Diffusible morphogen Neuroinduction. Intracellular Signaling through a Kinase Cascade; Signal...
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Transcript of Diffusible morphogen Neuroinduction. Intracellular Signaling through a Kinase Cascade; Signal...
![Page 1: Diffusible morphogen Neuroinduction. Intracellular Signaling through a Kinase Cascade; Signal Amplification and Multiple Control Points Ligand Receptor.](https://reader035.fdocuments.us/reader035/viewer/2022070411/56649cd55503460f9499c80d/html5/thumbnails/1.jpg)
Diffusible morphogen
Neuroinduction
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Intracellular Signaling through a Kinase Cascade;Signal Amplification and Multiple Control Points
Ligand
Receptor
Kinase Cascade
Gene Activation/Repression
Effector Proteins(transcription factors,ion channels,cytoskeletal proteins,enzymes, etc…)
A
BC
Scaffolding Proteins bindmultiple signaling moleculesto organize specific signaling pathways
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Endoderm and Mesoderm involute with gastrulation:Induction of the Neural Plate from Neuroectoderm,
by the underlying, closely apposed Mesoderm.
Ant
Post
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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.
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Hilde Mangold and Hans Spemann Experiments (1924)
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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
PresumptiveNeuroectoderm
Epidermis
Neurons
+”Epidermalfactor”
+”Neuronalfactor”
Model 1:
PresumptiveNeuroectoderm
Epidermis
Neurons+”Neuronalfactor”
(“default”)
Model 2:
PresumptiveNeuroectoderm
Epidermis
Neurons
+”Epidermalfactor”
(“default”)
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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A Dominant-Negative Receptor SubunitBlocks Activation of the Signaling Pathway
(Hemmati-Brivanlou and Melton, 1992)
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Blocking TGF- Signaling bya Dominant-Negative Receptor Causes
Isolated Neuroectoderm to Become Neuronal
(Intact)
+ TGF-Signaling
+ TGF-Signaling
Animal Cap(Intact)
(+Dominant-Negative Type II Receptor cRNA)
TFG- SignalingBlocked by expressionof Dom-Neg Type IIReceptor Subunit
Animal Cap(Intact)
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TGF-Transforming Growth Factor - BMP-4Bone Morphogenic Protein - 4
BMP-4 (TGF- Signaling Resultsin “Neural Epidermal Induction”
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Models for Neural Induction
+BMP-4+”Epidermalfactor”
PresumptiveNeuroectoderm
Epidermis
Neurons+”Neuronalfactor”
Model 1:
PresumptiveNeuroectoderm
Epidermis
Neurons+”Neuronalfactor”
(“default”)
Model 2:
Neurons
PresumptiveNeuroectoderm
Epidermis
(“default”)
Model 3:
+”Epidermalfactor”
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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)
+ BMP-4[BMP-4]
(Wilson and Hemmati-Brivanlou, 1995)
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Recombinant BMP-4 PromotesEpidermal Fate and Inhibits Neuronal Fate
NCAM(neuronalmarker)
(Wilson and Hemmati-Brivanlou, 1995)
Intact capsDispersed caps
Keratin(epidermal
marker)
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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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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 expresses 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)
(Sasai and DeRobertis. 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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Mechanism: Chordin / noggin / follistatin anatagonize BMP-4 activity by directly binding and inactivating BMP-4
(Stays in loading well)
(Migrates into gel)
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(Groppe, et al., 2002; Choe Lab)
Crystal structure of Noggin-BMP complex confirmsbiochemical/functional studies and identifies binding sites
noggin
BMP-7
Receptor(Type-II)
Receptor(Type-I)
BindingSites
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Molecular Mechanism of Neuralization
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TGF- proteins signal through heterodimericreceptors and Smad transcription factors
Multi-step pathway(kinases, scaffolding proteins)
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The mechanism for neural induction isevolutionarily conserved between
vertebrates and invertebrates
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.
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Neurogenesis: Inductive Mechanisms
1. Neuroectodermal cells choose either a neuronal or epidermal cell fate.
2. Interactions between mesoderm and neuroectoderm induce neuroectoderm to adopt the neural fate.
3. Induction acts through signaling by a secreted protein, Bone Morphogenic Protein-4 (BMP-4), made 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.
6. Neuroectodermal cells become neurons by suppression of BMP-4 activity by secreted antagonists from underlying mesodermal cells.
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Neurogenesis: Inductive Mechanisms (cont.)
7. The “default” state of neuroectodermal cells is neuronal.
8. This inductive mechanism is conserved between vertebrates and invertebrates.
9. BMP-4 is a member of the Transforming Growth Factor (TGF-) family of signaling molecules. Similar signaling events maybe selectively and focally re-employed later in the nervous system to mediate fine tuning at late developmental stages and
adult plasticity.