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Download Lamprey cranial neural crest migration (fore/midbrain)
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Formation, Migration and Evolution of the Neural Crest Marianne Bronner-Fraser California Institute of Technology [email protected] Amphioxus Lamprey Chicken stages Stage 5: 20 hours incubation Stage 10: 36 hours incubation Stage 16: 52 hours incubation Neural crest arises at neural plate border during neurulation Cell marking techniques: 1) Molecular markers 2) DiI labeling 3) Quail/chick (or duck!) chimerae 4) Image analysis Neural crest marker gene = Slug cranial trunk QuickTime™ and a Video decompressor are needed to see this picture. Neural crest derived quail cells (red) in trigeminal ganglion express trkA (green) Neural crest cell derivatives CRANIAL TRUNK Neurons and glia of cranial ganglia Pigment cells Neural Tube Sensory neurons/glia Cartilage and bone Connective tissue Sympatho adrenal cells Electroporation into chick neural tube Cell biological approaches: 1) Cell culture 2) Analysis of migratory behavior in vitro Soluble Slit enhances neural crest migration How do we think about neural crest formation? Neural Crest Induced by Neural Tube/Epidermis Interactions Wnt6 is expressed in the ectoderm Wg or Wnt induces neural crest cells Gain and loss of function approaches: 1) Antisense morpholino-mediated knock down 2) Over-expession by electroporation 3) Over-expression of dominant negative constructs 4) RNAi Morpholino antisense oligos (MO) knock-down Protein Protein Test the role of Pax7 in neural crest formation Ectopic expression on MSE55 Rho effector in neural tube causes of epithelial cells to be mesenchymal Control MSE55 Wnt6 is expressed in the ectoderm Dn Wnt inhibits Slug expression control The Good: 1) Cut/Paste Biology 2) Genome Available 3) Transgenesis Possible (though not simple) The Bad: 1) Genetics Difficult (some mutants but…) 2) No homologous recombination 3) Intermediate Visibility Amphioxus Lamprey Neural crest and placodes are present only in vertebrates Sea Lamprey: Petromyzon marinus Petromyzon marinus collection Lamprey as a useful experimental model • • • • • Important taxonomic position Manipulable embryos; egg size (1mm) Many embryos available (≈50,000 eggs/female) All developmental stages available Molecular/genomic tools becoming available – – – – – Lamprey genome project (NHGRI) BAC library (>150kb gDNA) Embryonic cDNA libraries (EST project) Perturb gene function (protein knockdown) Misexpression of foreign genes? Lamprey developmental stages 6d 10d 12d 4d 8d 10d 12d 6d 8d DiI labeling of neural crest in Petromyzon Neural crest migration comparison Knocking down PmSoxE1 protein using morpholino antisense oligos (MO) Protein Protein The Good: 1) DiI Labeling Possible 2) Genome Available Soon 3) Morpholinos Work The Bad: 1) Genetics Out of the Question 2) Opaque Embryo--Imaging is Out The Ugly: 1) Breed One Month/Year 2) Embryos Hard to Get Neural Crest Induction Via Cell Interactions at Neural Plate Border Steps: 1. Induction Signals 2. Neural Plate Border Specification 3. Neural Crest Specification Goal: Dissect Gene Regulatory Network Putative Vertebrate Gene Regulatory Network Putative Vertebrate Gene Regulatory Network Putative Vertebrate Gene Regulatory Network Summary Steps in Network: • INDUCTION: Cell interactions mediated by Wnts and other signals (BMP, FGF) induce neural crest • SPECIFICATION: Pax7 required for neural crest-- regulates Slug, Sox9, and Sox 10 FoxD3 • DOWNSTREAM TARGET: Slug represses Precrest Amphioxus Lamprey Question: What elements of gene network are conserved? What genetic changes drove neural crest evolution? Experimental Approach: Compare “neural crest gene” expression, function & regulation in amphioxus, agnathan & gnathostomes Amphioxus has a single AP-2 gene expressed in ectoderm Lamprey AP-2 Early lamprey AP-2 is expressed in ectoderm Later lamprey AP-2 is expressed in dorsal neural tube and neural crest Vertebrate neural crest markers are not co-expressed in Amphioxus Vertebrate Neural Plate Border Amphioxus Neural Plate Border Evolution of Neural Crest Likely Involved Co-option of Existing Genes to New Neural Crest Specifier Function AmphiSnail ? What could this mean? Gene expression implies cooption by vertebrate neural crest New gene deployment at neural plate border New function of “old genes” Martin Garcia-Castro David McCauley Martin Basch Lisa Taneyhill Dan Meulemans Ed Coles Celia Shiau Scott Fraser
