCES 371
APPENDIX 1. ABBREVIATIONS 373
APPENDIX 2. SUPPLIERS 381
INDEX 389
Key experiments in practical developmental biology /
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