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Neuroinduction. Diffusible morphogen. Ligand. Receptor. Kinase Cascade. A. C. B. Effector Proteins ( transcription factors , ion channels, cytoskeletal proteins, enzymes, etc…). Scaffolding Proteins bind multiple signaling molecules to organize specific signaling pathways.

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Diffusible morphogen

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Diffusible morphogen

Neuroinduction

Diffusible morphogen


Diffusible morphogen

Ligand

Receptor

Kinase Cascade

A

C

B

Effector Proteins

(transcription factors,

ion channels,

cytoskeletal proteins,

enzymes, etc…)

Scaffolding Proteinsbind

multiple signaling molecules

toorganize specific

signaling pathways

Gene Activation/Repression

Intracellular Signaling through a Kinase Cascade;

Signal Amplification and Multiple Control Points


Diffusible morphogen

Ant

Post

Endoderm and Mesoderm involute with gastrulation:

Induction of the Neural Plate from Neuroectoderm,

by the underlying, closely apposed Mesoderm.


Diffusible morphogen

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.


Diffusible morphogen

Hilde Mangold and Hans Spemann

Experiments (1924)


Diffusible morphogen

!

Explant Experiments with Animal Caps

from AmphibianBlastula: Puzzling Results…


Diffusible morphogen

+ Candidate

Neuroinducing

Factors

?

(Intact)

Isolating Inducing Factors that Promote Neuronal

Differentiation; “Sigma Catalog” Experiments

Result in Further Confusion…

(Many positives, including

apparently non-biological

factors!)


Diffusible morphogen

Model 2:

Epidermis

Presumptive

Neuroectoderm

(“default”)

Neurons

+”Neuronal

factor”

Model 3:

+”Epidermal

factor”

Epidermis

Presumptive

Neuroectoderm

Neurons

(“default”)

Models for Neural Induction

Model 1:

+”Epidermal

factor”

Epidermis

Presumptive

Neuroectoderm

Neurons

+”Neuronal

factor”


Diffusible morphogen

Vg1

(Melton, 1987; Weeks and Melton, 1987)

(K. Mowry Lab, Brown Univ.)

Multi-step pathway

(kinases, scaffolding

proteins)

TGF-b Proteins Signal Through Heterodimeric

Receptors and Smad Transcription Factors


Diffusible morphogen

A Dominant-Negative Receptor Subunit

Blocks Activation of the Signaling Pathway

(Hemmati-Brivanlou and Melton, 1992)


Diffusible morphogen

(+Dominant-Negative

Type II Receptor cRNA)

TFG-b Signaling

Blocked by expression

of Dom-Neg Type II

Receptor Subunit

Animal Cap

(Intact)

Animal Cap

(Intact)

+ TGF-b

Signaling

+ TGF-b

Signaling

(Intact)

Blocking TGF-b Signaling by

a Dominant-Negative Receptor Causes

Isolated Neuroectoderm to Become Neuronal


Diffusible morphogen

BMP-4 (TGF-b) Signaling Results

in “NeuralEpidermal Induction”

TGF-b: Transforming GrowthFactor - b

BMP-4: BoneMorphogenic Protein - 4


Diffusible morphogen

Model 1:

Epidermis

Presumptive

Neuroectoderm

Neurons

+”Neuronal

factor”

Model 2:

Epidermis

Presumptive

Neuroectoderm

(“default”)

Neurons

+”Neuronal

factor”

Model 3:

Epidermis

Presumptive

Neuroectoderm

Neurons

(“default”)

Models for Neural Induction

+”Epidermal

factor”

+”Epidermal

factor”

+BMP-4


Diffusible morphogen

+ BMP-4

[BMP-4]

BMP-4 (Secreted by Neuroectodermal Cells)

Inhibits Neuronal Fate and Promotes Epidermal Fate.

Tissue Dissociation dilutes BMP-4 activity

(Endogenous

BMP-4 Diluted)

(Wilson and Hemmati-Brivanlou, 1995)


Diffusible morphogen

Recombinant BMP-4Promotes

Epidermal Fate and InhibitsNeuronal Fate

Dispersed caps

Intact caps

Keratin

(epidermal

marker)

NCAM

(neuronal

marker)

(Wilson and Hemmati-Brivanlou, 1995)


Diffusible morphogen

Blastopore

Stg 14.0

Stg 11.5

Stg 11.0

A

D

D

D

D

V

V

P

V

V

Neural Crest

Stg 23.0

Stg 24.0

(Fainsod, et al., 1995)

A

A

D

A

P

P

V

P

BMP-4 mRNA is Expressed

in Presumptive Ectoderm


Diffusible morphogen

Are there native anatgonists of BMP-4?

Secreted from underlying mesoderm?

Yes… chordin / noggin / follistatin.

And they are enriched in the

Spemann-Mangold Organizer!


Diffusible morphogen

Noggin cRNA injections

rescue ventralized embryos

Chordin expresses

in mesoderm

+Noggin injection

1pg

10pg

(Sasai, et al., 1995)

100pg

(Smith and Harland, 1992)


Diffusible morphogen

Differential Substractive Screen

yields Chordin, a BMP-4 antagonist (1994)

Generate

cDNA library

from oocytes

Probe

cDNA library

with differential

probes

(Sasai and DeRobertis. 1994)


Diffusible morphogen

(Reiterate

with positive

fraction)

Functional Expression Cloning

yields noggin, a BMP-4 anatagonist (1992)

(Smith and Harland, 1992)


Diffusible morphogen

Mechanism: Chordin / noggin / follistatin anatagonize BMP-4

activity by directly binding and inactivating BMP-4

(Migrates

into gel)

(Stays in

loading well)


Diffusible morphogen

noggin

Receptor

(Type-II)

Receptor

(Type-I)

BMP-7

Crystal structure of Noggin-BMP complex confirms

biochemical/functional studies and identifies binding sites

Binding

Sites

(Groppe, et al., 2002; Choe Lab)


Diffusible morphogen

Molecular Mechanism of Neuralization


Diffusible morphogen

TGF-b proteins signal through heterodimeric

receptors and Smad transcription factors

Multi-step pathway

(kinases, scaffolding

proteins)


Diffusible morphogen

Vertebrates

Drosophila

Ligand

Receptor

Antagonist

Transcription

Factor

BMP-4

Type I

Type II

Type III

noggin

chordin

follistatin

Smad1

Smad2

Smad3

Smad4

Smad5

decapentaplegic (dpp)

punt

thick veins (tkv), saxophone (sax)

Short-gastrulation (sog)

Mothers against decapentaplegic (MAD)

Medea

The mechanism for neural induction is

evolutionarily conserved between

vertebrates and invertebrates


Diffusible morphogen

BMP-4 is only one member

of the large evolutionarily

conserved TGF-b gene

family, which mediates

many different tissue

inductive events.


Diffusible morphogen

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.


Diffusible morphogen

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.


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