Upload
tuvya
View
29
Download
3
Tags:
Embed Size (px)
DESCRIPTION
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 Presentation
Citation preview
Lecture IV. Mechanisms of Neural Development
Bio 3411 Wednesday
September 2, 2009
• T. Woolsey
• 3802 North Building
• 362-3601
September 2, 2009 2Lecture IV. Mechanisms of Neural Development
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
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
1) Cell Signaling
2) Discovery of the Organizer
3) How Could this Work?
4) The Answer
5) Blockers
6) Current View
7) Summary
September 2, 2009 5Lecture IV. Mechanisms of Neural Development
Cell Signaling
September 2, 2009 6Lecture IV. Mechanisms of Neural Development
Diffusible morphogen
Neuroinduction
September 2, 2009 7Lecture IV. Mechanisms of Neural Development
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
September 2, 2009 8Lecture IV. Mechanisms of Neural Development
Receptor
Ligand
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.
Discovery of the Organizer
September 2, 2009 10Lecture IV. Mechanisms of Neural Development
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
Mangold –Spemann Experiments (1924)
September 2, 2009 12Lecture IV. Mechanisms of Neural Development
How Could this Work?
September 2, 2009 13Lecture IV. Mechanisms of Neural Development
Explant Experiments with Animal Caps from Amphibian Blastula: Puzzling Results…
!
Isolating Inducing Factors that Promote Neuronal Differentiation; “Sigma Catalog” Experiments
Result in Further Confusion…
+ CandidateNeuroinducing
Factors?
(Intact)
(Many positives, including apparently non-biological factors!)
September 2, 2009 15Lecture IV. Mechanisms of Neural Development
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:
September 2, 2009 16Lecture IV. Mechanisms of Neural Development
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.)
September 2, 2009 17Lecture IV. Mechanisms of Neural Development
The Answer
September 2, 2009 18Lecture IV. Mechanisms of Neural Development
A Dominant-Negative Receptor SubunitBlocks Activation of the Signaling Pathway
September 2, 2009 19Lecture IV. Mechanisms of Neural Development
(Hemmati-Brivanlou and Melton, 1992)
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
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”
September 2, 2009 Lecture IV. Mechanisms of Neural Development
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
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
Recombinant BMP-4 PromotesEpidermal Fate and Inhibits Neuronal Fate
NCAM(neuronalmarker)
(Wilson and Hemmati-Brivanlou, 1995)
Intact capsDispersed caps
Keratin(epidermalmarker)
September 2, 2009 24Lecture IV. Mechanisms of Neural Development
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
September 2, 2009 25Lecture IV. Mechanisms of Neural Development
Blockers
September 2, 2009 26Lecture IV. Mechanisms of Neural Development
Are there native anatgonists of BMP-4?Secreted from underlying mesoderm?
Yes… chordin / noggin / follistatin.
And they are enriched in the Spemann-Mangold Organizer!
September 2, 2009 27Lecture IV. Mechanisms of Neural Development
Chordin expressed in mesoderm
(Sasai, et al., 1995)
Noggin cRNA injections rescue ventralized embryos
+Noggin injection
1pg
10pg
100pg
(Smith and Harland, 1992)
September 2, 2009 28Lecture IV. Mechanisms of Neural Development
Differential Substractive Screen yields Chordin, a BMP-4 antagonist (1994)
GeneratecDNA libraryfrom oocytes
ProbecDNA librarywith differentialprobes
September 2, 2009 29Lecture IV. Mechanisms of Neural Development
Functional Expression Cloning yields noggin, a BMP-4 anatagonist (1992)
(Smith and Harland, 1992)
(Reiteratewith positivefraction)
September 2, 2009 30Lecture IV. Mechanisms of Neural Development
(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
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)
Molecular Mechanism of Neuralization
September 2, 2009 33Lecture IV. Mechanisms of Neural Development
Current View
September 2, 2009 34Lecture IV. Mechanisms of Neural Development
TGF- proteins signal through heterodimericreceptors and Smad transcription factors
Multi-step pathway(kinases, scaffolding proteins)
September 2, 2009 35Lecture IV. Mechanisms of Neural Development
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
September 2, 2009 36Lecture IV. Mechanisms of Neural Development
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)
Summary
September 2, 2009 38Lecture IV. Mechanisms of Neural Development
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.
September 2, 2009 39Lecture IV. Mechanisms of Neural Development
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-) family of signaling molecules.
10. Similar signaling events in the nervous system mediate changes in later development stages and in adult plasticity.
September 2, 2009 40Lecture IV. Mechanisms of Neural Development
Neurogenesis: Inductive Mechanisms
END
September 2, 2009 Lecture IV. Mechanisms of Neural Development
41