Download + BMP-4

Neuroinduction
Diffusible morphogen
Endoderm and Mesoderm involute with gastrulation:
Induction of the Neural Plate from Neuroectoderm,
by the underlying, closely apposed Mesoderm.
Ant
Post
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.
Hilde Mangold and Hans Spemann
Experiments (1924)
Explant experiments with Animal Caps
from Amphibian Blastula: Puzzling results…
!
Isolating inducing factors that promote Neuronal
differentiation; The “Sigma catalog” experiments
further confusion…
?
(Intact)
+ Candidate
Neuroinducing
Factors
Models for Neural Induction
Model 1:
+”Epidermal
factor”
Epidermis
Presumptive
Neuroectoderm
+”Neuronal
factor”
Neurons
Model 2:
Epidermis
(“default”)
Presumptive
Neuroectoderm
+”Neuronal
factor”
Neurons
Model 3:
+”Epidermal
factor”
Epidermis
Presumptive
Neuroectoderm
Neurons
(“default”)
TGF-b Proteins Signal Through Heterodimeric
Receptors and Smad Transcription Factors
A Dominant-Negative Receptor Subunit
Blocks Activation of the Signaling Pathway
(Hemmati-Brivanlou and Melton, 1992)
Blocking TGF-b signaling by
a dominant-negative receptor causes
isolated Neuroectoderm to become Neuronal
(+Dominant-Negative
Type II
Receptor cRNA)
TFG-b Signaling
Blocked by expression
of Dom-Neg Type II
Receptor Subunit
Animal Cap
(Intact)
+ TGF-b
Signaling
Animal Cap
(Intact)
+ TGF-b
Signaling
(Intact)
BMP-4 (TGF-b) Signaling Results
in “Neural Epidermal Induction”
TGF-b: Transforming Growth Factor - b
BMP-4: Bone Morphogenic Protein - 4
Models for Neural Induction
Model 1:
Epidermis
Presumptive
Neuroectoderm
+”Neuronal
factor”
Neurons
Model 2:
Epidermis
(“default”)
Presumptive
Neuroectoderm
+”Neuronal
factor”
Neurons
Model 3:
+”Epidermal
+BMP-4
factor”
Epidermis
Presumptive
Neuroectoderm
Neurons
(“default”)
BMP-4 (Secreted by Neuroectodermal Cells)
Inhibits Neuronal Fate and Promotes Epidermal Fate.
Tissue Dissociation dilutes BMP-4 activity
[BMP-4]
+ BMP-4
(Endogenous
BMP-4 Diluted)
Recombinant BMP-4 promotes
epidermal fate and inhibits neuronal fate
Dispersed caps
Intact caps
Keratin
(epidermal
marker)
NCAM
(neuronal
marker)
(Wilson and Hemmati-Brivanlou, 1995)
BMP-4 mRNA is expressed in
presumptive ectoderm
Blastopore
(Fainsod, et al., 1995)
Are there native anatgonists of BMP-4?
Secreted from underlying mesoderm?
Yes… chordin / noggin / follistatin.
And they are enriched in the
Spemann-Mangold Organizer!
Chordin expresses
in mesoderm
Noggin cRNA injections
rescue ventralized embryos
+Noggin injection
1pg
(Sasai, et al., 1995)
10pg
100pg
(Smith and Harland, 1992)
Differential Substractive Screen
yields Chordin, a BMP-4 antagonist (1994)
(Sasai and DeRobertis. 1994)
Functional Expression Cloning
yields noggin, a BMP-4 anatagonist (1992)
(Smith and Harlan, 1992)
Mechanism: Chordin / noggin / follistatin anatagonize BMP-4
activity by directly binding and inactivating BMP-4
(Stays in
loading well)
(Migrates
into gel)
Crystal structure of Noggin-BMP complex confirms
biochemical/functional studies and identifies binding sites
Binding
Sites
noggin
Receptor
(Type-II)
BMP-7
Receptor
(Type-I)
(Groppe, et al., 2002)
Molecular Mechanism of Neuralization
TGF-b proteins signal through heterodimeric
receptors and Smad transcription factors
The mechanism for neural induction is
evolutionarily conserved between
vertebrates and invertebrates
Vertebrates
Drosophila
Ligand
BMP-4
decapentaplegic (dpp)
Receptor
Type I
Type II
Type III
punt
thick veins (tkv), saxophone (sax)
Antagonist
noggin
chordin
follistatin
Transcription
Factor
Smad1
Smad2
Smad3
Smad4
Smad5
Short-gastrulation (sog)
Mothers against decapentaplegic (MAD)
Medea
BMP-4 is only one member
of the large evolutionarily
conserved TGF-b gene
family, which mediates
many different tissue
inductive events.
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.
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-b)
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.