lecture iv. mechanisms of neural development
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Lecture IV. Mechanisms of Neural Development. Bio 3411 Wednesday September 2, 2009. T. Woolsey 3802 North Building 362-3601 [email protected]. Readings. NEUROSCIENCE: 4 th ed, pp 545-575 (sorta) References † : - PowerPoint PPT PresentationTRANSCRIPT
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Lecture IV. Mechanisms of Neural Development
Bio 3411 Wednesday
September 2, 2009
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• T. Woolsey
• 3802 North Building
• 362-3601
September 2, 2009 2Lecture IV. Mechanisms of Neural Development
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ReadingsNEUROSCIENCE: 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])
September 2, 2009 3Lecture IV. Mechanisms of Neural Development
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Embryogenesis1. 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. Notocord induces the Neural Plate.8. Neurulation forms the Neural Tube.9. Neural crest cells form the PNS.10. Segmentation & Cephalization (anterior enlargement) August 31, 2009 4Lecture III. Nervous System
Embryology
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1) Cell Signaling
2) Discovery of the Organizer
3) How Could this Work?
4) The Answer
5) Blockers
6) Current View
7) Summary
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Cell Signaling
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Diffusible morphogen
Neuroinduction
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Ligand
Receptor
Intracellular Signaling through a Kinase Cascade;Signal Amplification (Suppression) and Multiple Control Points
Kinase Cascade
GeneActivation/Repression
Effector Proteins(transcription factors,
ion channels,cytoskeletal proteins,
enzymes, etc…)
A
BC
Scaffolding Proteins bindmultiple signaling molecules to organizespecific signaling pathways
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Receptor
Ligand
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Ant
Post
September 2, 2009 9Lecture IV. Mechanisms of Neural Development
Endoderm and Mesoderm involute with gastrulation:Induction of the Neural Plate from Neuroectoderm,
by the underlying, closely apposed Mesoderm.
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Discovery of the Organizer
September 2, 2009 10Lecture IV. Mechanisms of Neural Development
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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.September 2, 2009 11Lecture IV. Mechanisms of Neural
Development
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Mangold –Spemann Experiments (1924)
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How Could this Work?
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Explant Experiments with Animal Caps from Amphibian Blastula: Puzzling Results…
!
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Isolating Inducing Factors that Promote Neuronal Differentiation; “Sigma Catalog” Experiments
Result in Further Confusion…
+ CandidateNeuroinducing
Factors?
(Intact)
(Many positives, including apparently non-biological factors!)
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Models for Neural Induction
+”Epidermalfactor”
+”Neuronalfactor”
PresumptiveNeuroectoderm
Epidermis
Neurons
Model 1:
+”Neuronalfactor”
(“default”)PresumptiveNeuroectoderm
Epidermis
Neurons
Model 2:
+”Epidermalfactor”
(“default”)
PresumptiveNeuroectoderm
Epidermis
Neurons
Model 3:
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TGF- Proteins Signal Through HeterodimericReceptors and Smad Transcription Factors
Multi-step pathway(kinases, scaffolding proteins)
Vg1(Melton, 1987; Weeks and Melton, 1987)
(K. Mowry Lab, Brown Univ.)
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The Answer
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A Dominant-Negative Receptor SubunitBlocks Activation of the Signaling Pathway
September 2, 2009 19Lecture IV. Mechanisms of Neural Development
(Hemmati-Brivanlou and Melton, 1992)
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Blocking TGF- Signaling bya Dominant-Negative Receptor Causes
Isolated Neuroectoderm to Become Neuronal
Animal Cap(Intact)
(Intact)
+ TGF-Signaling
(+Dominant-Negative Type II Receptor cRNA)
TFG- SignalingBlocked by expressionof Dom-Neg Type IIReceptor Subunit
Animal Cap(Intact)
September 2, 2009 20Lecture IV. Mechanisms of Neural Development
+ TGF-Signaling
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TGF-Transforming Growth Factor - BMP-4Bone Morphogenic Protein - 4
September 2, 2009 21Lecture IV. Mechanisms of Neural Development
BMP-4 (TGF- Signaling Resultsin “Neural Epidermal Induction”
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22
+”Epidermalfactor”
+”Neuronalfactor”
PresumptiveNeuroectoderm
Epidermis
Neurons
Model 1:
+”Neuronalfactor”
(“default”)PresumptiveNeuroectoderm
Epidermis
Neurons
Model 2:
(“default”)
PresumptiveNeuroectoderm
Epidermis
Neurons
Model 3:
Models for Neural Induction
+BMP-4
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BMP-4 (Secreted by Neuroectodermal Cells) Inhibits Neuronal Fate and Promotes Epidermal Fate.
Tissue Dissociation dilutes BMP-4 activity
(EndogenousBMP-4 Diluted)
(Wilson and Hemmati-Brivanlou, 1995)
September 2, 2009 23Lecture IV. Mechanisms of Neural Development
[BMP-4]
Neural
+ BMP-4
Epidermal
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Recombinant BMP-4 PromotesEpidermal Fate and Inhibits Neuronal Fate
NCAM(neuronalmarker)
(Wilson and Hemmati-Brivanlou, 1995)
Intact capsDispersed caps
Keratin(epidermalmarker)
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BMP-4 mRNA is Expressed in Presumptive Ectoderm
(Fainsod, et al., 1995)
Blastopore
Stg 11.5 Stg 11.0 Stg 14.0
Stg 23.0 Stg 24.0A P A P
D
V
D
V
A PD V
D
V
D
V
A
P
Neural Crest
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Blockers
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Are there native anatgonists of BMP-4?Secreted from underlying mesoderm?
Yes… chordin / noggin / follistatin.
And they are enriched in the Spemann-Mangold Organizer!
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Chordin expressed in mesoderm
(Sasai, et al., 1995)
Noggin cRNA injections rescue ventralized embryos
+Noggin injection
1pg
10pg
100pg
(Smith and Harland, 1992)
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Differential Substractive Screen yields Chordin, a BMP-4 antagonist (1994)
GeneratecDNA libraryfrom oocytes
ProbecDNA librarywith differentialprobes
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Functional Expression Cloning yields noggin, a BMP-4 anatagonist (1992)
(Smith and Harland, 1992)
(Reiteratewith positivefraction)
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(Stays in loading well)
(Migrates into gel)
September 2, 2009 31Lecture IV. Mechanisms of Neural Development
Chordin/Noggin/Follistatin directly bind to and inactivate BMP-4
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noggin
BMP-7
Receptor(Type-II) Receptor
(Type-I)
Structure of Noggin-BMP complex
BindingSites
September 2, 2009 32Lecture IV. Mechanisms of Neural Development
(Groppe, et al., 2002)
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Molecular Mechanism of Neuralization
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Current View
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TGF- proteins signal through heterodimericreceptors and Smad transcription factors
Multi-step pathway(kinases, scaffolding proteins)
September 2, 2009 35Lecture IV. Mechanisms of Neural Development
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Neural induction mechanisms are conserved:
Ligand
Receptor
Antagonist
TranscriptionFactor
BMP-4
Type IType IIType III
nogginchordin
follistatin
Smad1Smad2Smad3Smad4Smad5
Vertebrates
decapentaplegic (dpp)
puntthick veins (tkv), saxophone (sax)
Short-gastrulation (sog)
Mothers against decapentaplegic (MAD)
Medea
Drosophila
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BMP-4 is only one member of the large evolutionarily conserved TGF- gene family, which mediates many different tissue inductive events.
September 2, 2009 37Lecture IV. Mechanisms of Neural Development
Relationships between members of the TGF- super family. (After Hogan, 1996)
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Summary
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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.
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Neurogenesis: Inductive Mechanisms
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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-) family of signaling molecules.
10. Similar signaling events in the nervous system mediate changes in later development stages and in adult plasticity.
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Neurogenesis: Inductive Mechanisms
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