Description
The need to better understand the molecular, b- chemical, and cellular processes by which a developing neuronal system unfolds has led to the development of a unique set of experimental tools and organisms. Special emphasis was devoted to allowing us access, at the ear- est stages, to the genomic basis underlying the system’s ultimate complexity, as exhibited once its structures are fully formed. Yet, nerve cells are anatomically, physiolo- cally, and biochemically diverse. The multitude of d- tinctly different routes for their development thus makes the developing nervous system especially intriguing for molecular neurobiologists. In particular, the demands of modern molecular neuroscience call for the establishment of efficient yet versatile systems for studying these c- plex processes. Transgenic embryos of the frog Xenopus laevis offer an excellent system for approaching neuroscientific issues. Insertion of foreign genes is performed simply, by mic- injection under binocular observation; hundreds of in vitro-fertilized embryos can be microinjected in one experiment. Embryos develop in tap water, at room t- perature, and within a few days become independent swimming tadpoles with fully functioning neuromus- lar systems. Being relatively small, these organisms are amenable to detailed analyses at the levels of mRNA, protein, and cell. Their rapid development permits the study of morphogenetic processes involved in early development, such as myogenesis and neural induction, as well as those involved in organogenesis and formation of the brain, the musculature, and the interconnections between them. Foreign DNA remains predominantly extrachromosomal. Chapter 1: Scientific Background. Xenopus laevis as an Experimental Model System. Xenopus Development. Prefertilization. Fertilization. Postfertilization. Cortical Rotation. Cleavage (Stages 1-8 Leading to Blastula). Gastrulation (Stages 8-13). Neurulation (Stages 12-20). Myogenesis (Stage 10+ Onwards). Somitogenesis (Stage 17 to Tadpole). Hatching (Stage 25). Neuromuscular Junction Formation in Developing Xenopus Embryos. Xenopus Oocyte Microinjection. Xenopus Embryo Microinjection. Overview. Microinjection Strategies. General Considerations. Studying Gene Regulation. Studying Gene Function by Overexpression. Induction Assays in Animal Caps. Induction Assays in Whole Embryos. Other Gene Function Assays. Studying Gene Function by Downregulation. Targeted mRNA Destruction. Injection of Antibodies. Dominant-Negative Molecules. Host Transfer. Detection Strategies. Detection of RNA. Detection of Proteins. Histology. Artifacts. The Vertebrate Neuromuscular Junction. Neuromuscular Junction Structure. Aggregation of Acetylcholine Receptor/ Acetylcholinesterase. Synapse-Specific Transcription of Synaptic Proteins. Cholinergic Signaling and Neuromuscular Pathologies. Acetylcholinesterase. Biological Roles. Neuromuscular Junction Acetylcholinesterase. Acetylcholinesterase in the Central Nervous System. Embryonic Acetylcholinesterase. Hematopoietic Acetylcholinesterase. Acetylcholinesterase Gene. Acetylcholinesterase Gene mRNAs. Acetylcholinesterase-The Enzyme. Acetylcholinesterase Molecular Polymorphism. Heterologous Expression of Acetylcholinesterase. Chapter 2: Experimental Methodologies. Reagents, Buffers, and Solutions. Microinjections. Vectors. In Vitro-Transcribed RNA. DNA Expression Plasmids-Acetylcholinesterase. DNA Expression Plasmids-Acetylcholine Receptor. Xenopus Oocyte Microinjections. Xenopus Embryo Microinjections (see Appendix V for Detailed Protocol). Biochemical Analyses. Homogenizations. Total Homogenates. Subcellular Fractionation. Acetylcholinesterase Activity Assays. Sucrose Gradient Ultracentrifugation. Enzyme Antigen Immunoassay. Polyacrylamide Gel Electrophoresis. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis/Immunoblot. Nondenaturing Gel Electrophoresis. Histochemical Analyses. Whole-Mount Cytochemical Staining. Whole-Mount Immunocytochemical Staining. Electron Microscopy. RT-PCR Procedure and Primers. Chapter 3: Experimental Applications: Human Acetylcholinesterase as a Model Nervous System Protein. Xenopus Oocyte Microinjections. Human Acetylcholinesterase Expressed in mRNA-Injected Xenopus Oocytes. Heterologous Acetylcholinesterase Is Biochemically Indistinguishable from Native Human Acetylcholinesterase. Cytomegalovirus Promoter Directs Acetylcholinesterase Expression in DNA-Injected Xenopus Oocytes. Xenopus Embryo Microinjections. Transient Expression of Human Acetylcholinesterase in Microinjected Xenopus Embryos. Apparently Normal Development of Acetylcholinesterase-Overexpressing Xenopus Embryos. Recombinant Human Acetylcholinesterase Is Immunochemically Distinct from Xenopus Acetylcholinesterase. Oligomeric Assembly of Recombinant Human Acetylcholinesterase in Xenopus Embryos. Characterization of a Human Acetylcholinesterase Gene Promoter in Xenopus Embryos. Human Acetylcholinesterase Gene Promoter Composition. Transcription from the Human Acetylcholinesterase Gene Promoter in Xenopus Detected by RT-PCR . Microinjected Embryos Utilize Correct 5′ Splice Site. Unique Properties of an Alternative Acetylcholinesterase Expressed in Xenopus Embryos. A Novel AChE mRNA Species Characterized in Xenopus. Tissue-Specific Management of Human Acetylcholinesterases Derived from Alternative AChE mRNAs. Whole-Mount Cytochemical Staining Reveals Tissue-Specific Accumulations of Acetylcholinesterase. Electron Microscope Analysis Reveals Subcellular Compartmentalization of Human Acetylcholinesterase in Xenopus Muscle. Accumulation of Acetylcholinesterase in Neuromuscular Junctions of DNA-Injected Xenopus Embryos. C-Terminal Peptide Dictates Synaptic Localization of Heterologous Acetylcholinesterase. Polarized Accumulation of an Alternative Human Acetylcholinesterase in Epidermal Cells. A Model to Study the Role of Cholinergic Signaling in Neuromuscular Junction Development. Overexpression of Mouse Acetylcholine Receptor in Microinjected Xenopus Embryos. Developmental Implications of Acetylcholine Receptor-Overexpression in Xenopus. Nerve-Terminal Acetylcholinesterase Expression. Chapter 4: Conclusions. Characterization of Human Acetylcholinesterase Expressed in Xenopus. Native Properties of Heterologous Recombinant Human Acetylcholinesterase. Developmental Implications of Acetylcholinesterase Overexpression. A Role for Acetylcholinesterase in Cell Adhesion? Nonassembly of Recombinant Human Acetylcholinesterase in Xenopus. Regulation of Acetylcholinesterase Localization. Muscle Accumulation of Acetylcholinesterase. Neuromuscular Junction Localization of Acetylcholinesterase. Stabilization of Acetylcholinesterase in Neuromuscular Junctions. A Novel Secretory Human Acetylcholinesterase? A Morphogenic Role for Acetylcholinesterase. Implications of Acetylcholinesterase Overexpression for Synaptogenesis. Acetylcholine Metabolism and Acetylcholinesterase Overexpression. A Trophic Role for Acetylcholine? Clinical Implications of Perturbations in Cholinergic Signaling. Concluding Remarks. Future Directions. Appendices to Experimental Methodologies. Appendix I: Worldwide Xenopus Suppliers. Appendix II: Laboratory Maintenance of Xenopus laevis Frogs. Appendix III: Preparation of Buffers/Reagents for Microinjections. Appendix IV: Microinjection Equipment. Appendix V: In Vitro Fertilization and Microinjection of Xenopus Embryos. Appendix VI: Whole-Mount Staining for Catalytically Active Acetylcholinesterase (Based on the Original Method Described by Karnovsky and Roots [1964]). Appendix VII: Isolation of Xenopus Oocytes Using Collagenase. References. Index.
