Lecture IV. Mechanisms of Neural. Neural Development

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1 Lecture IV. Mechanisms of Neural Bio 3411 Monday 1 Readings NEUROSCIENCE: 5 th ed, pp (sorta) 4 th ed, pp (sorta) References : Fainsod, A., Steinbeisser, H., & De Robertis, E. M. (1994). EMBO J, 13(21), Hemmati-Brivanlou, A., & Melton, D. (1997). Annu Rev Neurosci, 20, Melton, D. A. (1987). Nature, 328(6125), Sasai, Y., & De Robertis, E. M. (1997). Dev Biol, 182(1), Smith, W. C., & Harland, R. M. (1992). Cell, 70(5), Weeks, D. L., & Melton, D. A. (1987). Proc Natl Acad Sci U S A, 84(9), Wilson, P. A., & Hemmati-Brivanlou, A. (1995). Nature, 376(6538), Xanthos, J. B., Kofron, M., Wylie, C., & Heasman, J. (2001)., 128(2), Zimmerman, L. B., de Jesus-Escobar, J. M., & Harland, R. M. (1996). Cell, 86(4), (pdfs on course websites: [[ 2 1

2 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. Gastrulation. 6. Spemann organizer creates anterior-posterior axis. 7. Notocord induces the Neural Plate. 8. Neurulation forms the Neural Tube. 9. Neural crest cells form the PNS. 10. Segmentation & Cephalization (anterior enlargement) 3 1) Cell Signaling 2) Discovery of the Organizer 3) How Could this Work? 4) The Answer 5) Blockers 6) Current View 7) Summary 4 2

3 Cell Signaling 5 Neuroinduction 6 3

4 Intracellular Signaling through a Kinase Cascade; Signal Amplification (Suppression) and Multiple Control Points 7 Endoderm and Mesoderm involute with gastrulation: Induction of the Neural Plate from Neuroectoderm, by the underlying, closely apposed Mesoderm. 8 4

5 Discovery of the Organizer 9 Hilde Mangold and Hans Spemann Key experiments performed in 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

6 Mangold Spemann Experiments (1924) 11 How Could this Work? 12 6

7 Explant Experiments with Animal Caps from Amphibian Blastula: Puzzling Results! 13 Isolating Inducing Factors that Promote Neuronal Differentiation; Sigma Catalog Experiments Result in Further Confusion (Many positives, including apparently non-biological factors!)? + Candidate Neuroinducing Factors (Intact) 14 7

8 Models for Neural Induction Model 1: Presumptive Neuroectoderm + Epidermal factor + Neuronal factor Epidermis Neurons Model 2: Presumptive Neuroectoderm + Neuronal factor Epidermis ( default ) Neurons Model 3: Presumptive Neuroectoderm + Epidermal factor Epidermis Neurons ( default ) 15 TGF-β Proteins Signal Through Heterodimeric Receptors and Smad Transcription Factors 16 8

9 The Answer 17 A Dominant-Negative Receptor Subunit Blocks Activation of the Signaling Pathway (Hemmati-Brivanlou and Melton, 1992) 18 9

10 Blocking TGF-β Signaling by a Dominant-Negative Receptor Causes Isolated Neuroectoderm to Become Neuronal (+Dominant-Negative Type II Receptor crna) Animal Cap (Intact) Animal Cap (Intact) TFG-β Signaling Blocked by expression of Dom-Neg Type II Receptor Subunit + TGF-β Signaling (Intact) + TGF-β Signaling 19 BMP-4 (TGF-β) Signaling Results in Neural Epidermal Induction TGF-β: Transforming Growth Factor - β BMP-4: Bone Morphogenic Protein

11 Models for Neural Induction Model 1: Presumptive Neuroectoderm + Epidermal factor + Neuronal factor Epidermis Neurons Model 2: Presumptive Neuroectoderm + Neuronal factor Epidermis ( default ) Neurons Model 3: Presumptive Neuroectoderm +BMP-4 Epidermis Neurons ( default ) 21 BMP-4 (Secreted by Neuroectodermal Cells) Inhibits Neuronal Fate and Promotes Epidermal Fate. Tissue Dissociation dilutes BMP-4 activity (Wilson and Hemmati-Brivanlou, 1995) Neural [BMP-4] (Endogenous BMP-4 Diluted) + BMP-4 Epidermal 22 11

12 Recombinant BMP-4 Promotes Epidermal Fate and Inhibits Neuronal Fate (Wilson and Hemmati-Brivanlou, 1995) 23 BMP-4 mrna is Expressed in Presumptive Ectoderm 24 12

13 Blockers 25 Are there native anatgonists of BMP-4? Secreted from underlying mesoderm? Yes chordin / noggin / follistatin. And they are enriched in the Spemann-Mangold Organizer! 26 13

14 27 Differential Substractive Screen yields Chordin, a BMP-4 antagonist (1994) 28 14

15 Functional Expression Cloning yields noggin, a BMP-4 anatagonist (1992) 29 Chordin/Noggin/Follistatin directly bind to and inactivate BMP

16 Structure of Noggin-BMP complex 31 Molecular Mechanism of Neuralization 32 16

17 Current View 33 TGF-β proteins signal through heterodimeric receptors and Smad transcription factors 34 17

18 Neural induction mechanisms are conserved: Ligand Receptor Antagonist Transcription Factor Vertebrates BMP-4 Type I Type II Type III noggin chordin follistatin Smad1 Smad2 Smad3 Smad4 Smad5 Drosophila decapentaplegic (dpp) punt thick veins (tkv), saxophone (sax) Short-gastrulation (sog) Mothers against decapentaplegic (MAD) Medea 35 BMP-4 is only one member of the large evolutionarily conserved TGF-β gene family, which mediates many different tissue inductive events. Relationships between members of the TGF-β super family. (After Hogan, 1996) 36 18

19 Summary 37 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

20 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-β) family of signaling molecules. 10. Similar signaling events in the nervous system mediate changes in later development stages and in adult plasticity. 39 END 40 20