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Lecture IV. Mechanisms of Neural Development

Lecture IV. Mechanisms of Neural Development. Bio 3411 Wednesday September 2, 2009. T. Woolsey 3802 North Building 362-3601 woolseyt@medicine.wustl.edu. Readings. NEUROSCIENCE: 4 th ed, pp 545-575 (sorta) References † :

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Lecture IV. Mechanisms of Neural Development

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  1. Lecture IV. Mechanisms of Neural Development Bio 3411 Wednesday September 2, 2009

  2. T. Woolsey • 3802 North Building • 362-3601 • woolseyt@medicine.wustl.edu Lecture IV. Mechanisms of Neural Development

  3. Readings NEUROSCIENCE: 4th ed, pp 545-575 (sorta) References†: • Fainsod, A., Steinbeisser, H., & De Robertis, E. M. (1994). EMBO J, 13(21), 5015-5025. • Hemmati-Brivanlou, A., & Melton, D. (1997). Annu Rev Neurosci, 20, 43-60. • Melton, D. A. (1987). Nature, 328(6125), 80-82. • Sasai, Y., & De Robertis, E. M. (1997). Dev Biol, 182(1), 5-20. • Smith, W. C., & Harland, R. M. (1992). Cell, 70(5), 829-840. • Weeks, D. L., & Melton, D. A. (1987). Proc Natl Acad Sci U S A, 84(9), 2798-2802. • Wilson, P. A., & Hemmati-Brivanlou, A. (1995). Nature, 376(6538), 331-333. • Xanthos, J. B., Kofron, M., Wylie, C., & Heasman, J. (2001). Development, 128(2), 167-180. • Zimmerman, L. B., De Jesus-Escobar, J. M., & Harland, R. M. (1996). Cell, 86(4), 599-606. ______________________ †(pdfs on course website: [http://artsci.wustl.edu/~sdanker/index.html]) Lecture IV. Mechanisms of Neural Development

  4. Embryogenesis 1. Maternal cytoplasmic determinants. 2. Fertilization creates dorsal-ventral axis. 3. Cell division. 4. Blastula created. 6. Ectoderm, mesoderm, endoderm created. by molecular signals along the Animal/Vegetal axis. 5. Gastulation. 6. Spemann organizer creates anterior-posterior axis. 7. Notocordinduces the Neural Plate. 8. Neurulation forms the Neural Tube. 9. Neural crest cells form the PNS. 10. Segmentation & Cephalization (anterior enlargement) Lecture III. Nervous System Embryology

  5. Cell Signaling • Discovery of the Organizer • How Could this Work? 4) The Answer 5) Blockers 6) Current View 7) Summary Lecture IV. Mechanisms of Neural Development

  6. Cell Signaling Lecture IV. Mechanisms of Neural Development

  7. Neuroinduction Diffusible morphogen Lecture IV. Mechanisms of Neural Development

  8. Ligand Receptor A C B Gene Activation/Repression Intracellular Signaling through a Kinase Cascade; Signal Amplification (Suppression) and Multiple Control Points Ligand Receptor Kinase Cascade Effector Proteins (transcription factors, ion channels, cytoskeletal proteins, enzymes, etc…) Scaffolding Proteins bind multiple signaling molecules to organize specific signaling pathways Lecture IV. Mechanisms of Neural Development

  9. Ant Post Endoderm and Mesoderm involute with gastrulation: Induction of the Neural Plate from Neuroectoderm, by the underlying, closely apposed Mesoderm. Lecture IV. Mechanisms of Neural Development

  10. Discovery of the Organizer Lecture IV. Mechanisms of Neural Development

  11. Hilde Mangold and Hans Spemann • Key experiments performed in 1921-1923 at the University of Freiburg, Germany. • Hilde Mangold was a 24 year old graduate student when she performed these experiments. She died tragically in an accidental alcohol heater explosion. • Hans Spemann was awarded the Nobel Prize in 1935. Lecture IV. Mechanisms of Neural Development

  12. Mangold –Spemann Experiments (1924) Lecture IV. Mechanisms of Neural Development

  13. How Could this Work? Lecture IV. Mechanisms of Neural Development

  14. ! Explant Experiments with Animal Caps from AmphibianBlastula: Puzzling Results…

  15. + Candidate Neuroinducing Factors ? (Intact) Isolating Inducing Factors that Promote Neuronal Differentiation; “Sigma Catalog” Experiments Result in Further Confusion… (Many positives, including apparently non-biological factors!) Lecture IV. Mechanisms of Neural Development

  16. Models for Neural Induction +”Epidermal factor” Epidermis Model 1: Presumptive Neuroectoderm Neurons +”Neuronal factor” (“default”) Model 2: Epidermis Presumptive Neuroectoderm +”Neuronal factor” Neurons +”Epidermal factor” Model 3: Epidermis Presumptive Neuroectoderm (“default”) Neurons Lecture IV. Mechanisms of Neural Development

  17. Vg1 (Melton, 1987; Weeks and Melton, 1987) (K. Mowry Lab, Brown Univ.) Multi-step pathway (kinases, scaffolding proteins) TGF-b Proteins Signal Through Heterodimeric Receptors and Smad Transcription Factors Lecture IV. Mechanisms of Neural Development

  18. The Answer Lecture IV. Mechanisms of Neural Development

  19. A Dominant-Negative Receptor Subunit Blocks Activation of the Signaling Pathway (Hemmati-Brivanlou and Melton, 1992) Lecture IV. Mechanisms of Neural Development

  20. (+Dominant-Negative Type II Receptor cRNA) TFG-b Signaling Blocked by expression of Dom-Neg Type II Receptor Subunit Animal Cap (Intact) Animal Cap (Intact) + TGF-b Signaling (Intact) Blocking TGF-b Signaling by a Dominant-Negative Receptor Causes Isolated Neuroectoderm to Become Neuronal + TGF-b Signaling Lecture IV. Mechanisms of Neural Development

  21. BMP-4 (TGF-b) Signaling Results in “NeuralEpidermal Induction” TGF-b: Transforming GrowthFactor - b BMP-4: BoneMorphogenic Protein - 4 Lecture IV. Mechanisms of Neural Development

  22. Models for Neural Induction +”Epidermal factor” Epidermis Model 1: Presumptive Neuroectoderm Neurons +”Neuronal factor” (“default”) Model 2: Epidermis Presumptive Neuroectoderm +”Neuronal factor” Neurons +BMP-4 Model 3: Epidermis Presumptive Neuroectoderm (“default”) Neurons Lecture IV. Mechanisms of Neural Development

  23. [BMP-4] BMP-4 (Secreted by Neuroectodermal Cells) Inhibits Neuronal Fate and Promotes Epidermal Fate. Tissue Dissociation dilutes BMP-4 activity (Wilson and Hemmati-Brivanlou, 1995) + BMP-4 (Endogenous BMP-4 Diluted) Neural Epidermal Lecture IV. Mechanisms of Neural Development

  24. Recombinant BMP-4Promotes Epidermal Fate and InhibitsNeuronal Fate Dispersed caps Intact caps Keratin (epidermal marker) NCAM (neuronal marker) (Wilson and Hemmati-Brivanlou, 1995) Lecture IV. Mechanisms of Neural Development

  25. Blastopore Stg 14.0 Stg 11.5 Stg 11.0 D D D D A V V P V V Neural Crest Stg 23.0 Stg 24.0 (Fainsod, et al., 1995) A A D A P P V P BMP-4 mRNA is Expressed in Presumptive Ectoderm Lecture IV. Mechanisms of Neural Development

  26. Blockers Lecture IV. Mechanisms of Neural Development

  27. Are there native anatgonists of BMP-4? Secreted from underlying mesoderm? Yes… chordin / noggin / follistatin. And they are enriched in the Spemann-Mangold Organizer! Lecture IV. Mechanisms of Neural Development

  28. Noggin cRNA injections rescue ventralized embryos Chordin expressed in mesoderm +Noggin injection 1pg 10pg (Sasai, et al., 1995) 100pg (Smith and Harland, 1992) Lecture IV. Mechanisms of Neural Development

  29. Differential Substractive Screen yields Chordin, a BMP-4 antagonist (1994) Generate cDNA library from oocytes Probe cDNA library with differential probes Lecture IV. Mechanisms of Neural Development

  30. (Reiterate with positive fraction) Functional Expression Cloning yields noggin, a BMP-4 anatagonist (1992) (Smith and Harland, 1992) Lecture IV. Mechanisms of Neural Development

  31. Chordin/Noggin/Follistatin directly bind to and inactivate BMP-4 (Stays in loading well) (Migrates into gel) Lecture IV. Mechanisms of Neural Development

  32. Structure of Noggin-BMP complex noggin Binding Sites (Groppe, et al., 2002) Receptor (Type-II) Receptor (Type-I) BMP-7 Lecture IV. Mechanisms of Neural Development

  33. Molecular Mechanism of Neuralization Lecture IV. Mechanisms of Neural Development

  34. Current View Lecture IV. Mechanisms of Neural Development

  35. TGF-b proteins signal through heterodimeric receptors and Smad transcription factors Multi-step pathway (kinases, scaffolding proteins) Lecture IV. Mechanisms of Neural Development

  36. Vertebrates Drosophila Ligand Receptor Antagonist Transcription Factor BMP-4 Type I Type II Type III noggin chordin follistatin Smad1 Smad2 Smad3 Smad4 Smad5 decapentaplegic (dpp) punt thick veins (tkv), saxophone (sax) Short-gastrulation (sog) Mothers against decapentaplegic (MAD) Medea Neural induction mechanisms are conserved: Lecture IV. Mechanisms of Neural Development

  37. BMP-4 is only one member of the large evolutionarily conserved TGF-b gene family, which mediates many different tissue inductive events. Relationships between members of the TGF-b super family. (After Hogan, 1996) Lecture IV. Mechanisms of Neural Development

  38. Summary Lecture IV. Mechanisms of Neural Development

  39. Neurogenesis: Inductive Mechanisms 1. Neuroectodermal cells choose either a neuronal or epidermal fate. 2. Interactions between mesoderm and neuroectoderm induce neuroectoderm to adopt the neural fate. 3. Induction is signaled by Bone Morphogenic Protein-4 (BMP-4), a protein made and secreted by neuroectodermal cells. 4. BMP-4 inhibits neuralization and promotes the epidermal fate in neighboring cells. 5. Mesodermal cells secrete proteins (Chordin, Noggin, Follistatin) which directly bind and antagonizes BMP-4 activity. Lecture IV. Mechanisms of Neural Development

  40. Neurogenesis: Inductive Mechanisms 6. Neuroectodermal cells become neurons by suppression of BMP-4 activity by secreted antagonists from underlying mesodermal cells. 7. The “default” state of neuroectodermal cells is neuronal. 8. This mechanism is conserved between vertebrates and invertebrates. 9. BMP-4 is a member of the Transforming Growth Factor (TGF-b) family of signaling molecules. 10. Similar signaling events in the nervous system mediate changes in later development stages and in adult plasticity. Lecture IV. Mechanisms of Neural Development

  41. END Lecture IV. Mechanisms of Neural Development

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