lecture iv. mechanisms of neural development

Lecture IV. Mechanisms of Neural Development

Bio 3411 Monday

September 13, 2010

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 websites: [http://artsci.wustl.edu/~bio3411/] & [http://www.nslc.wustl.edu/courses/Bio3411/bio3411.html]

September 13, 2010 2Lecture IV. Mechanisms of Neural Development

Embryogenesis

1. 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)

September 13, 2010 3Lecture IV. Mechanisms of Neural

Development

1) Cell Signaling

2) Discovery of the Organizer

3) How Could this Work?

4) The Answer

5) Blockers

6) Current View

7) Summary


Cell Signaling


Diffusible morphogen

Neuroinduction


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


Receptor

Ligand

Ant

Post


Endoderm and Mesoderm involute with gastrulation:Induction of the Neural Plate from Neuroectoderm,

by the underlying, closely apposed Mesoderm.

Discovery of the Organizer


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.


Mangold –Spemann Experiments (1924)


How Could this Work?


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

+”Epidermalfactor”

+”Neuronalfactor”

PresumptiveNeuroectoderm

Epidermis

Neurons

Model 1:


(“default”)PresumptiveNeuroectoderm

Epidermis

Neurons

Model 2:


(“default”)


Epidermis

Neurons

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.)


The Answer


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

Animal Cap(Intact)

(Intact)

+ TGF-Signaling

(+Dominant-Negative Type II Receptor cRNA)

TFG- SignalingBlocked by expressionof Dom-Neg Type IIReceptor Subunit

Animal Cap(Intact)


+ TGF-Signaling

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


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

September 13, 2010 Lecture IV. Mechanisms of Neural Development

21




Epidermis

Neurons

Model 1:


(“default”)PresumptiveNeuroectoderm

Epidermis

Neurons

Model 2:

(“default”)


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)


[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(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


Blockers


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 expressed 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)

GeneratecDNA libraryfrom oocytes

ProbecDNA librarywith differentialprobes


Functional Expression Cloning

yields noggin, a BMP-4 anatagonist (1992)

(Smith and Harland, 1992)

(Reiteratewith positivefraction)


(Stays in loading well)

(Migrates into gel)


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


(Groppe, et al., 2002)

Molecular Mechanism of Neuralization


Current View


TGF- proteins signal through heterodimericreceptors and Smad transcription factors

Multi-step pathway(kinases, scaffolding proteins)


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


BMP-4 is only one member of the large evolutionarily conserved TGF- gene family, which mediates many different tissue inductive events.


Relationships between members of the TGF- super family. (After Hogan, 1996)

Summary


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.


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.


Neurogenesis: Inductive Mechanisms

END

September 13, 2010 Lecture IV. Mechanisms of Neural Development

40

lecture iv. mechanisms of neural development

Documents

neural plate

neural tube

neural crest cells

hans spemann key experiments

mesoderm involute

spemann organizer

apposed mesoderm

anteriorposterior axis