Diffusible morphogen

Neuroinduction

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

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; “Sigma Catalog” Experiments

Result in Further Confusion…

+ CandidateNeuroinducing

Factors?

(Intact)

(Many positives, including apparently non-biological factors!)

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:

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.)

A Dominant-Negative Receptor SubunitBlocks Activation of the Signaling Pathway

(Hemmati-Brivanlou and Melton, 1992)

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)

TGF-Transforming Growth Factor - BMP-4Bone Morphogenic Protein - 4

BMP-4 (TGF- Signaling Resultsin “Neural Epidermal Induction”

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”

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)

Recombinant BMP-4 PromotesEpidermal Fate and Inhibits Neuronal Fate

NCAM(neuronalmarker)

(Wilson and Hemmati-Brivanlou, 1995)

Intact capsDispersed caps

Keratin(epidermal

marker)

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

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

(Sasai, et al., 1995)

Noggin cRNA injections rescue ventralized embryos

+Noggin injection

1pg

10pg

100pg

(Smith and Harland, 1992)

Differential Substractive Screen yields Chordin, a BMP-4 antagonist (1994)

(Sasai and DeRobertis. 1994)

GeneratecDNA libraryfrom oocytes

ProbecDNA librarywith differentialprobes

Functional Expression Cloning

yields noggin, a BMP-4 anatagonist (1992)

(Smith and Harland, 1992)

(Reiteratewith positivefraction)

Mechanism: Chordin / noggin / follistatin anatagonize BMP-4 activity by directly binding and inactivating BMP-4

(Stays in loading well)

(Migrates into gel)

(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

Molecular Mechanism of Neuralization

TGF- proteins signal through heterodimericreceptors and Smad transcription factors

Multi-step pathway(kinases, scaffolding proteins)

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

BMP-4 is only one member of the large evolutionarily conserved TGF- 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-) 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.